Physiol Rev 94: 1249 –1285, 2014
doi:10.1152/physrev.00008.2014
NEURONAL SUMOYLATION: MECHANISMS,
PHYSIOLOGY, AND ROLES IN NEURONAL
DYSFUNCTION
Jeremy M. Henley, Tim J. Craig, and Kevin A. Wilkinson
School of Biochemistry, University of Bristol, Bristol, United Kingdom
Henley JM, Craig TJ, Wilkinson KA. Neuronal SUMOylation: Mechanisms, Physiology, and Roles in Neuronal Dysfunction. Physiol Rev 94: 1249 –1285, 2014;
doi:10.1152/physrev.00008.2014.—Protein SUMOylation is a critically important
posttranslational protein modification that participates in nearly all aspects of cellular
physiology. In the nearly 20 years since its discovery, SUMOylation has emerged as a
major regulator of nuclear function, and more recently, it has become clear that SUMOylation has
key roles in the regulation of protein trafficking and function outside of the nucleus. In neurons,
SUMOylation participates in cellular processes ranging from neuronal differentiation and control of
synapse formation to regulation of synaptic transmission and cell survival. It is a highly dynamic and
usually transient modification that enhances or hinders interactions between proteins, and its
consequences are extremely diverse. Hundreds of different proteins are SUMO substrates, and
dysfunction of protein SUMOylation is implicated in a many different diseases. Here we briefly
outline core aspects of the SUMO system and provide a detailed overview of the current understanding of the roles of SUMOylation in healthy and diseased neurons.
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INTRODUCTION
DISCOVERY AND RELEVANCE OF...
SUMOYLATION AND DESUMOYLATION
SUMOYLATION CONSENSUS MOTIFS
MOLECULAR CONSEQUENCES OF...
SIMS AND RELATED SUMO ...
REGULATION OF PROTEIN ...
PARALOG SPECIFICITY OF ...
SUMOYLATION IN NEURONS
SUMOYLATION IN NEURONAL ...
SUMOYLATION IN ROD AND CONE...
SUMOYLATION IN SYNAPSE...
REGULATION OF ION CHANNELS...
SUMOYLATION IN POLARIZED ...
SYNAPTIC SUMOYLATION AND ...
SUMOYLATION AND SYNAPTIC...
SUMOYLATION AND SYNAPTIC...
POSSIBLE FUTURE DIRECTIONS...
SUMOYLATION AND NEURONAL...
SUMMARY
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I. INTRODUCTION
The ability of cells to relay and respond to extracellular
signals is a fundamental aspect of physiology. While in the
long term, cells may respond to stimuli through alterations
in gene expression, initially cells must transmit and react to
signals through modification of the behavior and function
of preexisting proteins. This is particularly relevant in the
nervous system, where communication between cells typi-
cally takes place on a timescale on the order of milliseconds.
To rapidly respond in such a manner, cells utilize a diverse
toolbox of tags that, when conjugated to target proteins,
alter the properties of the substrate. These tags can take
many forms, and include chemical modifications, such as
phosphate or acetyl groups, lipids, or even the covalent attachment of other polypeptides. Alone, or in combination,
these posttranslational modifications (PTMs) rapidly coordinate changes in the behavior of their target proteins, setting in
motion the complex cellular response to the stimulus.
The prototypical protein-based modifier is ubiquitin, a 76amino acid protein covalently conjugated to lysine residues
in target proteins. Initially reported in 1975, ubiquitin is
now considered to be a central regulator of cellular function
and is best understood for its role in targeting substrates for
proteasomal degradation (97). Subsequently, a whole family of ubiquitin-like proteins (UBLs) have been discovered
which, while not necessarily sharing high sequence homology with ubiquitin, share a similar three-dimensional structure, and also function as protein-based modifiers (130,
282). UBLs share analogous conjugation pathways to ubiquitin and are ultimately attached to substrates through a
COOH-terminal glycine residue. One member of this family, small ubiquitin-like modifier (SUMO), was initially
characterized for its role in regulating nuclear function.
However, SUMO has subsequently been demonstrated to
modify proteins throughout the cell and, in particular, has
emerged as central regulator of neuronal and synaptic function. Moreover, defective protein SUMOylation is impli-
0031-9333/14 Copyright © 2014 the American Physiological Society
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HENLEY ET AL.
cated in a growing number of disorders of the nervous system.
Here, we briefly review the basic cell biology and regulation of
the SUMO pathway and discuss in detail the emerging roles of
SUMOylation in neuronal function and dysfunction.
II. DISCOVERY AND RELEVANCE OF
PROTEIN SUMOYLATION
SUMOylation is a lysine-targeted PTM in which members of
the ⬃11-kDa SUMO family of proteins are covalently conjugated to target proteins to alter their function. SUMO1 was
originally identified by several different groups in the mid
1990s as a novel UBL that bound the DNA repair enzymes
RAD51/52 (246), interacted with the PML protein (16), and
protected cells from Fas-induced cell death (194). Subsequently, SUMO1 was confirmed as a genuine UBL modifier
that regulated the partitioning of the nuclear pore protein
RanGAP1 (166, 177). Through homology screening, these
studies also predicted the existence of other SUMO paralogs,
designated SUMO2 and SUMO3, which were subsequently
also shown to be functional UBL proteins (124, 125).
Concerted effort by several groups identified the machinery
required for SUMO conjugation. As for other UBL modifiers,
SUMO is conjugated to proteins via a three-step enzymatic
pathway involving an ATP-dependent activation step, performed by an E1 enzyme, transfer of the activated UBL to an
E2 enzyme and, finally, E3-mediated conjugation of the UBL
to the substrate (282). For SUMO, there is a single E1 enzyme,
a heterodimer of SAE1 and SAE2 in humans (79), and in stark
contrast to the ubiquitin system, a sole E2, Ubc9 (51, 79, 146,
237). A number of proteins with E3 or E3-like activity that
catalyze the transfer of SUMO from the E2 to the substrate
have also been identified as well as a growing number of enzymes which remove SUMO proteins from substrates (for reviews, see Refs. 57, 69, 94, 99, 119, 299).
Early studies indicated that SUMOylation is a predominantly nuclear modification, targeting many proteins involved in nuclear organization and function (126). Subsequent proteomic studies have identified several hundred targets of SUMOylation, highlighting it as a crucial
regulator of nuclear function (15, 78, 104, 176, 235, 286,
287). This is underlined by the fact that knockout of the sole
SUMO conjugating enzyme Ubc9, which prevents all protein
SUMOylation, causes many major nuclear defects resulting in
embryonic lethality in mice (187). Importantly, however,
SUMOylation also targets many proteins in cellular compartments outside the nucleus, including plasma membrane proteins (for reviews, see Refs. 73, 299). Taken together, these
findings have led to the realization that SUMOylation plays a
key role in a wide variety of essential extranuclear cellular
processes.
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A. SUMO Proteins
There are four reported SUMO paralogs in humans, designated SUMO1– 4. SUMO1 shares ⬃50% identity to
SUMO2, while SUMO2 and 3 differ in only three NH2terminal residues and are thus generally collectively referred
to as SUMO2/3 (119). SUMO4 possesses 86% homology
with SUMO2 (18), but its role is unclear because a COOHterminal proline residue unique to SUMO4 appears to prevent the generation of conjugatable SUMO4 from its precursor pro-SUMO4 (199). However, one study has suggested that under basal conditions SUMO4 is rapidly
degraded but can be matured by a stress-induced endogenous hydrolase and conjugated to substrate proteins to regulate intracellular stress pathways (295). Notably, this
study only examined the ability of recombinant or overexpressed SUMO4 to be matured. Thus, despite high mRNA
expression in kidney (18), the functional relevance of
SUMO4 as a genuine UBL modifier remains controversial.
SUMO1 and SUMO2/3 target overlapping sets of substrate
proteins (286) but differ in their abundance and properties.
At rest, mammalian cells contain very little unconjugated
SUMO1 but a large pool of free SUMO2/3, which is also
severalfold more abundant than SUMO1 (232). Furthermore, SUMO1 and SUMO2/3 differ in their conjugation
dynamics throughout the cell cycle and in response to cellular stressors such as heat shock or hypoxia (6, 232). These
findings suggest that, despite utilizing the same conjugation
machinery, the SUMO proteins are differentially regulated
in vivo, strongly supporting a functional distinction between the paralogs (6, 232). Most notably, SUMO2/3, but
not SUMO1, are able to form SUMO chains on substrate
proteins as a result of internal lysine residues that can be
SUMOylated (268). Thus, similar to ubiquitination, substrates can undergo mono- or poly-SUMOylation to promote distinct functional consequences.
B. SUMO Knockout Mice
There have been mixed results from different studies that
have generated SUMO1 knockout mice. Alkuraya et al. (2)
generated transgenic mice in which the SUMO1 gene was
interrupted by a ␤-galatosidase insertion. These mice exhibited cleft palate defects and increased rates of late embryonic and early postnatal death, supporting a role for
SUMO1 in development (2). In apparent contrast, SUMO1
knockout mice generated in two other studies were initially
reported to show normal phenotypes, consistent with
SUMO2/3 compensating for loss of SUMO1 during mouse
development (64, 317). However, more detailed analysis of
these mice has revealed differences in body weight and adipogenesis (181), heart function (290), and inflammatory
responses (285), suggesting that, while SUMO2/3 may
compensate for many of the developmental roles of
SUMO1, there are more subtle paralog-specific functions in
which SUMO2/3 cannot compensate for SUMO1.
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NEURONAL SUMOYLATION
III. SUMOYLATION AND DESUMOYLATION
adenylation of the SUMO COOH terminus. The second
step of the reaction proceeds through a dramatic reorganization of the E1 active site, resulting in the attack of
the SUMO adenylate by a catalytic cysteine residue
in SAE2, forming a high-energy thioester bond between
the catalytic cysteine and the COOH terminus of SUMO
(195).
SUMO proteins are initially synthesized as nonconjugatable precursors that are processed by SUMO-specific proteases to expose a COOH-terminal diglycine motif that is
required for for covalent conjugation. The mature SUMO
proteins are then attached to target proteins through the
actions of enzymes termed E1, E2, and E3 (FIGURE 1).
B. The E2, Ubc9
A. The E1
Once activated, SUMO is passed from E1 to the active site
cysteine of the SUMO-specific conjugating enzyme Ubc9.
Ubc9 binds the thioester-loaded E1 complex through a
ubiquitin fold domain in SAE2 (275). Once loaded with
SUMO, Ubc9, either directly or in conjunction with an E3
Mature SUMO is activated by the E1 complex, which in
humans is a heterodimer of SAE1 and SAE2 (80). This
enzymatic complex stimulates a two-step reaction in
which the E1 binds ATP-Mg2⫹ and SUMO to catalyze the
SUMO
SENP cleavage
Maturation
ATP
AMP
SUMO
Substrate
FIGURE 1. The SUMO pathway. All SUMO
proteins are first synthesized as inactive precursors that are cleaved by SUMO proteases
(SENPs) to reveal mature (conjugatable)
SUMO. SUMO is then activated in an ATPdependent step by the E1 enzyme, a heterodimer of the subunits SAE1 and SAE2.
SUMO is then passed to the active site cysteine of the sole SUMO conjugating enzyme,
Ubc9, which, in combination with one of a
growing number of described E3 enzymes,
mediates target recognition and conjugation
of SUMO to a lysine residue in the substrate
protein. Broadly speaking, SUMOylation can
have 4 nonmutually exclusive consequences:
the attached SUMO can change the interaction profile of the substrate, either by blocking
binding of an interacting protein, or by the
attached SUMO acting as a recruitment factor for new proteins. Alternatively, SUMOylation can control substrate stability, through
SUMO chain-mediated recruitment of SUMOtargeted ubiquitin ligases (STUbLs), or regulate substrate activity, through inducing a
change in protein conformation.
MO
SAE1
SU
SAE2
Activation
deSUMOylation
E3
Substrate
MO
SU
Ubc9
MO
SU
Conjugation
MO
SU
Substrate
Substrate
MO
SU
MO
SU
STUbL
MO
U
S
Substrate
Substrate
MO
SU
MO
SU
Ub
Ub
Ub
Interactor
Alters substrate
conformation
Regulation of substrate
stability through recruitment of
SUMO-targeted ubiquitin ligases
Promotes
interactions
Interactor
Blocks
interactions
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HENLEY ET AL.
enzyme, catalyzes conjugation of SUMO to the ⑀-amino
group of a lysine residue in the substrate via an isopeptide
bond. Ubc9 plays a role in target specificity by recognizing
and binding to a SUMOylation consensus site in substrate
proteins, defined as ␺-K-x-D/E, where ␺ represents a large
hydrophobic residue (usually isoleucine, leucine or valine),
K represents the target lysine, x is any residue, and D/E are
acidic residues (227, 233).
C. E3 Ligases
E3 enzymes catalyze the transfer of UBL modifiers from thioester loaded E2s to the substrate. Since Ubc9 is sufficient for
SUMO transfer to occur, at least in vitro, it was initially unclear whether E3 enzymes were required for SUMOylation.
However, Ubc9 binding to the consensus motif is relatively
weak, and SUMOylation of most substrates proceeds inefficiently in in vitro reactions lacking E3s.
1. PIAS proteins and RanBP2
The first E3s identified for the SUMO system were the Siz
proteins, which mediate SUMO transfer to septins in the
yeast S. cerevisiae, (120). The mammalian counterparts of
Siz proteins are the PIAS (protein inhibitor of activated
STAT) family that have E3 activity for a wide range of
SUMO targets (for a comprehensive review of the PIAS
proteins, see Ref. 230). There are four PIAS proteins expressed in humans, PIAS1, PIASx (which is alternatively
spliced to give PIASx␣ and x␤), PIAS3, and PIASy.
The E3 activity of the Siz and PIAS proteins is mediated by
a Siz/PIAS-RING (Really Interesting New Gene) motif that
forms a zinc finger structural domain of 30 – 40 amino acid
residues. In addition to the SP-RING, the PIAS E3s also
contain a number of other motifs, including a SAP domain,
a PINIT motif, a characteristic COOH-terminal domain
(SP-CTD), and a SUMO interacting motif (SIM; see below).
Together, these domains mediate protein-protein interactions and subcellular localization of the PIAS family. Mechanistically, the E3 ligase activity of the PIAS family is restricted to the SP-RING and SP-CTD domains, which bind
Ubc9 and SUMO, respectively, and together are proposed
to position thioester-loaded Ubc9 in a conformation conducive to SUMO transfer (316).
Interestingly, a number of proteins that lack the SPRING motif have also been identified as SUMO E3s. For
example, the SP-RING-lacking nuclear pore protein
RanBP2 binds SUMO and Ubc9, and possesses SUMO E3
activity (205). As for the PIAS proteins, the minimal region required for E3 activity contains SUMO and Ubc9
binding sites, but does not appear to bind the substrate,
suggesting a similar model whereby RanBP2 positions
thioester-loaded Ubc9 in a position optimal for SUMO
ligation to the substrate (205, 207, 223). However, in
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vivo, RanBP2 forms part of a much larger complex that
also contains SUMO-modified RanGAP1, and two copies
of Ubc9 (296). Together, this complex has been proposed
to function as a novel multisubunit E3 ligase that couples
substrate binding to facilitation of SUMO transfer, similar to those found in the ubiquitin system (296).
2. Other SUMO ligases
The PIAS family and RanBP2 are generally considered bona
fide E3 enzymes because they are capable of enhancing
SUMO transfer to a wide variety of substrates at substochiometric concentrations. In addition, a growing number
of proteins are being identified that can enhance the
SUMOylation of specific substrates, and thus function either as E3 ligases or cofactors of SUMOylation. For example, the histone deacetylases (HDACs) 4 and 7 have been
reported to enhance SUMOylation of the androgen receptor and PML, respectively (71, 313). Furthermore, members of the tripartite motif (TRIM) family of proteins that
contain a RING finger domain, B-box zinc finger domain(s), and a coiled coil region can stimulate SUMOylation in vitro (39, 71, 313). The RING domain-containing protein TOPORs, which acts as an E3 ligase for ubiquitin (219), also enhances SUMOylation of a number of
targets, including p53 (294), topoisomerase I (90), and the
NF␬B regulator IKK⑀ (221), suggesting it may function as a
dual-specificity E3 for both the ubiquitin and SUMO systems. Furthermore, the protein RSUME has been reported
to potentiate the SUMOylation of I␬B, HIF1␣, and glucocorticoid receptor (28, 58), although this effect appears to
be due to a general ability to enhance SUMO loading of
Ubc9, suggesting RSUME may function as a generalized
cofactor of SUMOylation rather than as an E3 (28).
Since all SUMOylation events are mediated by only one
E1 complex and one E2, the diversity of E3s may provide
a mechanism for subtle, substrate-specific regulation of
SUMOylation. Therefore, the regional and subcellular
distributions of SUMO E3 ligases are likely to define
which substrates are SUMOylated in those areas and
compartments. For example, the protein MAPL (mitochondrial-associated protein ligase) has been proposed to
be a SUMO E3 exclusively localized at mitochondria
and has been reported to enhance SUMOylation of the
GTPase dynamin-related protein 1 (Drp1) at the mitochondrial membrane (21). On a wider scale, tissue-specific expression patterns of SUMO ligases/cofactors may
underlie the differences in substrate SUMOylation between tissues. The protein Rhes is enriched in the striatum and has been reported to specifically enhance
SUMOylation of mutant Huntingtin, leading to the observed striatal-specific degeneration observed in patients
with Huntington’s disease (262).
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NEURONAL SUMOYLATION
D. DeSUMOylation: SUMO Proteases
SUMO proteases perform two core functions in the
SUMOylation pathway: 1) they are responsible for the
initial COOH-terminal cleavage of pro-SUMO to mature, conjugatable SUMO, via their endopeptidase activity; and 2) they deconjugate substrate-bound SUMO via
their isopeptidase activity (for focused reviews, see Refs.
99, 186, 314).
1. SENP proteins
The SUMOylation status of a specific substrate is a delicate
balance between E1/Ubc9/E3-mediated conjugation and
SUMO protease-mediated deconjugation (FIGURE 2). Regulation of the distribution and activities of SUMO proteases
therefore provides a mechanism to tightly control the location and levels of protein SUMOylation. In yeast, two
SUMO proteases have been identified, Ulp1 and Ulp2 (151,
152). Their mammalian counterparts, the SENPs (sentrinproteases), were subsequently isolated through homology
screening. There are six SENPs in humans, designated
SENP1, 2, 3, 5, 6, and 7. Individual SENPs differ in their
subcellular localization, SUMO paralog specificity, the ef-
A
ficiency of their endopeptidase versus isopeptidase activities, and their ability to cleave monomeric SUMO versus
SUMO chains (TABLE 1; for reviews, see Refs. 99, 186,
314). All SENPs contain a conserved COOH-terminal catalytic domain and an NH2-terminal domain that likely dictates their localization and protein interactions. SENP1 and
SENP2 appear to preferentially cleave proSUMO to mature
SUMO1 and SUMO2, respectively, while SENP5 appears
most efficient at maturation of SUMO3 (52, 82, 222, 306).
In terms of deconjugation, SENP1 removes both SUMO1
and SUMO2/3 from substrate proteins in vivo, whereas
SENPs 2, 3, and 5 show a preference towards SUMO2/3
(52, 82, 136, 222). SENP6 and 7 show preferential isopeptidase activity against SUMO2/3 chains (154, 185, 244). As
for SUMO, the subcellular localization of SENPs is predominantly nuclear, and the different SENPs exhibit differing
subnuclear localizations. SENP1 and 2 localize to the nuclear pore and discrete nuclear dots (8, 81, 91), SENP3 and
5 have a mainly nucleolar localization (82, 191), and
SENP6 and SENP7 are found throughout the nucleoplasm
(185, 244). Importantly, however, although highly enriched, SENPs are certainly not exclusively restricted to the
nucleus. SENP3 and SENP5 are present at mitochondria
B
Substrate
Substrate
SUMO1
SUMO2/3
E3
Ubc9
E3
Ubc9
SENP1/2
E3
Ubc9
SENP3/5
FIGURE 2. Properties of SUMO1 and SUMO2/3 conjugation. Schematic highlighting the main differences between SUMO1 and SUMO2/3 modification. A: SUMO1 is
conjugated to substrates via Ubc9 and cleaved mostly by
SENP1/2. SUMO1 does not contain an internal SUMOylation site and therefore cannot form polySUMO chains.
B: SUMO2/3 is conjugated by Ubc9 and cleaved by SENP3
or SENP5. Since SUMO2/3 contains SUMOylation sites,
SUMO2/3 can form polySUMO chains of n length. SUMO
chains are shortened by cleavage by SENP6/7. Conjugation of SUMO1 to SUMO2/3 can cap SUMO2/3 chains to
terminate further elongation.
SENP6/7
n
SUMO1
E3
Ubc9
SENP1/2
?
n
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HENLEY ET AL.
Table 1. Localizations and actions of SUMO proteases
Cleavage of
ProSUMO
Removal of SUMO From
Target Substrates
SUMO Protease
Main Cellular Location
SUMO Selectivity
SENP1
SUMO1 & SUMO2/3
Yes SUMO1 &
SUMO2
Yes
SUMO2/3 ⬎ SUMO1
Yes SUMO1 &
SUMO2
Yes
SUMO2/3
ND
Yes
SUMO2/3
Yes SUMO3
Yes
SENP6
Mainly nucleus and subnuclear structures
but also present in cytoplasm and other
extranuclear compartments
Mainly nucleus and subnuclear structures
but also present in cytoplasm and other
extranuclear compartments
Nucleolus as well as extranuclear
compartments including mitochondria
Nucleolus as well as extranuclear
compartments including mitochondria
Nucleoplasm
SUMO2/3 chains
No
SENP7
Nucleoplasm
SUMO2/3 chains
No
DeSI1
DeSI2
USPL1
Nucleus and cytoplasm
Nucleus and cytoplasm
Cajal bodies in nucleus
SUMO1 & SUMO2/3
ND
SUMO2/3 ⬎ SUMO1
Possibly
No
Possibly
Yes, preferentially SUMO2/3
chains
Yes, preferentially SUMO2/3
chains
Yes
ND
Yes
SENP2
SENP3
SENP5
See text for details and references. ND, not determined.
(89, 328), and in neurons, SENP1, 3, and 6 have a widespread distribution throughout neuronal processes and at
synapses, albeit at much lower levels than present in the
nucleus (89, 159, 160, 174).
2. Other SUMO proteases
In addition to the SENP enzymes, two novel SUMO-specific
proteases, DeSI1 and USPL1, have recently been reported
(236, 248). DeSI1 was initially identified as an interactor of
the transcription factor BZEL (248). When heterologously
expressed, DeSI1 forms homodimers that distribute
throughout both the cytosol and nucleus. DeSI1 can cleave
SUMO1 and SUMO2/3 from BZEL, both in vivo and in
vitro, and also deconjugates SUMO2/3 chains in vitro
(248). However, DeSI1 appears to lack endopeptidase activity, since it does not mature SUMO1. Furthermore, in
contrast to the SENPs, DeSI1 does not deSUMOylate the
well-characterized SUMO substrates PML or ⌬Np63, and
knockdown of DeSI1 does not overtly alter the pattern of
cellular SUMOylation. Thus these results suggest that
DeSI1 has a much more restricted selection of substrates
than the SENPs. From sequence alignment analysis, a similar protein, named DeSI2, has also been identified (248);
however, at the time of writing, whether this protein also
shows deSUMOylating activity has not yet been reported.
The other novel SUMO-specific protease, USPL1, was identified in a screen for proteins with deSUMOylating activity
(236). It is primarily localized at Cajal bodies within the
nucleus, binds SUMO, and deconjugates SUMO from the
prototypic substrates RanGAP1 and Sp100, favoring deconjugation of SUMO2/3 over SUMO1 (236). However,
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the in vivo functions and targets of USPL1 remain to be
elucidated.
IV. SUMOYLATION CONSENSUS MOTIFS
Early analysis of the modified lysine residues in SUMO
substrates led to identification of a SUMOylation consensus
site, ␺-K-x-D/E, to which Ubc9 binds directly, albeit with
relatively low affinity (227, 233). Subsequent studies have
built on these findings to define more extended SUMOylation target motifs, which appear to promote substrate
SUMOylation by enhancing binding to Ubc9. For example,
the negative charge-dependent SUMOylation motif (NDSM)
contains an acidic patch located downstream of the modified consensus lysine, that binds to a cognate basic patch on
Ubc9 (310). Similarly, the phosphorylation-dependent
SUMOylation motif (PDSM) or phosphorylated SUMOylation motif (pSuM) contain phosphorylatable serine residues COOH-terminal to the modified lysine, or in place of
the acidic residue of the consensus motif, respectively (100,
204). When phosphorylated, these residues provide the negative charge required to enhance substrate recognition by
Ubc9 and provide an elegant mechanism to explain how
substrate phosphorylation can regulate SUMOylation
(100).
In addition to these “extended consensus motifs,” a number
of other SUMO target sites have been defined by proteomic
studies. For example, in some cases, methionine, phenylalanine, and cysteine can be tolerated in place of the large
hydrophobic residue of the consensus motif. Furthermore,
an inverted consensus motif (E/D-x-K-␺) also functions for
some substrates (176).
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NEURONAL SUMOYLATION
An important note of caution, however, is that while the
presence of a consensus sequence in a protein of interest
indicates the potential for that protein to undergo SUMO
modification, the context of the consensus sequence is
critical. Ubc9 binding and SUMOylation can only occur
at consensus sequences located in unstructured regions of
the target protein (206), and many, perhaps most, apparently consensus lysines are not modified. Equally importantly, many SUMO substrate proteins can be modified at
nonconsensus lysines. How the SUMO machinery recognizes nonconsensus sites remains to be fully resolved and
is an active area of investigation, but it is likely that
E3 ligases may play a crucial role in recognition of these
sites (69).
V. MOLECULAR CONSEQUENCES OF
PROTEIN SUMOYLATION
The majority of reported SUMO substrates are located
within the nucleus, and there are clear roles for SUMO
modification in DNA repair, transcription, and nuclear
organization. However, increasing numbers of SUMO
substrates are being identified in compartments outside
the nucleus and have been implicated in almost every
aspect of cell biology. This diversity of substrate proteins
means that it is difficult to predict the overall effects of
global SUMOylation. Nonetheless, for specific substrates
where the effects of SUMO conjugation have been defined, SUMOylation can be broadly considered to have
four nonmutually exclusive effects on the target protein.
Briefly, SUMOylation can do the following:
1) reduce interactions at the substrate protein by blocking protein-protein interaction sites;
2) enhance interactions at the substrate protein by providing a platform to recruit proteins that noncovalently bind the attached SUMO;
3) control target protein stability and turnover, especially via the recruitment of members of the SUMOtargeted ubiquitin ligase (STUbL) family of proteins;
and
4) cause conformational changes in the target protein,
which in turn regulate its function and/or activity (see
FIGURE 1).
VI. SIMS AND RELATED SUMO BINDING
MOTIFS
Many ubiquitin-binding motifs have been defined that mediate the downstream consequences of ubiquitin modification of the substrate. For SUMOylation, at least three distinct SUMO interacting motifs (SIMs) have been described
in proteins that bind SUMO noncovalently. The first to be
defined was the “classical” SIM, which consists of a hydrophobic core, often flanked COOH- or NH2-terminally by
acidic residues (95, 182, 252, 253). Proteins containing this
motif can bind to a hydrophobic groove present in all
SUMO paralogs (95) and can bind SUMO in either orientation (95, 253). Although most classical SIMs do not show
paralog preference, there are reported examples of paralog
specificity (180, 198, 321).
Additional “nonclassical” SIMs have been discovered in
Ubc9 (27, 53, 135) and in dipeptidyl peptidase 9 (DDP9),
an enzyme which cleaves NH2-terminal dipeptides from
substrates that have a proline residue at the second position (Xaa-Pro) (208). The SIMs in these proteins do not
recognize the classical SIM binding groove in SUMO;
rather, they bind to a surface surrounding Glu67 in
SUMO1, which has been called the E67 interacting loop
(EIL). Ubc9 binds equally to this EIL surface in all SUMO
paralogs, whereas DDP9 binding is highly specific to
SUMO1 (208). In addition, a zinc finger motif in the
ubiquitin ligase HERC2 has recently been identified that
binds SUMO1 and, to a lesser extent SUMO2, and this
binding requires its ability to chelate zinc (48). Thus,
similar to ubiquitin, we anticipate that beyond these
three already identified SUMO interacting motifs, multiple other motifs capable of recognizing SUMO paralogs
await discovery, and this emerging family of interacting
domains will provide a mechanism for the versatile control of SUMOylation and its downstream pathways.
A. Functions of SIMs
SIMs can dictate the downstream effects of substrate
SUMOylation by recruiting SIM-containing effector proteins to SUMOylated target proteins. For example, recruitment of the SIM-containing transcriptional repressor Daxx
to many SUMOylated transcription factors mediates the
repressive effects of SUMOylation on transcription (33,
142, 155). Furthermore, SUMO-SIM interactions can act as
molecular “glue”, driving protein complex assembly, such
as the formation of PML nuclear bodies (155, 245) or the
recruitment of the DNA repair machinery to double strand
breaks (217).
Beyond a role in the engagement of effector proteins, a
number of identified SUMO substrates contain SIMs, including the deubiqutinating enzyme ubiquitin-specific peptidase 25 (USP25) (180) and the RecQ DNA helicase mutated in Bloom syndrome (BLM) (321). In these cases, the
SIM recruits SUMO-loaded Ubc9 to the substrate, enhancing substrate SUMOylation. Alternatively, the SIM within
the substrate can bind to the SUMO after it has been conjugated, leading to a change in conformation, as is the case
for the DNA repair enzyme thymine D-glycosylase (TDG)
(7, 92).
A recent advance in our understanding of how SIMs mediate the downsteam effects of SUMOylation has come
from the discovery of SUMO-targeted ubiquitin ligases
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HENLEY ET AL.
(STUbLs; for more detailed reviews, see Refs. 74, 202,
255). These proteins, of which RNF4 is the prototype,
are ubiquitin ligases that harbor several tandem SIMs,
allowing them to recognize SUMO chains. Recruitment
of STUbLs to substrates via SUMO chains results in ubiquitination and degradation of the substrate. These findings highlight how mono-SUMOylation and polySUMOylation can have different effects and they provide
an elegant example of the cooperation between the
SUMO and ubiquitin pathways.
B. Regulation of SUMO-SIM Interactions
A number of studies have investigated the posttranslational
modifications of SIMs and the SIM binding groove on
SUMO that regulate their interaction properties (FIGURE 3).
The SIM of the E3 enzyme PIAS1 can be phosphorylated
by casein kinase 2 (CK2), which enhances its interaction
with SUMO1 both in vitro and in vivo, and alters the
ability of PIAS1 to function as a transcriptional coregulator (258). CK2-regulated SIMs have also been identified in PML, the exosome component PMSCL1, and the
transcriptional repressor Daxx, suggesting that SUMOSIM interactions may be generally regulated by phosphorylation (258). More recent studies have extended
these findings to show that SUMO-SIM interactions can
A
CK2
MO
P
VII. REGULATION OF PROTEIN
SUMOYLATION
As described above, SUMOylation proceeds with a highly
restricted number of enzymes compared with the analogous
ubiquitin system. Therefore, a key question in the field
is how is substrate-specific regulation of SUMOylation
achieved? As discussed below, recent advances revealing the
roles of other PTMs of both substrate proteins and of specific components of the SUMO conjugation machinery are
starting to provide partial answers. One particularly interesting emergent concept is that, in addition to regulation at
the level of individual substrates, SUMOylation can also be
regulated on a global level, as exemplified by the observation that various cellular stresses, including heat shock, osmotic shock, and ischemic challenge, lead to a global increase in SUMOylation, and dramatically alter the profile of
cellular SUMOylation (88).
SI
M
SU
also be regulated through modification of the SIM-binding groove on SUMO (278). Acetylation of K37 in
SUMO1, or K33 in SUMO2, blocked SUMO binding to
various SIM-containing interactors. Furthermore, acetylation of SUMO dramatically reduced its capacity to repress transcription and the assembly of PML nuclear
bodies, functions both dependent on SUMO-SIM interactions (278). We anticipate that other kinases and sites
of phosphorylation, as well as other posttranslational
modifications, regulate SUMO-SIM interactions and
that, taken together, these processes provide the mechanisms to determine the extent and outcome of substrate
protein SUMOylation under different conditions and circumstances.
PIAS1
A. Interplay Between SUMOylation and
Other PTMs of Substrate Proteins
B
M
P
SI
MO
Ac
SU
PIAS1
Class I
HDACs
?
MO
SU
FIGURE 3. Regulation of SUMO-SIM interactions. SIM-containing
proteins mediate the downstream effects of SUMOylation through
binding to the SUMOylated substrate. Furthermore, SIMs are present in several components of the SUMOylation machinery, where
they act to coordinate substrate SUMOylation. Intriguingly, SUMOSIM interactions can be reciprocally regulated. A: phosphorylation of
the SIM in the E3 ligase PIAS1 by casein kinase 2 (CK2) enhances its
capacity to bind SUMO. B: acetylation of SUMO within the SIMbinding groove, which is reversed by class I histone deacetylases
(HDACs), blocks SUMO-SIM interactions.
1256
A common theme in the regulation of substrate SUMOylation
lies with crosstalk between SUMOylation and other
PTMs. The best studied example is phosphorylation of
the substrate protein, which can either enhance or reduce
SUMOylation at a nearby lysine, depending on the circumstances and the specific substrate (FIGURE 4). As outlined
above, the SUMO target lysine in a number of SUMO substrates lies within a PDSM motif which, when phosphorylated, enhances substrate recognition by Ubc9 (100). Furthermore, phosphorylation can also enhance substrate
SUMOylation when not part of a PDSM motif, as is the case
for the kainate receptor subunit GluK2, for which PKC-mediated phosphorylation of a serine 18 residues upstream of
the SUMO target lysine promotes SUMOylation (32, 137).
Conversely, phosphorylation of the transcription factor
Elk-1 (311) and the DNA binding protein SATB1 (265)
reduce SUMOylation, the latter through reducing the binding of the E3 PIAS1 to the substrate.
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A
Ubc9
P
K
Substrate
– EE
K
D
E
Substrate
– EE
Phosphorylationdependent SUMOylation
K
D
E
Substrate
SU
MO
B
K
Substrate
SU
MO
P
O
M
SU
SU
MO
NEURONAL SUMOYLATION
K
K
SATB1
SATB1
Negative chargedependent SUMOylation
FIGURE 4. Interplay between substrate
SUMOylation and phosphorylation. A: phosphorylation of some substrates, such as
MEF2A and GATA-1, promotes SUMOylation via enhaced recruitment of Ubc9. This is
due to the negative charge of the phosphate
group interacting with a cognate basic patch
on Ubc9. Other substrates, such as Elk-1,
are efficiently modified by SUMO due to the
presence of a negatively charged region immediately COOH-terminal to the modified
lysine, mimicking the effect of phosphorylation, a motif known as an NDSM (negatively
charged SUMOylation motif). B: conversely,
phosphorylation can also reduce SUMOylation of some substrates. For example, phosphorylation of SATB1 reduces its SUMOylation through blocking binding to the E3,
PIAS1.
PIAS1
Ubc9
MO
SU
P
K
SATB1
Another emerging regulatory mechanism is the interrelationship between substrate SUMOylation and other
lysine-targeted PTMs, most notably by ubiquitin or acetylation. While these PTMs were originally suspected to
compete with SUMOylation for the target lysine, it is
now clear that the interactions can be far more nuanced,
and that the interplay between different PTMs at the
same lysine allows complex regulation of substrate properties and functions (for review, see Ref. 279). These
different PTMs may be elicited by distinct stimuli or in
different cellular locations and can be interchangeable
and/or occur sequentially to regulate the substrate without necessarily being directly competitive. One example
of this lies with the transcription factor MEF2A. MEF2A
contains a PDSM-type SUMOylation site that, when
phosphorylated, drives MEF2A SUMOylation (100,
240). Neuronal activation, however, leads to MEF2A
dephosphorylation, deSUMOylation, and acetylation of
the target lysine to regulate MEF2A transcriptional
activity (240). Thus, while not in direct competition,
SUMOylation and acetylation target the same lysine to
tightly control the transcriptional response of MEF2A to
neuronal activity. Analogous examples have emerged
from the interplay between ubiquitin and SUMO. For
example, the NF␬B regulator NEMO is sequentially
modified by SUMO and ubiquitin at the same lysine residue to control its nuclear localization (109). Furthermore, perhaps the most complex and elegant examples of
the cross-regulation of substrate proteins through the
interplay of PTMs are the STUbLs mentioned above (74,
202, 255).
B. Regulation Through Targeting of the
SUMO Machinery
SUMOylation of all substrates uses the same core pathway and a very limited repertoire of enzymes. One consequence is that regulation of these SUMO machinery
enzymes offers the potential to influence SUMOylation
on a global level, or at least to spatiotemporally alter the
SUMOylation of a subset of targets within a particular
area or cellular compartment. Indeed, regulation of the
SUMOylation machinery, either posttranslationally or
through regulating enzyme turnover, is an important
emerging concept in the field.
1. Regulation of the E1
High levels of H2O2 induce oxidative stress and increase
SUMO conjugation (232), whereas low H2O2 levels rapidly decrease in SUMO conjugation (19). This dramatic
biphasic effect is due to the direct and reversible inhibition of SUMO conjugating enzymes by low levels of reactive oxygen species. Inhibition occurs via the formation
of a disulfide bond between the catalytic cysteines of the
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HENLEY ET AL.
MO
SU
K
K
SI
M
K
Ubc9
B
Ubc9
K
In contrast, acetylation of Ubc9 reduces its ability to modify
substrates that contain an NDSM. Interestingly, enhanced
SUMOylation during hypoxia correlates with SIRT1-mediated deacetylation of Ubc9, which relieves the inhibition to
facilitate enhanced SUMOylation of NDSM containing
substrates (105).
These are still relatively early days in this field, but it is
already clear that, depending on the precise stimuli and
pathways involved, PTMs can initiate global changes in E1
and Ubc9 activity, and it seems PTMs may also be capable
of mediating more subtle changes to alter SUMOylation at
certain sets of substrates.
3. Regulation of deSUMOylation
As outlined above, nine SUMO proteases have been reported that vary in their ability to process and/or deconju-
K
Substrate
Cdk1
Phosphorylation of Ubc9 by CDK1/cyclin B also regulates
SUMOylation in in vitro assays by enhancing thioester
loading (260). As yet, however, the role of Ubc9 phosphorylation in regulating SUMO modification in vivo remains
undetermined.
MO
SU
Substrate
2. Regulation of Ubc9
1258
MO
SU
Ubc9
MO
SU
P
C
MO
Because it is the only E2, Ubc9 represents a prime target for
global regulation of protein SUMOylation, and several
PTMs of Ubc9 have been reported to modulate its target
specificity (FIGURE 5). Mammalian Ubc9 is SUMOylated at
a single lysine (K14), which, while not affecting its ability to
modify the substrates HDAC4, PML, or E2–25K, impairs
its ability to modify RanGAP1 and potentiates its activity
against Sp100, in a manner dependent on the SIM in Sp100
(134). SUMOylation of Ubc9 thus acts to regulate its target
specificity, skewing its activity to favor particular subsets of
proteins.
Substrate
SU
MO
The SUMO E1 itself is also a SUMOylation substrate (261,
275). SUMOylation of the E1 subunit SAE2 occurs at multiple sites within both the Cys domain and near the COOH
terminus of the protein (275). Cys domain SUMOylation
appears to inhibit the transfer of activated SUMO from the
E1 to the catalytic cysteine of Ubc9, while SUMOyation at
the COOH terminus is required for nuclear retention of the
E1 complex (275). Notably, heat shock, which increases
SUMO2/3 modification of many substrate proteins, reduces
SUMO modification of the E1 Cys domain (275). An attractive possibility is that SUMOylated E1 represents an inactive cellular reserve of E1 that, under stress conditions, can
be activated by deSUMOylation to mediate the global increase in SUMO conjugation (275).
SU
MO
Substrate
O
Ubc9 SUM
Ac
– EE
SU
K
Ubc9
SU
MO
A
SI
M
E1 subunit SAE2 and Ubc9 (19), providing an elegant
mechanism to prevent SUMO conjugation without affecting deconjugation, and thereby reduce SUMOylation.
K
D
E
Substrate
Hypoxia
SIRT1
Ubc9
MO
SU
FIGURE 5. Posttranslational regulation of Ubc9 can alter global
SUMOylation, or modification of particular subsets of proteins.
A: SUMOylation of some target proteins, such as USP25 and BLM,
is enhanced by the presence of a SIM in the substrate, which
recruits SUMO-loaded Ubc9 through noncovalent binding to SUMO.
However, Ubc9 can also be covalently modified by SUMO at lysine14, and SUMOylation of Ubc9 acts to enhance its activity towards a
subset of SIM-containing proteins, presumably due the covalently
attached SUMO promoting recruitment to the substrate. B: phosphorylation of Ubc9 at serine-71 by the kinase Cdk1 enhances its
activity in vitro. C: acetylation of Ubc9 at lysine-65 specifically reduces its activity towards NDSM-containing substrates. However,
hypoxia results in SIRT1-mediated deacetylation of this residue, enhancing SUMOylation.
gate SUMO, as well as in their paralog and substrate specificity. Together with E3s, deSUMOylating enzymes provide the most diverse aspects of the SUMO pathway, and
modulation of individual SUMO proteases offers the opportunity to regulate the paralog-specific SUMOylation of
distinct subsets of substrate proteins. This has been most
extensively examined in the field of ischemic and oxidative
stress. In particular, regulation of the SUMO2/3-specific
protease SENP3 has emerged as a central player in cellular
stress responses. Under basal conditions, SENP3 is constitutively degraded through the ubiquitin-proteasome sys-
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NEURONAL SUMOYLATION
tem. However, low doses of H2O2 that induce mild oxidative stress rapidly stabilize SENP3 protein levels (107). One
important component of the cellular response to oxidative
stress is the transcription factor hypoxia inducible factor 1␣
(HIF1␣) which, along with its coactivator p300, drives expression of stress response genes (242). SENP3 stabilization
following mild oxidative stress promotes deSUMOylation
of p300, enhancing its interaction with HIF1␣ and promoting transcription (107). The ubiquitin ligase CHIP [COOH
terminus of heat shock cognate protein (Hsc70) interacting
protein] has been identified as the mediator of SENP3 ubiquitination and turnover (309). Interestingly, mild oxidative
stress also causes oxidative modification of cysteines C243
and C274 in SENP3, indicating that SENP3 itself functions
as a redox sensor. Redox modification of these cysteines
recruits Hsp90, which prevents CHIP-mediated ubiquitination of SENP3 and stabilizes SENP3 levels (309). Thus direct oxidative modification of SENP3 can lead to differential interactions with chaperone proteins to ultimately stabilize SENP3 and drive the cellular response to oxidative
stress.
Another study has also identified SENP3 as a central regulator of the cellular response to oxygen and glucose deprivation (OGD), a widely used model of ischemic insult (88,
89). This is described in more detail in the ischemia section
below but, briefly, during OGD SENP3 is rapidly degraded
by the lysosomal protease cathepsin B, and this loss correlates with increased SUMO2/3 conjugation (89). Thus cellular SUMOylation of subsets of proteins can be regulated
by targeted degradation of deSUMOylating enzymes.
VIII. PARALOG SPECIFICITY OF
SUMOYLATION
Ubc9 is capable of conjugating both SUMO1 and SUMO2/3
with similar efficiency (268). This raises the question as
to the differences between SUMO1 versus SUMO2/3
conjugation, and how the paralog-specific modification
observed for some substrates is achieved. While much
remains to be discovered, a number of mechanisms are
beginning to emerge. As outlined above, while many
SIMs appear unable to distinguish between SUMO paralogs, several SIMs that can differentiate between SUMO1
and SUMO2/3 have been defined. For example, USP25
(180) and BLM (321) both contain SIMs that selectively
recruit Ubc9 thioester-loaded with SUMO2/3 and promote consequent SUMO2/3-ylation.
The prototypic SUMO substrate RanGAP1, which is primarily modified by SUMO1, provides another example of
paralog specificity. Intriguingly, this occurs via a separate
regulatory mechanism since the conjugation machinery
does not appear to distinguish between the covalent attachment of SUMO1 versus SUMO2/3 to RanGAP1. Instead,
SUMO1 modification of RanGAP1 recruits it into a complex
with the multifunctional protein RanBP2 that protects it from
SENP-mediated deconjugation. In contrast, SUMO2/3-modified RanGAP1 is rapidly deconjugated, thereby generating effective SUMO1-specificity at equilibrium (323).
RanBP2 also provides a different mechanism for SUMO paralog specificity. One role of RanBP2 is as an E3 that conjugates
both SUMO1 and SUMO2 to the substrates PML and Sp100,
but conjugation is more efficient for SUMO2 (269). RanBP2
binds noncovalently to both Ubc9 and SUMO1, but not to
SUMO2 and, like many E3s in the ubiquitin system, is also
capable of catalyzing nonproductive automodification (autoSUMOylation). Essentially, autoSUMOylation versus substrate modification can be viewed as a competitive process.
Because RanBP2 binds SUMO1, it preferentially autoSUMOylates with this paralog; however, because it does not
directly bind to SUMO2, it preferentially conjugates
SUMO2 to the substrate (269). Thus, while the E1 and E2
of the SUMO system seem to show little preference in paralog conjugation (268, 270), it is becoming increasingly clear
that, for some targets, E3s can elegantly regulate paralog
specificity.
IX. SUMOYLATION IN NEURONS
A. Distribution and Developmental
Expression of the SUMO Machinery
Despite a growing appreciation of the importance of
SUMOylation in neuronal function, the distribution and
developmental profiles of the SUMOylation machinery
have not yet been extensively characterized. However, this
is an active area of investigation and a number of studies
have begun to address this issue.
Investigation of the developmental profile of Ubc9 at the
mRNA and protein levels in rat brain revealed highest expression between E13–18; thereafter, Ubc9 expression decreased following birth and remained low in adults. Total
levels of substrate SUMOylation showed a corresponding
developmental profile, consistent with an important role for
SUMOylation in late embryonic development (292). The
Ubc9 transcript was detected embryonically throughout the
brain by in situ hybridization but was particularly concentrated in areas containing proliferating neural stem cells,
with moderate expression elsewhere. After birth, Ubc9 expression was more restricted, being highest in dentate granular neurons and pyramidal neurons in the hippocampus, as
well as large pyramidal neurons of the cerebral cortex
(292). These results were interpreted to suggest key roles for
Ubc9/SUMOylation in neuronal differentiation in the developing brain and, given the high expression levels in the
adult hippocampus, synaptic plasticity in the adult (292).
A more recent study has also examined the developmental
regulation and spatiotemporal distribution of the SUMO-
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HENLEY ET AL.
ylation machinery with broadly similar results (160). Total
protein conjugation by SUMO1 and SUMO2/3 peaked at
E12, while levels of the deconjugating enzymes SENP1 and
SENP6 were highest early in development and decreased
thereafter. The highest levels of Ubc9 were detected in the
developing brain (E15–18), with a decrease after birth. Interestingly, this study also investigated levels of SUMOylated Ubc9, and observed that this displayed a different
profile to non-SUMOylated Ubc9, being low before birth,
peaking at P14, and remaining high in the adult (160). The
physiological consequences of this developmental switch
from non-SUMOylated to predominantly SUMOylated
Ubc9 are currently unclear. However, since Ubc9 SUMOylation is known to regulate target discrimination (134), an
attractive possibility is that SUMOylation allows Ubc9 to
target distinct sets of substrates during development.
B. Subcellular Localization of the SUMO
Machinery
Because of the abundance of both SUMO and the conjugation machinery in nuclear compartments, historically
SUMOylation has been regarded as a nuclear modification. Indeed, in cultured neurons, SUMO is predominantly
localized in the nucleus, but significant SUMO immunoreactivity is also present in axons and dendrites, and at synapses (36, 67, 76, 113, 159, 160, 174, 210). In rat brain,
SUMO1 and SUMO2/3 conjugation show similar profiles
during development, with a peak at E12 in both nuclear and
cytosolic fractions (160). Furthermore, SUMO1, SUMO2/3,
and the conjugation machinery are readily detectable in
synaptosome fractions, indicating a synaptic role for
SUMOylation (67, 160, 174). Consistent with this, immunostaining of cultured neurons shows that SUMO proteins,
the E1 complex, Ubc9, SENPs, and the PIAS proteins are
localized in dendrites and axons as well as at synapses (113,
159, 160, 174). Interestingly, these proteins show differential localization patterns at synapses during neuronal development. In cultured hippocampal neurons, presynaptic levels
of the E1 subunit SAE1 decreased between DIV10 and 20,
whereas Ubc9 remained unchanged and SENP1 and SENP6
decreased. In contrast, postsynaptic levels of SAE1, Ubc9, and
SENP6 all increased between DIV10 and DIV20, while
SENP1 decreased (160). These findings illustrate that the
SUMOylation machinery at synapses is dynamically regulated
in a complex manner throughout neuronal development.
Targeting E3s or SENPs to a particular location at a particular time can locally alter SUMOylation to regulate complex cellular processes, such as cell cycle progression (120,
327) or DNA repair (217). However, exactly how SUMOylation and deSUMOylation enzymes are targeted to the
right place at the right time in neurons is unknown. SUMO
enzymes have been reported to undergo developmental and
KCl depolarization-dependent redistribution (159) but, as
yet, no experiments investigating the mechanisms and con-
1260
sequences of enzyme translocation under physiological and
pathophysiological conditions have been reported.
An interesting concept that has emerged recently is that in
yeast, anchoring of the E3 Siz2 catalyzes a local “SUMO
spray” on nearby proteins to potentiate physical interactions and accelerate DNA repair (118). Neurons are far
more morphologically complex cells, and spatiotemporally
regulated events require sophisticated coordinatation of
protein trafficking, retention, and turnover. Nonetheless,
the concept that enzymes might be trafficked to cellular
compartments to mediate global SUMOylation of substrate
proteins present in a specific location at a particular time is
an intriguing avenue for future study.
C. Activity-Dependent Regulation of
Synaptic SUMOylation
As well as being developmentally regulated, it is also becoming clear that the synaptic localization of SUMO paralogs and the SUMOylation machinery is under tight activity-dependent control. The pre- and postsynaptic distribution of the SUMO proteins and SUMO machinery in cultured
neurons following depolarization with KCl has been investigated. KCl stimulation caused a sustained (at least 30 min)
increase in presynaptic, but no change in postsynaptic, protein
conjugation by SUMO1. In contrast, KCl stimulation caused a
transient decrease in postsynaptic levels of SUMO2/3 conjugation but left presynaptic levels unchanged (159). KCl stimulation also led to complex and differential changes in the preand postsynaptic distribution of the E1 subunits SAE1, Ubc9,
SENP1, and SENP6 (159).
Similarly, treatment of synaptosomes with AMPA has
been shown to recruit Ubc9 to the presynaptic fraction
(67), and stimulation of neurons with glycine, a coagonist of NMDA receptors, resulting in chemically induced
LTP (Chem-LTP), also leads to an increase in colocalization of Ubc9 and SUMO1 with the postsynaptic marker
PSD95 (113). Taken together, these results indicate that
the dynamic recruitment and removal of the SUMO machinery provides a mechanism for the sophisticated control of substrate-specific SUMOylation at synapses in response to neuronal activity.
In addition to the localization of SUMO proteins and the
SUMO machinery at synapses, an important role for
SUMOylation in synaptic physiology has been inferred
from the growing number of SUMO substrates identified in
dendrites, axons, and synapses and the observation that
perturbing SUMOylation has major consequences for synaptic function and plasticity (32, 36, 43, 113, 164, 174,
239, 301). In rather stark contrast, however, a report examining the distribution of SUMO reactivity in a His-HAtagged SUMO1 knock-in mouse failed to detect any synaptic SUMOylation (274). Consistent with previous studies,
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NEURONAL SUMOYLATION
the majority of SUMO in brains from these mice was present in the nuclear fraction but, in contrast to previously
published immunostaining data, no colocalization was observed between His-HA-SUMO and the synaptic markers
synapsin (all synapses), VIAAT (inhibitory synapses), or
vGluT1 (excitatory synapses) (274). The precise reasons for
these differences are unclear; however, it is notable that
SUMO1 conjugation is decreased in the transgenic animals
compared with wild-type mice, perhaps suggesting that any
synaptic SUMO may be below the detection sensitivity of
the techniques used (274). Notwithstanding the differences with transgenic mouse data, overall, a very substantial body of data suggests that, in addition to its roles
in nuclear function, SUMOylation occurs throughout
neurons, can be readily detected at synapses, and is integral to proper synaptic function.
X. SUMOYLATION IN NEURONAL
DEVELOPMENT AND DIFFERENTIATION
The terminal differentiation of neurons requires complex
transcriptional remodeling to control the activation of neuronal-specific genes in cells destined to become neurons.
Soon after its discovery it was realized that SUMOylation is
ideally placed to control the kind of transcriptional programming that drives neuronal differentiation. Indeed, a
number of transcription factors involved in the expression
of neuronal-specific genes, or in the repression of neuronal
genes in nonneuronal cells, have been shown to be subject
to control by SUMOylation. This is a research field of intense activity that is rapidly advancing. Therefore, to illustrate some of the pathways involved, we have selected several examples in which SUMOylation plays a key role in
neuronal differentiation and outgrowth.
A. Pax Proteins
Pax proteins are a family of nine transcription factors that
control embryonic neural development, several of which
are regulated by SUMOylation (273). For example, Pax-6
performs an evolutionarily conserved role in eye and brain
development (26). There are multiple isoforms of Pax-6 generated mainly by alternative splicing. Interestingly, however,
the isoform p43 Pax-6 is a SUMOylated form of the smaller
p32 isoform rather than a distinct alternative splice isoform
(308). More specifically, Pax-6 SUMOylation at K91 enables p32 Pax-6 to bind DNA and become transcriptionally
active. Furthermore, in developing mouse eyes, Pax-6 colocalizes with SUMO1 in a manner dependent on the presence
of K91 (308). Thus, while the exact role of Pax-6 SUMOylation in regulating eye development is unclear, these data
suggest that through modification of Pax-6, SUMOylation
may play an important role in this process.
Pax-7 is required for neural crest development in mice and
chicks and, in the adult, for muscle homeostasis and regen-
eration (24). Pax-7 is SUMOylated at a single nonconsensus
lysine, K85, and while SUMOylation is not essential for
DNA binding activity, it is required for normal neural crest
development (163). Knockdown of Pax-7 in developing
chick embryos leads to defective expression of neural crest
markers that can be rescued by reexpression of wild-type
Pax-7, but not a non-SUMOylatable Pax-7 mutant (163).
Thus, although the exact mechanism of action remains unclear, Pax-7 SUMOylation is required for normal neural
crest development.
B. BRAF35
LSD1-CoREST is a histone demethylase complex involved
in the repression of neuronal genes in nonneuronal cells via
recruitment to target promoters by the repression factor
REST (5, 9). SUMOylation of one member of this complex,
BRAF35, inhibits neuronal differentiation (31). BRAF35
can be SUMOylated at four lysine residues (K31, 125, 156,
and 157), and overexpression of a mutant in which these
residues have been mutated to non-SUMOylatable arginines (BRAF35– 4KR) impaired the ability of the LDS1CoREST complex to repress the expression of neuronalspecific sodium channel ␣-subunit genes in HeLa cells (31),
suggesting that SUMO modification of BRAF35 is required
for the repressive activity of the LSD1-CoREST complex.
With the use of chromatin immunoprecipitation assays,
overexpression of non-SUMOylatable BRAF35 was shown
to decrease chromatin binding by the LSD1-CoREST, suggesting this modification is required to either recruit or stabilize the complex on chromatin (31). The P19 embryonic
carcinoma stem cell line can be differentiated into neurons
through overexpression of the transcription factor NeuroD2 and its dimerization partner E12 (65). Neuronal differentiation was strongly inhibited by coexpression of
BRAF35, but not non-SUMOylatable BRAF35, demonstrating the requirement for BRAF35 SUMOylation on the repression of neuronal genes. Interestingly, mature neurons express
an inhibitor of BRAF35, iBRAF, which heterodimerizes with
BRAF35, sequestering it from the LSD1-CoREST complex
and reducing BRAF35 SUMOylation (31). Thus SUMOylation of BRAF35 mediates the repression of neuronal genes in
nonneuronal cells and, through targeting BRAF35 SUMOylation, iBRAF can effectively inhibit BRAF35 function to drive
neuronal differentiation.
C. Verloren
Olfactory projection neurons extend dendrites that synapse onto a specific class of olfactory receptor neurons,
and project axons to higher brain centers in a manner
that has been extensively mapped in Drosophila (115–
117, 171, 305). The protein Verloren (Velo) was origi-
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HENLEY ET AL.
nally identified in a genetic screen for proteins that regulated the specificity of dendrite targeting from olfactory
projection neurons. Velo-deficient flies exhibit numerous
axonal targeting defects (13) and, interestingly, sequence
analysis suggested Velo encodes a SUMO protease, most
similar to the human protease SENP7. In contrast to
effective rescue with wild-type Velo, expression of a mutant Velo lacking the predicted active site cysteine did not
rescue the effects of Velo perturbation, suggesting that its
catalytic activity is essential for dendrite and axon targeting of olfactory projection neurons (13). The effects of
Velo perturbation on dendritic targeting could be partially rescued by other Drosophila SUMO proteases, but
the axonal defects could not, suggesting that Velo is specifically required for axonal targeting. Further underscoring a crucial role for SUMOylation, loss-of-function
mutants of the Drosophila equivalents of both SUMO
and Ubc9 also exhibited targeting defects (13). These
results are particularly interesting because they demonstrate that targeted deSUMOylation is essential for the
regulation of neuronal outgrowth, and future work will
focus on identifying the specific substrate(s) involved.
Overall, these examples illustrate how SUMO regulation
of transcription is an indispensible regulator of neuronal
differentiation. The sophistication of this system is only
just beginning to emerge, and the regulatory pathways
are subtle and complex. SUMOylation of some transcription factors drives neuronal differentiation, whereas
SUMOylation of others prevents transcription of neuronal specific genes. Nonetheless, it is already evident that
differential substrate-specific modification participates in
the control of highly complex cellular processes to ensure
coordinated cellular reprogramming to a neuronal fate.
XI. SUMOYLATION IN ROD AND CONE
DEVELOPMENT
The vertebrate retina is a widely used model system for investigating neuronal differentiation. The retina contains two distinct types of photoreceptor, rods and cones, which arise from
a common progenitor. For progenitor cells to become rods,
they must express rod-specific genes, and repress the expression of cone-specific genes. These cell fate decisions are regulated by expression of relatively few transcription factors such
as Nrl and Nr2e3, which are expressed primarily in rod precursors, drive the expression of rod-specific genes and, in the
same cells, repress cone-specific genes. Interestingly, SUMOmediated regulation of Nrl and Nr2e3 is an important factor
in rod differentiation in the retina (196, 228).
A. PIAS3 and Nr2e3
Expression of the SUMO E3 PIAS3 in the developing retina
is almost exclusively restricted to Nr2e3-positive rod pre-
1262
cursors (196). Furthermore, overexpression or knockdown
of PIAS3 in the developing mouse retina led to an increase
or decrease, respectively, in the number of cells showing
mature rod phenotype. However, further examination of
retinas in which PIAS3 had been knocked down revealed
that over 60% of cells expressing the rod marker rhodopsin
also expressed cone markers. The effect of PIAS3 knockdown was therefore attributed to defective repression of
cone-specific genes in developing rods, a phenotype also
observed upon mutation of Nrl or Nr2e3 (196). Following
this line of investigation, PIAS3 was shown to bind to and
occupy the same promoters as Nr2e3, and to catalyze the
SUMO1 modification of Nr2e3 at three lysines: K178,
K315, and K322 (196). Mice with a spontaneous null mutation in Nr2e3 exhibit a phenotype characterized by excessive numbers of cells expressing cone markers, and this can
be partially rescued by expression of wild-type Nr2e3 but
not by expression of an Nr2e3 mutant lacking the major
SUMO acceptor site, K178, suggesting that SUMOylation
of Nr2e3 is required for the effective repression of conespecific genes. Taken together, these data have been interpreted to show that PIAS3-mediated SUMOylation of
Nr2e3 on cone-specific promoters turns it from an activator
of transcription into a potent repressor. Interestingly, expression of the viral protein Gam1, which specifically inhibits SUMOylation through targeting the E1 complex for degradation (17), caused defective repression of cone-specific
genes in rods and defective expression of the rod marker
rhodopsin. Thus, while PIAS3-mediated SUMOylation of
Nr2e3 is required for the repression of cone markers,
SUMOylation per se is also required to drive the expression
of rhodopsin, suggesting both negative and positive regulation of gene expression by SUMOylation is required to
drive rod differentiation (196).
B. Nrl
The rod-specific transcription factor Nrl is also SUMOylated,
primarily at K20 (228). Like Nr2e3, Nrl represses cone-specific genes but, in addition, it also drives expression of many
rod-specific genes, including rhodopsin and Nr2e3 itself. NonSUMOylatable Nrl is less effective than wild-type Nrl at activating rhodopsin and Nr2e3 promoters (228). Furthermore,
Nrl knockout mice show reduced numbers of rhodopsin expressing cells and a loss of Nr2e3 expression. While expression
of wild-type Nrl could almost completely rescue this phenotype, expression of non-SUMOylatable Nrl was much less
effective (228). Thus SUMOylation of Nrl controls its ability
to drive expression of rod-specific genes and is required for
proper retinal development. Overall, these reports illustrate
how SUMOylation of distinct targets on specific promoters
can lead to the complex alterations in gene expression patterns
required to determine cell fate and differentiation in the mammalian retina.
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XII. SUMOYLATION IN SYNAPSE
FORMATION AND SPINE
MORPHOGENESIS
A. MEF2A SUMOylation and Synapse
Formation
synaptic sites. By microarray analysis, the authors identified
synaptotagmin-1 as a target of MEF2A transcriptional activity and concluded that SUMOylation of MEF2A suppresses orphan presynapse number through repression of
synaptotagmin-1 expression (307).
B. MeCP2 and Synapse Formation
Activity-dependent synapse formation and remodeling is
critical for neuronal development and circuit formation. In
cerebellar granule neurons, specialized structures called
dendritic claws act as postsynaptic targets for mossy fiber
and Golgi neuron axons to form synapses onto. Dendritic
claw formation is activity-dependently regulated by the
transcription factor MEF2A. When activated, MEF2A promotes transcription of a set of genes, including Arc and
synGAP, that suppress synapse formation (68). As discussed above, MEF2 transcriptional activity is controlled
by the interplay between several posttranslational modifications including phosphorylation, acetylation, and
SUMOylation (86, 127, 226, 240, 319). In brief, constitutive
phosphorylation of S408 under basal conditions promotes
MEF2A SUMOylation, negatively regulating its transcriptional activity. Calcium influx following neuronal activation causes calcineurin-mediated dephosphorylation of
S408, which promotes a switch from SUMOylation to acetylation, leading to activation of MEF2A and the consequent
transcription of genes that repress postsynaptic differentiation (240).
The role of SUMOylation in MEF2A transcriptional activity is further supported by the identification of the critical
role of the SUMO E3 PIASx in dendritic claw formation
(239). Knockdown of PIASx in cultured cerebellar granule
neurons increases transcription of a MEF2A reporter gene
and reduced the number of dendritic claws in cerebellar
slices. Correspondingly, overexpression of PIASx increased
the number of dendritic claws, and this effect was completely suppressed upon knockdown of MEF2A (239), indicating that MEF2A is the target of PIASx E3 activity that
underlies dendritic claw formation.
As well as controlling postsynaptic specialization, MEF2A
SUMOylation also plays a role in presynaptic differentiation (307). During development, immature neurotransmitter release sites assemble independently of postsynaptic
contact. As networks mature, these “orphan” presynaptic
sites are eliminated and their vesicles and proteins are redistributed to presynaptic terminals that have formed mature
synapses with postsynaptic neurons (325). In cultured cerebellar granule neurons, knockdown of MEF2A caused an
increase in these orphan presynaptic sites without affecting
the number of mature synapses (307). The MEF2A knockdown phenotype was rescued by expression of wild-type
MEF2A, but not by a non-SUMOylatable MEF2A mutant,
indicating that the SUMOylated transcriptional repressor
form of MEF2A mediates the suppression of orphan pre-
Methyl CpG binding protein 2 (MeCP2) is an X-linked
DNA binding protein that functions as a transcriptional
repressor through the recruitment of HDACs (188). Mutations in MeCP2 are the primary cause of Rett Syndrome (3),
a neurodevelopmental disorder that almost exclusively affects females and is characterized by developmental regression, loss of hand skills, impaired mobility and speech, and
other symptoms commonly associated with autism (70).
SUMOylation of MeCP2, primarily at K223, has been reported to be involved in the regulation of synapse development (37). Interestingly, non-SUMOylatable MeCP2 did
not bind HDAC1 and 2, suggesting a role for MeCP2 SUMOylation in transcriptional repression. Knockdown of
MeCP2 in neurons caused upregulation of multiple genes in
a whole genome cDNA microarray assay. More detailed
analysis of five of these genes revealed that their upregulation caused by MeCP2 knockdown was returned to normal
levels by exogenous expression of an RNAi-insensitive
wild-type MeCP2. However, expression of an RNAi-resistant non-SUMOylatable MeCP2 did not reverse the upregulation, suggesting that SUMOylation of MeCP2 is required for its repressive capability (37). Furthermore,
knockdown of MeCP2 reduced synaptic density both in
cultured hippocampal neurons and in the hippocampus in
vivo after in utero electroporation with an shRNA targeting
MeCP2. Reexpression of wild-type MeCP2 rescued this
phenotype, but expression of non-SUMOylatable MeCP2
did not (37). These data highlight how SUMOylation of
MeCP2 acts as a regulator of its transcriptional activity and
synapse formation and open new possibilities for defining
mechanistically how defects in SUMOylation and/or
MeCP2 are involved in the cellular pathology of Rett Syndrome.
C. SUMOylation of CASK Regulates
Dendritic Spine Morphogenesis
Calcium/calmodulin-dependent serine protein kinase (CASK)
is a member of the membrane-associate guanylate kinase
(MAGUK) family of proteins that includes the postsynaptic
density protein PSD95. CASK binds to cell adhension molecules including syndecan-2 and synaptic cell adhesion molecule (SynCAM) and plays a key role in the formation of
dendritic spines by connecting transmembrane adhesion
molecules to the actin cytoskeleton via interaction with the
scaffold protein 4.1 (106). Knockdown of CASK in cultured
hippocampal neurons reduces spine size and number, sug-
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HENLEY ET AL.
gesting that CASK stabilizes or maintains spine morphology. The knockdown phenotype can be rescued by
expression of wild-type CASK but not CASK lacking the
protein 4.1 binding site (36). Interestingly, CASK has been
shown to undergo activity-dependent SUMOylation at
K679, and this modification regulates binding to protein
4.1, since a CASK-SUMO1 fusion construct or SUMO1
overexpression reduced the CASK-protein 4.1 interaction
(36). CASK overexpression increases spine size and density,
whereas overexpression of the CASK-SUMO1 fusion protein has opposite effects, leading to a decrease in both spine
size and number. These results suggest that SUMOylation
of CASK downregulates spine formation via uncoupling
membrane adhesion molecules from protein 4.1 and the
cytoskeleton (36).
XIII. REGULATION OF ION CHANNELS
AND CELL EXCITABILITY
Modulation of neuronal excitability can profoundly affect
synaptic properties and neuronal networking. SUMOylation
plays a fundamental role in controlling membrane potential
and cell excitability via regulation of ion channels in neurons,
as well as cells isolated from different organs including heart
and pancreas. SUMOylation has been proposed to regulate a
wide range of channels but, as outlined below, in many cases
the evidence is not yet conclusive.
A. SUMO Regulation of K2P1 (TWIK1)
Activity
The K2P family of potassium channels helps maintain negative resting membrane potentials below the firing threshold of excitable cells by contributing to the leak K⫹ conductance. Most K2P channels are actively modulated by neurotransmitters, and within the wider family, channel
subunits mediate time- and voltage-independent potassium
leak currents whereas other subtypes, including TASK
channels, increase channel open probability with depolarization (211).
The K2P1 channel that regulates the background potassium
current in many cells has been reported to be a target for
SUMOylation. When wild-type K2P1 channel is heterologously expressed at the plasma membrane of Xenopus
oocytes, its deSUMOylation is necessary to generate an outward potassium current. This led to the suggestion that
SUMOylation of K274 maintains the channel in an inactive
state (218). Notably, mutation of K274 to glutamic acid or
overexpression of SENP1 allows the channel to operate,
exhibiting K⫹ selectivity and sensitivity to extracellular pH
(218). However, the role of K274 SUMOylation has been
challenged by another group who reported that the mutation K274E caused an increase in K2P1 current density,
while the mutation K274R, which would also prevent
1264
SUMOylation, did not (66). These data suggested a charge
effect of amino acid substitution rather than an effect of
SUMOylation was responsible for the observed alteration
in K2P1 activity. Furthermore, they also suggest that endocytosis rather than SUMO-mediated inactivation accounts
for K2P1 silencing (66). However, the authors of the original Rajan et al. (218) report have disputed these findings in
a subsequent publication in which they demonstrate that
silent K2P1 channels in excised patches of plasma membrane can be activated by application of recombinant
SENP1 and resilenced by addition of SUMO1 (209). In
these experiments, mutation of K274 to glutamine, arginine, glutamic acid, aspartic acid, cysteine, or alanine residues all resulted in constitutively active K2P1 channels,
which were insensitive to SUMO1 and SENP1 (209). Thus,
as things stand, whether the K2P1-mediated current is controlled by SUMOylation remains a matter of contention.
Interestingly, K2P1 can form heteromeric assemblies with two
P domain, acid-sensitive K⫹ channel (TASK) subunits K2P3 or
K2P9, and the resultant channels mediate the noninactivating
standing outward K⫹ current (IKso). In addition to controlling
the resting membrane potential, IKso defines cerebellar granule
neuron responses to, among other things, membrane stretching, intracellular acidosis, and volatile anesthetics such as
halothane (161). Inclusion of picomolar concentrations of recombinant SENP1 in the recording patch pipette revealed rapidly increased IKso in rat cerebellar granule neurons, suggesting
that the endogenous SUMO pathway suppresses a fraction of
IKso (212). However, channels formed exclusively by K2P3 or
K2P9 were shown to be insensitive to SENP1. These results
suggest that SUMOylation in cerebellar granule neurons silences IKso channels containing K2P1 and that deSUMOylation by infusion of SENP1 releases these channels from suppression to reduce their excitability (212). Extraplolation of
these data suggests that SUMOylation likely impacts on the
many homeostatic mechanisms mediated via TASK channels
containing K2P1.
B. Voltage-Gated Potassium Channels and
SUMOylation
The voltage-gated potassium channel Kv2.1 is widely expressed in many tissues including brain, heart, muscle, and
pancreas where it controls membrane excitability by mediating the majority of the delayed inwardly rectifying current
(IDR). During periods of increased activity, the voltage-dependent activation of Kv2.1 is shifted to more negative
membrane potentials, resulting in homeostatic suppression
of excitability (59, 60). Inclusion of SUMO1 in the recording electrode in current clamp experiments on hippocampal
neurons increased action potential firing rates, whereas
SENP1 decreased action potentials (210). The increased
excitability mediated by SUMO1 was attributed to decreased voltage sensitivity of the delayed inwardly-rectifying current and shown to be due to SUMOylation of K470
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NEURONAL SUMOYLATION
of Kv2.1 (210). SUMO conjugation makes the channels more
sensitive to depolarization by shifting the half-maximal activation voltage of the channels. Kv2.1 channels comprise four
subunits, but only two nonadjacent subunits can be simultaneously SUMOylated. SUMOylation of one subunit shifts
voltage for half-activation (V1/2) by 15 mV, whereas SUMOylation of two subunits shifts the V1/2 by 35 mV. Thus direct
SUMOylation of Kv2.1 provides a graded regulatory system
for controlling the excitability of hippocampal neurons, and
most likely other excitable cells (210).
Kv2.1 also regulates pancreatic ␤-cell excitability and insulin secretion (165). In HEK 293 cells, infusion of recombinant SUMO1 inhibited current through exogenously expressed Kv2.1 by 80%, and expression of the YFP-SUMO1
in human ␤-cells or insulinoma cells significantly inhibited
native voltage-gated potassium currents (47). In HEK 293
cells, SUMOylation of Kv2.1 accelerated inactivation and
inhibited recovery from inactivation of the Kv2.1-mediated
current. These properties result in the widening of action
potentials and a decreased firing frequency, suggesting
SUMOylation has a strong inhibitory action on Kv2.1 to
regulate cellular excitability in native ␤-cells and, by extrapolation, other cell types (47).
Another voltage-gated potassium channel, Kv1.5, is present
in brain (284) but is best characterized for mediating the K⫹
current (IKur) central to action potential repolarization in
atrial myocytes (190). Kv1.5 is directly SUMOylated at two
sites located close to plasma membrane spanning domains
(12). SUMOylation alters the biophysical properties of the
channel to accelerate inactivation and inhibit recovery from
inactivation, resulting in decreased firing frequency (12). In
Cos-7 cells, deSUMOylation of the Kv1.5 channel, either by
disruption of the SUMOylation sites or by overexpression
of SENP2, causes a hyperpolarizing shift in the steady-state
inactivation of the related IKur current. This may represent a
significant mechanism to fine tune electrical activity. Although no direct experimental evidence has yet been reported, multiple other Kv channel ␣-subunits contain consensus SUMOylation motifs in similar positions, suggesting
that SUMOylation may represent a general mechanism of
channel regulation. In this respect it may also be significant
that KChAP, a protein originally described as a potassium
channel chaperone that increases expression (298), was
subsequently identified as the SUMO E3 ligase PIAS3 (297).
C. TRPM4
Transient receptor potential melastatin (TRPM) ion channel
type 4 (TRPM4) is a nonselective cation channel that is activated by, but is not permeable to, intracellular Ca2⫹. It is
widely expressed in neurons as well as in heart, pancreas, and
kidney and is particularly abundant in cardiac Purkinje fibers
(141). TRPM4 is a SUMO substrate, although the specific
lysine residue(s) involved have not yet been defined (289).
However, a glutamate to lysine mutation at residue 7 in the
NH2 terminus of TRPM4, which enhances channel SUMOylation and increases plasma membrane expression and consequent channel activity, has been linked to the autosomal dominant disease progressive familial heart block type I (141).
Further genetic linkage analyses of families with autosomal
dominant isolated cardiac conduction block revealed other
missense mutations in the TRPM4 gene. Each of these mutations resulted in an increased current density gain of function, which was suggested to be attributable to impaired
endocytosis caused by misregulation of SUMOylation
(156). However, given that the first reported mutation adds
a lysine residue to the NH2 terminus and the other three do
not involve lysines (R164W, A432T, and G844D), how this
would occur is unclear.
XIV. SUMOYLATION IN POLARIZED mRNA
AND TRAFFICKING
Many neuronal processes, including synapse formation and
maintenance, and synaptic plasticity, require local protein synthesis (288) which, in turn, requires the transport of mRNA to
distal processes by mRNA binding proteins. SUMOylation of
one such transport protein, La (283), regulates its interaction
with the motor proteins kinesin and dynein, which are required for retrograde and anterograde transport, respectively
(101). In a seemingly simple and elegant “switch” system,
SUMOylated La binds only dynein, whereas non-SUMOylated La binds only kinesin, thus determining the direction of
the axonal transport of La and its associated mRNAs (283). It
remains unclear how the SUMOylation of La is regulated, and
it is possible that other mRNA binding proteins could also be
subject to such directional regulation. It is therefore likely that
SUMOylation plays important roles in the delivery of mRNAs
throughout the neuron.
XV. SYNAPTIC SUMOYLATION AND
NEUROTRANSMISSION
A. Presynaptic SUMOylation and
Neurotransmitter Release
Timely and appropriate neurotransmitter release is fundamental to synaptic function. Moreover, activity-dependent changes in release properties underlie some
forms of synaptic plasticity (30). The fusion of synaptic
vesicles with the presynaptic membrane is very precisely
controlled by the interplay between active zone proteins.
While many of these proteins have been identified, it is
unclear how the complex web of interactions is regulated, particularly since several of these proteins are multifunctional. Intriguingly, there are multiple, mainly
unidentified, SUMO substrate proteins present in synaptosomes and purified presynaptic membrane fractions
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HENLEY ET AL.
(67). Furthermore, presynaptic SUMOylation appears to
be activity-dependently regulated since application of
AMPA to isolated synaptosomes increases the extent of
protein SUMOylation, while kainate application decreases synaptosomal protein SUMOylation and NMDA
stimulation has no discernible effect (67).
During the preparation of isolated synaptosomes, presynaptic boutons are sheared from axons and then reseal. It is
therefore possible to entrap compounds, peptides, or even
recombinant proteins inside the synaptosomes during the
resealing process. Synaptosomes loaded with SENP1 displayed increased KCl-evoked glutamate release, whereas
synaptosomes loaded with SUMO1 exhibited decreased
KCl-evoked release. Conversely, presynaptic release evoked
by stimulation with kainate was enhanced by SUMO1 (but
not SUMO1⌬GG, which lacks the two COOH-terminal
glycines required for conjugation), and reduced by SENP1
(67). Thus SUMOylation of presynaptic proteins can either
up- or downregulate neurotransmitter release depending on
the precise stimulus. In synaptosomes containing SENP1,
KCl- or AMPA-induced Ca2⫹ influx was enhanced,
whereas in synaptosomes containing SUMO1, KCl- or
AMPA-induced Ca2⫹ influx was reduced. Consistent with
the release assays, the opposite effects were observed following kainate stimulation (67).
These results illustrate the complexity of the roles of
SUMOylation in neurotransmitter release. Overall, the data
suggest that SUMO conjugation to distinct subsets of presynaptic proteins acts to tune presynaptic release in an activity-dependent and stimulus-specific manner.
Exactly which proteins mediate these effects is the subject of
a concerted research effort, and several presynaptic proteins
known to regulate release have been identified as SUMO
substrates (FIGURE 6). For example, the syntaxin 1A interacting protein tomosyn is a SUMO2/3 substrate at K730
(303). Mutation of K730 enhances tomosyn inhibition of
presynaptic exocytosis in PC12 cells, but does not alter the
interaction with syntaxin1A, suggesting that SUMOylation
of this protein could downregulate release (303).
Some neurotransmitter receptors that are partly or exclusively localized at the presynaptic terminal have also been
proposed as SUMOylation targets. These include and cannabinoid receptor type 1 (CB1) (85) and group III mGluRs
(62, 266, 302), but as yet no compelling functional evidence
has been reported to support a physiological role for
SUMOylation of these receptor proteins.
1. Rab3 interacting molecule 1␣
To date, the only active zone protein that has been extensively
characterized as a SUMO substrate is Rab3 interacting molecule (RIM1␣). RIM1␣ is a large, multifunctional protein
which coordinates many of the critical stages of neurotrans-
1266
mitter release (25), including docking and priming (50) of
synaptic vesicles, presynaptic LTP (29), and clustering of Ca2⫹
channels (123) to allow coordindated Ca2⫹ entry in response
to an action potential. Despite its critical role in neurotransmitter release, little is known about how the many functions of
RIM1␣ are coordinated. In a recent study, Girach et al. (76)
demonstrated that RIM1␣ is SUMOylated in neurons at a
single lysine, K502, and that SUMOylation of this site is required for normal presynaptic exocytosis. Specifically, knockdown of RIM1␣ and replacement with a non-SUMOylatable
mutant eliminates the fast component of synaptic vesicle exocytosis, due to a defect in depolarization-induced calcium entry. SUMOylation of RIM1␣ makes its PDZ domain available
for interaction with CaV2.1 and is required for the Ca2⫹-channel clustering function of RIM1␣, although docking of synaptic vesicles appears to be unimpaired by a lack of SUMOylation. Therefore, RIM1␣ SUMOylation acts as a molecular
switch, coordinating the diverse functions of this protein. This
study found no activity dependence of RIM1␣ SUMOylation,
implying instead that there may be two distinct populations of
SUMOylated and non-SUMOylated RIM1␣, which carry out
distinct functions at the presynapse.
As outlined above, the effects of altering SUMOylation levels on presynaptic glutamate release are diverse and stimulus dependent (67). This kind of SUMO-mediated molecular switch may represent a general mechanism for the coordination of multifunctional presynaptic proteins, possibly
explaining how many proteins (e.g., syntaxin1A, synaptotagmin, synapsins) participate in different stages of the
synaptic vesicle cycle. Interestingly, syntaxin1A has been
suggested to be a SUMO substrate in pancreatic ␤-cells
(170) where it acts as a “brake” on exocytosis. If this were
the case in neurons, it would mean that SUMOylation has
different effects on synaptic vesicle exocytosis depending on
the substrate protein, consistent with a highly sophisticated
level of regulation of presynaptic function by SUMOylation. Clearly, this is an underdeveloped field with many
intriguing questions remaining to be answered.
2. Collapsin response mediator 2
Collapsin response mediator 2 (CRMP2) is a microtubulebinding protein involved in axon guidance and the establishment of neuronal polarity (112). CRMP2 can regulate presynaptic release through interactions with the calcium channel
subunit CaV2.2 (304). In hippocampal neurons, CRMP2
promotes neurotransmitter release through enhanced
surface expression of CaV2.2. Interestingly, CRMP2 has
been identified as a SUMO substrate, which is modified at
a single lysine residue, K374, in the CAD cell line (121).
In adult dorsal root ganglion (DRG) neurons, overexpression of CRMP2, SUMO, and Ubc9 reduced KCl depolarization-induced calcium influx. Conversely, overexpression of a non-SUMOylatable CRMP2 mutant along
with SUMO and Ubc9 increased KCl-induced calcium
influx, suggesting CRMP2 SUMOylation negatively
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NEURONAL SUMOYLATION
Reserve vesicle pool
A
Synapsins
SUMO1
Kainate receptors
Recycling vesicle pool
CB1 receptors
RIM1α
α
B
Synaptotagmin I, II
VAMP2
γ β
D
Munc18a
CRMP2
Nav1.7
SUMO1
SNAP-25
C
Cav2.2
Syntaxin1A
Neurotransmitter release
Ca2+
Primed vesicle
FIGURE 6. The roles of protein SUMOylation at the presynapse. Schematic highlighting proteins involved in
key stages of presynaptic vesicle exocytosis. A: the reserve pool of synaptic vesicles is tethered to the actin
cytoskeleton by synapsin proteins. On depolarization, calcium influx activates protein kinases, e.g., CaMKII and
PKA, which phosphorylate synapsins allowing migration of vesicles from the reserve to the recycling pool.
B: vesicles in the recycling pool are docked and primed for release. This process involves many proteins
including the SNARE complex (VAMP2, SNAP-25, and syntaxin-1A), munc18a, synaptotagmins, munc13–1,
and RIM1␣. C: calcium influx through voltage-gated CaV channels (clustered by RIM1␣) triggers exocytosis of
neurotransmitter by binding to synaptotagmin. D: collapsin response mediator protein 2 (CRMP2) is reported
to be SUMOylated in neurons. SUMOylation of CRMP2 has been implicated in inhibition of Ca2⫹ entry through
CaV2.2 channels, and increasing surface expression of NaV1.7 channels. Named proteins are potential or
confirmed SUMO substrates. For confirmed SUMO substrates, the role of SUMOylation is shown, i.e.,
SUMOylation of RIM1␣ is required to cluster CaV channels and allow coordinated Ca2⫹ entry, and GluK2
SUMOylation triggers kainate receptor endocytosis.
regulates calcium influx (121). However, exactly how
SUMOylation of CRMP2 affects CaV2.2 trafficking and
calcium influx in DRG neurons has not yet been investigated.
In addition to CaV2.2, CRMP2 has also been reported
to regulate surface expression of the voltage-gated sodium channel subunit NaV1.7 in DRG neurons (61). The
ability of CRMP2 to regulate NaV1.7 trafficking is also
regulated by SUMOylation. CRMP2 mutants lacking the
SUMOylation site impair sodium currents in CAD cells, and
decrease surface trafficking of NaV1.7 (61). This effect was
specific to NaV1.7, since NaV1.1 and NaV1.3 currents were
unaffected. Furthermore, in DRG neurons, overexpression
of non-SUMOylatable CRMP2 reduced sodium current
density (61). Together, these data suggest that SUMOylation of CRMP2 is required for correct surface trafficking
of NaV1.7 sodium channels. However, whether CRMP2
interacts directly with NaV1.7, and the mechanism underlying how SUMOylaton of CRMP2 regulates NaV1.7 channel trafficking, is currently unclear. Furthermore, in addition to these examples, CRMP2 has also been reported to
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HENLEY ET AL.
regulate the trafficking and signaling of NMDA and kainate-type glutamate receptors (22, 23, 172). Further work
will be necessary to test if SUMOylation of CRMP2 also
controls glutamate receptor function.
B. Postsynaptic SUMOylation
As for the presynaptic terminal, there has been rapid development in the identification and understanding of the roles of
SUMOylation of a growing number of predominantly postsynaptic proteins (FIGURE 7). As outlined below, the best characterized of these is the GluK2 subunit of kainate receptors.
1. Kainate receptors
Kainate receptors (KARs) are tetrameric glutamate-gated
cation channels composed of the subunits GluK1–5 (formerly GluR5–7, KA1, KA2) (83). They are present at preand postsynaptic membranes at many synapses throughout
the brain. Presynaptic KARs control neurotransmitter release, extrasynaptic KARs regulate neuronal excitability,
and postsynaptic KARs contribute to postsynaptic depolarization (42, 83). Furthermore, KARs are highly expressed in
many neuronal circuits during development where they regulate synapse formation and stabilization, after which their
expression is reduced (131, 143, 144, 267).
The KAR subunit GluK2 is strongly linked with autism
(133, 203, 250) and intellectual disability (183). GluK2 is
SUMOylated at a single lysine residue (K886) in its intracellular COOH terminus in response to agonist activation,
and this modification is required for agonist-induced receptor internalization (175). Furthermore, postsynaptic KAR
responses at mossy fiber-CA3 synapses are decreased by
infusion from the recording electrode of SUMO1, or increased by infusion of the catalytic domain of the SUMOspecific deconjugating enzyme SENP1 (175).
Protein kinase C (PKC)-mediated phosphorylation of
KARs can enhance or depress KAR surface expression in
cultured hippocampal neurons (173). Subsequent work
demonstrated that PKC phosphorylation of GluK2 at
S868 precedes and regulates receptor SUMOylation. The
PKC activator PMA enhances GluK2 SUMOylation and
consequent internalization, whereas mutation of S868 to
a nonphosphorylatable alanine prevents kainate-induced
GluK2 SUMOylation at K886 and endocytosis (137,
300). Further analysis of the functional interrelationship
between phosphorylation and SUMOylation of GluK2
was performed using patch-clamp electrophysiology of
HEK 293 cells expressing recombinant wild-type or mutated GluK2. Infusion of conjugatable SUMO1 from the
electrode caused a decrease in the KAR current amplitude
in HEK 293 cells expressing wild-type GluK2. However,
SUMO1 infusion did not decrease the KAR current am-
1268
plitude evoked from HEK 293 cells expressing K886R
GluK2 or in cells expressing a nonphosphorylatable alanine at serine-868 (32).
The physiological importance of GluK2 SUMOylation was
highlighted by the observation that it is required for longterm depression (LTD) of postsynaptic KARs (32). At
mossy fiber-CA3 synapses, inclusion of nonconjugatable
SUMO1-⌬GG had no effect on electrophysiologically induced LTD compared with control conditions. However,
infusion of conjugatable SUMO1 decreased KAR EPSCs
and completely occluded LTD. Furthermore, infusion of
SUMO1 into CA3 neurons virally expressing wild-type
GluK2 depressed KAR EPSCs, and induction of KAR
LTD was indistinguishable between wild-type GluK2transduced and nontransduced neurons. In contrast, infusion of SUMO1 into neurons virally expressing either
non-SUMOylatable K886R GluK2 or nonphosphorylatable S868A GluK2 did not depress KAR EPSCs, and KAR
LTD was completely blocked (32). Taken together, these
results indicate that SUMOylation of GluK2 at K886 and
phosphorylation at S868 are required for KAR LTD.
These studies on the GluK2 subunit provide a clear example
of how SUMOylation and phosphorylation can combine to
modulate synaptic function by orchestrating the temporal
and spatial regulation of KARs. While the underlying mechanics of how phosphorylation and SUMOylation modulate KAR surface expression is an area of active investigation, these findings illustrate the complexity and refinement
of receptor trafficking pathways and provide insight into
the mechanisms that control synaptic transmission, and
consequently network activity and brain function. More
generally, these results underscore the importance of the
interplay between posttranslational modifications in controlling substrate protein behavior.
XVI. SUMOYLATION AND SYNAPTIC
PLASTICITY
A. AMPARs
AMPA receptors (AMPARs) mediate most fast excitatory
neurotransmission in the CNS and are key determinants
of neuronal function and dysfunction (96, 110). While
there have been investigations looking for AMPAR
SUMOylation in recombinant systems (302) and investigating the effects of modulating synaptic SUMOylation
on basal AMPAR responses (174), to date, there are no
reports of AMPARs themselves being directly SUMOylated. Nonetheless, as discussed below, it is becoming
increasingly apparent that SUMOylation may play import roles in activity-dependent AMPAR trafficking
through SUMOylation of AMPAR interacting proteins.
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NEURONAL SUMOYLATION
Glutamate
NMDA
receptor
AMPA
receptor
Kainate
receptor
P
Ca2+
S1
?
Arc
S1
Ubc9
Arc
S1
S1
SENP1
TTX
A
S1
Ubc9
B
S1
SUMO1
S2
SUMO2
S1
K2P1
Ubc9
S1
FIGURE 7. SUMOylation at the postsynapse. SUMOylation regulates postsynaptic
structure and function through modification
of multiple substrates. A: agonist activation
of the kainate receptor (KAR) leads to PKCmediated phosphorylation and SUMOylation
of the GluK2 receptor subunit, ultimately resulting in KAR endocytosis. Although not direct targets themselves, SUMOylation also
regulates the trafficking of AMPA receptors.
Induction of LTP with the NMDAR coagonist
glycine recruits Ubc9 and SUMO to the postsynapse, and SUMOylation is essential for
AMPAR insertion into the postsynaptic membrane. Suppression of synaptic activity by
TTX blockade of voltage-gated sodium channels causes increased AMPAR insertion and
synaptic upscaling. This treatment also
causes the loss of the SUMO protease
SENP1, promoting SUMOylation of the synaptic protein Arc, which in turn reduces
AMPAR endocytosis, resulting in increased
AMPAR surface expression. B: SUMOylation
modulates neuronal excitability through modification of the potassium channels K2P1 and
Kv1.5, and potentially regulates the ability of
synapses to meet energy demands through
modification of the mitochondrial GTPase
Drp1, which controls mitochondrial number.
S1
S1
Ubc9
K+
K+
Drp1
Drp1
S2
Mitochondria
SENP3
B. Long-Term Potentiation
Synaptic plasticity generally refers to alterations in the number, subunit composition, and/or properties of synaptic
AMPARs that potentiate (long-term potentiation; LTP) or
depress (LTD) the efficiency of synaptic transmission. This
AMPAR-mediated synaptic plasticity occurs at synapses
throughout the CNS and can last minutes, hours, or years
and underlies many neuronal, network, and systems level
functions including learning, memory, and intellectual pro-
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HENLEY ET AL.
cessing (96, 110, 167). The most extensively studied form of
LTP is the NMDAR-dependent increase in the functional
activity and number of postsynaptic AMPARs (129).
NMDARs require both agonist activation and membrane
depolarization to remove an intracellular channel block by
Mg2⫹ to allow Ca2⫹ gating (200). The increased intracellular Ca2⫹ due to NMDAR opening activates calcium/calmodulin-dependent protein kinase II (CaMKII), which triggers the phosphorylation events necessary for AMPAR insertion into the postsynaptic membrane (158, 168, 169).
The possible roles of SUMOylation in plasticity have been
investigated in several experimental models. In cultured rat
hippocampal neurons, increased AMPAR surface expression mediated by application of the NMDAR coagonist
glycine (ChemLTP) (138, 162) recruits SUMO1 to synapses
and increases colocalization of SUMO1 with Ubc9 (113).
Interestingly, SUMO1 and Ubc9 mRNAs were also increased and trafficked to dendrites following ChemLTP,
highlighting SUMO1 and Ubc9 as activity-dependent plasticity-related genes (113). Preventing SUMOylation by expression of dominant negative Ubc9 or the catalytic domain
of SENP1 prevented the ChemLTP-induced increase in
AMPAR surface expression, indicating that SUMOylation
is required for this form of plasticity (113). However, the
SUMO substrates responsible for AMPAR trafficking during LTP are currently unclear.
C. Synaptic Scaling
Another form of plasticity is homeostatic, or synaptic, scaling in which neurons modulate the sensitivity of their synaptic inputs in response to prolonged changes in network
activity. This process is important because it allows neurons
to remain excitable despite large changes in background
network activity (215, 276). Chronic blockade of neuronal
activity with tetrodotoxin (TTX) or NMDAR and AMPAR
antagonists, or increasing neuronal network activity by
blocking inhibitory transmission with the GABAA receptor
antagonist bicuculline, results in compensatory increases or
decreases in AMPAR surface expression and synaptic
strength, respectively.
The signaling mechanisms underpinning synaptic scaling
are the focus of intense investigation, and a number of secreted signaling molecules have been implicated as key mediators including brain-derived neurotrophic factor
(BDNF) (229) and tumor necrosis factor (TNF)-␣ (259).
Furthermore, scaling has been reported to be dependent on
various CaMK isoforms (111, 272). Perhaps the best-characterized protein involved in synaptic scaling is the activityregulated immediate early gene product Arc/Arg3.1 (Arc)
(247), which has been proposed to mediate AMPAR endocytosis. Levels of Arc protein are decreased during TTXmediated upscaling, and this decrease reduces AMPAR internalization resulting in increased surface expression (38,
1270
224). Interestingly, in parallel with TTX-induced increases
in AMPAR surface expression, levels of the deSUMOylating enzyme SENP1 are reduced, and there is a consequent
increase in protein SUMOylation. Furthermore, overexpression of the catalytic domain of SENP1 prevents the
TTX-induced increase in surface AMPARs (45), suggesting
that SUMOylation is required for AMPAR upscaling. Mutation of two consensus SUMOylation sites in Arc (Arc⌬KK) disrupts Arc localization to dendrites, which has been
interpreted to suggest that Arc SUMOylation plays a role in
structural changes required for some forms of LTP consolidation (20). In cultured neurons, viral overexpression of
Arc-⌬KK, but not wild-type Arc, prevents TTX-induced
synaptic scaling, indicating that Arc SUMOylation is required for scaling and that non-SUMOylatable Arc-⌬KK
acts as a dominant negative (45). These initial data are
particularly interesting because Arc-dependent synaptic
scaling has been implicated in intellectual disability (251)
and neurodegenerative diseases (216).
XVII. SUMOYLATION AND SYNAPTIC
SIGNALING CASCADES
A. Kinases and Phosphatases as SUMO
Substrates
As outlined above, phosphorylation can regulate substrate
SUMOylation. However, these two systems also participate
in intricate reciprocal regulation, since important components of the phosphorylation machinery that play direct
roles in synaptic regulation are themselves subject to SUMO
modification.
1. GSK3␤
The multifunctional proline-directed serine/threonine kinase glycogen synthase kinase 3␤ (GSK3␤) plays an important role in the phosphorylation cascade that initiates
NMDAR-dependent LTD (132, 201). Interestingly, inhibition of GSK-3␤ has no effect on NMDAR-dependent LTP,
but GSK-3␤ overexpression blocks LTP, and LTP induction
inhibits the activity of GSK-3␤ (102, 322). These data have
been interpreted to suggest that GSK-3␤ may act as a molecular bridge between LTP and LTD pathways (102, 201).
GSK3␤ is SUMOylated at a single lysine residue (K292),
and mutating this site to a non-SUMOylatable arginine decreased its kinase activity and protein stability, and reduced
its nuclear localization (145). The conditions that promote
GSK3␤ SUMOylation and the physiological relevance to
synaptic plasticity have not yet been established. However,
given that SUMOylation decreases GSK3␤ activity, it is
tempting to speculate that SUMOylation will act to modify
the control of plasticity exerted by GSK3␤. We expect that
defining exactly how SUMOylation regulates GSK3␤ activity is likely to be an active and potentially fruitful area of
future research.
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2. PTEN
Phosphatase and tensin homolog deleted on chromosome
ten (PTEN) is a lipid phosphatase that dephosphorylates
phosphatidylinositol 3,4,5-trisphosphate (PIP3) to generate
phosphatidylinositol 4,5-bisphosphate (PIP2). This downregulates the PI3K-Akt pathway, a major cell regulatory
system, and acts as a brake on cell growth and division
(254). Thus PTEN is a tumor suppressor, and its mutation
is implicated in a large number of cancers (256).
Interestingly, PTEN is highly expressed in adult brain,
and defects in PTEN have been proposed to be a potential
cause of autism spectrum disorders (189). Downregulating PTEN negatively regulates mTOR and promotes
axon branching (56) and neuronal regeneration (157).
Furthermore, activation of NMDA receptors enhances
PDZ interactions between PTEN and PSD95 leading to
the recruitment of PTEN to spines where its phosphatase
activity decreases AMPAR-mediated synaptic responses
(122). Importantly, inhibition of PTEN activity prevents
NMDAR-dependent LTD, but not mGluR-dependent
LTD or LTP. Thus PTEN appears to play a key role as a
synaptic signaling molecule required for NMDAR-dependent LTD (122).
Separate groups have independently reported that PTEN
can be SUMOylated at K254 and K266, and that SUMOylation increases PTEN membrane association and activity
(10, 84, 108). Because PTEN is a negative regulator, its
SUMOylation acts to downregulate the Akt pathway. In
mitotic cells, this has been proposed to suppress cell proliferation and tumor growth in vivo (108). As yet, there have
been no reports on the effects of PTEN SUMOylation in
neurons, but we believe that this too represents an exciting
avenue for future investigation.
XVIII. POSSIBLE FUTURE DIRECTIONS FOR
THE ANALYSIS OF PROTEIN
SUMOYLATION IN NEURONS
Although the techniques used so far have advanced understanding of neuronal and synaptic SUMOylation, and provided some insights into the SUMO-regulation of synaptic
transmission, there are limitations in the approaches currently available. Inclusion of either SUMO1 or SENP1 in
recording electrodes when studying ion channels or synaptic plasticity (e.g., Refs. 32, 210) has been fairly widely
used. This nongenetic approach to manipulating SUMOylation has the advantage that, where an acute effect is seen,
it is highly likely to be due to changes in SUMOylation of
extranuclear proteins, rather than an alteration in gene expression. However, the disadvantage of this approach is a
lack of specificity and fine control, in that the SUMOylation
of many different substrate proteins will be either up- or
downregulated simultaneously. To draw conclusions relat-
ing to specific substrates, therefore, it is essential to show
the insensitivity of a non-SUMOylatable mutant to these
manipulations. Furthermore, it is not possible to empirically determine the extent of up- or downregulation of
SUMOylation of particular proteins using this approach.
As a result, considerable care needs to be taken in interpreting such data, and safe conclusions can only be drawn when
this approach is used in combination with other techniques.
Similarly, when SUMOylation levels in neurons are manipulated by overexpression of either SENPs or SUMO (e.g.,
using transfection or viral infection), many different SUMO
substrates will be affected, and thus it is not possible to
ascribe a functional effect to the modification of a particular
protein without adopting a combinatorial approach (e.g.,
Refs. 44, 113). An advantage of this overexpression approach over inclusion of SUMO or SENP proteins in recording electrodes is that it allows direct biochemical analysis of the changes in SUMOylation levels of individual
proteins, and the SUMO state of the cell as a whole. However, a disadvantage is that, due to the time scale of transfection/viral infection, effects of SUMOylation at a transcriptional level cannot be discounted. It also needs to be
recognized that proteins are substrates for different SUMO
paralogs and that the SUMOylated forms are preferentially
deSUMOylated by different SENPs. Clearly, these parameters should ideally be established before such studies are
performed.
Increasingly elegant techniques are being utilized to offer a
more precise analysis of the effect of SUMOylation of individual proteins on neuronal function. One of these is the
application of an acute molecular replacement (knockdown-rescue) strategy (e.g., Refs. 76, 89). This approach
involves the replacement of endogenous protein with a nonSUMOylatable mutant, allowing much more precise analysis. However, there are caveats: the level of knockdown is
rarely 100%, diluting the observed effect by the continuing
presence of the wild-type protein. Additionally, rigorous
controls must be conducted to ensure that the rescue protein
is expressed to the same level as the wild-type. Since the
substrate cannot be SUMOylated at any stage, this approach offers limited insights into the temporal and spatial
regulation of SUMOylation. A powerful parallel approach
will be to generate knock-in transgenic mice in which the
endogenous protein is replaced with a non-SUMOylatable
mutant. To our knowledge, this has not yet been attempted,
but it has the potential to yield valuable insight into the role
of individual SUMO substrates, not just at a cellular level,
but also on whole animal behavior.
One major drawback to the genetically encoded approaches
listed above is that it is currently only possible to reduce or
eliminate SUMOylation. As yet, there is no SUMO equivalent of the phosphomimetic mutations, which have provided key insights in the study of synaptic regulation by
phosphorylation. While directly fusing SUMO to the NH2
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HENLEY ET AL.
or COOH terminus of the target protein has been utilized
for a number of substrates (e.g., Refs. 36, 226, 257), this
approach is highly substrate dependent, since the fused
SUMO does not necessarily replicate the functional effects
of SUMO conjugation to an internal lysine residue. One
potential answer is to substitute the SUMOylation site of a
neuronal protein with that from a protein that is an intrinsically good SUMO substrate. However, this technique has
not yet been tested and is likely to have many potential
problems and caveats to overcome. Another potential solution is to use an inducible Ubc9/substrate dimerization system in which SUMOylation of a specific substrate can be
induced by addition of rapamycin (324). Again, this system
has yet to be validated, and extensive and rigorous controls
will be needed.
In summary, there are many potential emergent techniques
for the analysis of the role of protein SUMOylation on
neuronal function, but all have limitations and drawbacks.
However, when used in combination, we anticipate that
these approaches will continue to drive rapid progress towards understanding the roles and consequences of protein
SUMOylation in neurons.
XIX. SUMOYLATION AND NEURONAL
DYSFUNCTION
In addition to being a critical regulator of neuronal and
synaptic function under normal conditions, an increasing
body of evidence is accumulating demonstrating that protein SUMOylation is an important factor in a very wide
range of diseases. As well as aberrant SUMOylation being
implicated in disease etiology, an emergent theme is that
SUMOylation can be a powerful cytoprotective mechanism
in cells subjected to severe stress. This is a very active and
rapidly advancing field of investigation, especially in elucidating roles of SUMOylation in transcriptional regulation
and cancer biology (14, 46, 69, 118) and in cardiac dysfunction (289, 326). In addition, the potential roles of SUMOylation in neurological and neurodegenerative diseases are
receiving increased attention, and genetic linkage studies in
particular have implicated the SUMO pathway in multiple
hereditary brain disorders (4, 140). Here we summarize
some of the latest developments linking SUMOylation with
different forms of brain disease (TABLE 2).
A. Polyglutamine Disorders
Polyglutamine (polyQ) disorders are gain-of-function disorders caused by expansion of a CAG repeat in diseasespecific genes (for review, see Ref. 72). These disorders are
generally dominantly inherited, with age of onset dependent on the length of the CAG expansion. The polyQ expansion results in neuronal dysfunction and degeneration,
as well as the formation of neuronal inclusions of the dis-
1272
ease protein in question, although there is some doubt as to
whether these inclusions are directly pathogenic or represent a cellular response to the pathogenic protein. Examples
of polyQ disorders include Huntington’s disease, spinal and
bulbar muscular atrophy (SBMA), dentatorubral-pallidoluysian atrophy (DRPLA), and spinocerebellar ataxias
(SCA). All of these disorders show enhanced neuronal staining for SUMO1, implicating SUMOylation in the pathology
of these diseases (277).
1. Huntington’s disease
Probably the most studied of the polyQ disorders, Huntington’s disease, is caused by a polyQ expansion in the NH2terminal domain of the Huntingtin protein (Htt). It is an
incurable genetic neurodegenerative disorder that causes
cognitive impairment and psychiatric problems as well as
affecting muscle coordination (75). Typically, this disease
manifests in mid-adulthood, and early damage is predominantly in the striatum. However, in some cases mutant proteins containing 60 or more CAG repeats can manifest earlier (before 20 years of age) (75). Huntingtin is a substrate
for both ubiquitination and SUMOylation on the same
lysine residue (257), and the effect of these two modifications appears to be antagonistic; in a Drosophila model,
ubiquitination of Httex1p (a pathogenic fragment of mutant Htt) reduces neurodegeneration, whereas SUMOylation exacerbates it (257). In cultured cells, SUMOylation
stabilizes Httex1p, reducing its ability to form aggregates
and promoting its capacity to repress transcription.
SUMOylation of Htt has also been postulated as an explanation for the specific degeneration of the corpus
striatum observed in Huntington’s disease; the small
GTP-binding protein Rhes is specifically expressed in the
corpus striatum and acts as a SUMO E3 ligase for mutant, but not wild-type Htt, enhancing its SUMOylation
and cytotoxicity (262). This study showed that, in both
HEK 293T cells and striatal neurons, mutant Htt expression was not cytotoxic in the absence of Rhes. Therefore,
it seems likely that SUMOylation of Htt is required for its
cytotoxic effects, and the expression pattern of Rhes
seemingly explains how the ubiquitously expressed mutant Htt leads to the characteristically specific pathology
of Huntington’s disease. This hypothesis is further supported by a recent study showing that genetic ablation of
Rhes is neuroprotective in the striatal-specific 3-nitropropionic acid (3-NP) mouse model of HD (179). However, as this model elicits striatal lesion via 3-NP toxicity
rather than mutant Htt, it remains unclear whether the
neuroprotective effect of the Rhes knockout is due to its
interaction with Htt and SUMO E3 ligase activity. Furthermore, the mechanism of SUMO-Htt toxicity is unlikely to be a simple antagonism of ubiquitination, as
removal of the target lysines for both results in a reduced
mutant Htt toxicity (257).
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Table 2. SUMOylation and disease
Disease
SUMOylated Proteins
Huntington’s disease
Spinal and bulbar muscular
atrophy (SBMA)
Htt (mediated by Rhes)
Androgen receptor
Dentatorubral-pallidoluysian
atrophy (DRPLA)
Spinocerebellar ataxias (SCAs)
Intranuclear inclusions are SUMO1
positive, no substrate yet identified
Ataxin-1
Ataxin-3
Ataxin-7 (by SUMO1 and SUMO2)
Alzheimer’s disease
APP
Tau
Parkinson’s disease
␣-Synuclein
Parkin
DJ-1
Ischemia
General increase in
SUMO2/3-ylation
Drp1
Effect
Reference Nos.
Toxic
Uncertain, possibly protective?
Attenuates AR aggregation and
transcriptional activity
SUMOylation possibly increases
nuclear inclusions and apoptosis
Unclear, possibly increases
aggregation and cytotoxicity
Possibly increases cytotoxicity of
polyQ protein
Reduces protein aggregation and
cytotoxicity
Uncertain. Increasing cellular
SUMOylation increases A␤
production, but SUMOylation of
APP reduces A␤ production.
Further studies show increasing
cellular SUMOylation rescues
cytotoxicity
Controversial. Tau aggregates in
AD model mice stain for SUMO1,
but this has not been shown in
human brain. Tau can be
SUMOylated at K340, but there is
no in vivo evidence
Uncertain. Has been reported to
reduce aggregation and toxicity.
Another study has shown it to
increase its aggregation.
Noncovalent interaction with
SUMO1 increases ubiquitin E3
ligase activity
L166P mutation leads to aberrant
SUMOylation and loss of oxidative
stress response
Likely to be neuroprotective
178, 256, 261
183, 191, 212
SUMO2/3-ylation is anti-apoptotic
during reperfusion
270
224, 230
319
113
54, 102, 152,
314, 317
53, 213, 263
138, 192
279
248
41, 147, 149,
310, 311
88
See text for details.
2. Spinal and bulbar muscular atrophy
Spinal and bulbar muscular atrophy (SBMA) is a disease
that causes degeneration of the motor neurons in the
brain stem and spinal cord resulting in muscle cramp and
progressive weakness (128). Unlike the other polyQ disorders, which are all autosomal, SBMA is an X-linked
disorder caused by a polyQ expansion in the androgen
receptor (AR). The AR is a transcription factor, which is
responsive to androgenic hormones, e.g., testosterone
(11). Like Htt, the AR can be SUMOylated and ubiquitinated at the same lysine residue (213), and this SUMOylation represses the transcriptional activity of the AR
(192). Additionally, SUMOylation of polyQ AR inhibits
its aggregation (184), whereas overexpression of a nondeconjugatable SUMO3 in HeLa cells attenuates polyQ
AR aggregation, and this effect is dependent on the
SUMOylation sites in AR. This effect was not seen for an
NH2-terminal fusion of SUMO3 with polyQ AR, indicating it is the branched peptide nature of SUMOylation
that disrupts aggregation (184). These data have led to
suggestions that enhancing SUMOylation might be a
therapeutic strategy for SBMA. However, it is still unclear whether the aggregation of polyQ AR is cytotoxic in
itself, or whether it represents a cellular response to a
toxic protein. Indeed, in several SBMA models, no correlation has been found between large intracellular inclusions and neurotoxicity (11).
3. Dentatorubral-pallidoluysian atrophy
Dentatorubral-pallidoluysian atrophy (DRPLA) is an autosomal dominant progressive neurological disorder caused
by a polyQ expansion in the atrophin-1 gene. The disease
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HENLEY ET AL.
usually manifests in midlife and has a similar pathology to
Huntington’s disease, including ataxia, tremors, and dementia (234). The function of atrophin-1 is not currently understood, but the polyQ expansion associated with DRPLA
leads to characteristic intranuclear inclusions, which are cytotoxic and stain positively for SUMO1 (271). Coexpression of
SUMO1 with polyQ atrophin-1 in PC12 cells leads to an increase in nuclear inclusions and subsequent apoptosis (271).
Expression of a nonconjugatable mutant of SUMO1 had the
opposite effect, thus implicating SUMOylation in the toxicity
of polyQ atrophin-1. However, whether this is by direct modification of the disease protein or another cellular substrate
remains unclear.
B. Alzheimer’s Disease
4. Spinocerebellar ataxias
Spinocerebellar ataxias (SCAs) are progressive neurodegenerative disorders characterized by atrophy of cerebellar Purkinje cells (197). There are six polyQ spinocerebellar ataxias: SCA types 1, 2, 3, 7 are caused by polyQ expansions in
the proteins ataxin 1, 2, 3 and 7, respectively; SCA type 6 is
caused by a polyQ expansion in the calcium channel subunit CACNA1A, and the polyQ expansion in SCA17 is in
the TBP gene (197). Interestingly, there is mounting evidence that SUMOylation can play a role in the toxicity of
the polyQ mutant protein in SCAs 1, 3, and 7.
SUMOylation of ataxin-1 is inhibited by the polyQ expansion (225), and overexpression of either Ubc9 or SUMO1
increases aggregation of polyQ ataxin-1. Since the protein
aggregates seen in SCAs are presumed to be cytotoxic, protein SUMOylation is implicated in the pathology of SCA
type 1. Additionally, hydrogen peroxide-induced oxidative
stress increases SUMOylation of ataxin-1, resulting in
higher levels of aggregation of the polyQ but not the wildtype protein (231). This oxidative stress-induced aggregation seems to involve the JNK pathway, although the
relationship between this and SUMOylation is unclear.
Indeed, this proposed pathway is unusual because SUMOylation is generally perceived to reduce aggregation
and enhance protein solubility. Interestingly, however, a
phosphorylation site mutant of ataxin-1, S776A, prevents oxidative stress-induced aggregation in a similar
manner to JNK inhibition (225).
Ataxin-3 interacts with SUMO1 (243), and a recent study
has also implicated the SUMOylation of polyQ ataxin-3 in
the pathology of SCA type 3 (320). In HEK 293 cells,
SUMOylation of ataxin-3 at K166 did not affect its cellular
localization or ubiquitination; however, the non-SUMOylatable mutant polyQ ataxin-3 showed reduced stability.
Additionally, cells expressing non-SUMOylatable polyQ
ataxin-3 showed reduced rates of early apoptosis compared
with cells expressing SUMOylatable polyQ ataxin-3 (320).
Thus SUMOylation may have a role in SCA type 3 pathology by increasing the toxicity of polyQ ataxin-3.
1274
In contrast, SUMOylation of ataxin-7 may play the opposite role in SCA type 7. Ataxin-7 is modified by both
SUMO1 and SUMO2, and ataxin-7 inclusions stain positively for both of these proteins (114). The polyQ expansion
in mutant ataxin-7 does not alter the levels of SUMOylation, but expression of a SUMO-deficient polyQ ataxin-7 in
Cos-7 cells resulted in greater ataxin-7 aggregation and cell
death. Thus, in contrast to ataxin-1 and -3, SUMOylation
of ataxin-7 appears to ameliorate the cytotoxic effects of the
polyQ expansion, a pathway more in line with the known
roles of SUMOylation in other models of protein aggregation.
Alzheimer’s disease (AD) is the most prevalent neurodegenerative disorder of the elderly. It is characterized clinically
by progressive dementia and histologically by amyloid
plaques and neurofibrillary tangles. Amyloid plaques are
composed of aggregated amyloid-beta protein (A␤), a
cleavage product of the amyloid precursor protein (APP),
and neurofibrillary tangles consist of a hyperphosphorylated form of the microtubule-associated protein tau. As
with other progressive dementias, some doubt still remains
as to whether these plaques and tangles are the direct cause
of neurodegeneration, or represent a cellular response to the
disease. Intriguingly, several genome-wide association studies have identified polymorphisms in genes involved in SUMOylation as risk factors in sporadic AD, including SAE2
(87), SENP3 (293), and Ubc9 (1). Furthermore, there is
growing evidence of a general involvement of SUMOylation in AD pathology (147), and both APP (318) and tau
(54, 77) have been identified as potential SUMO substrates.
1. SUMO and A␤
The role of SUMOylation in the generation of cytotoxic A␤
from APP is somewhat controversial, with several different
studies reaching opposing conclusions. Li et al. (153) noted
in 2003 that SUMO3 overexpression reduced A␤ production, whereas overexpression of the K11R mutant, which
cannot form polySUMO chains, had the opposite effect. In
contrast, Dorval et al. (55) showed that overexpression of
SUMO3 increased A␤ secretion, but that this was independent of SUMO conjugation. That study also found that
SUMO3 overexpression increased levels of BACE1 (␤secretase, the A␤ cleaving enzyme). Interestingly, a recent
study has reported similar findings, that SUMO1 modification increases BACE1 levels and consequently increases A␤
generation (315). Conversely, a 2008 study showed that
APP itself can be SUMOylated in vivo at two sites, K587
and K595, which are directly adjacent to the sites of
␤-secretase cleavage (318) and that this SUMOylation reduced levels of A␤ aggregates. This study further reported
that overexpression of SUMO1 and Ubc9 reduced levels of
A␤ aggregates in cells expressing the AD-associated V642F
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NEURONAL SUMOYLATION
APP mutant (318). Another recent study demonstrated that
increasing levels of SUMO1 conjugation in astrocytes decreased A␤-induced astrocyte reactivity (103), which has
been implicated in neuronal loss in AD. Clearly further
incisive work is required to resolve these apparently contradictory lines of evidence pointing to SUMOylation either
reducing or exacerbating the neurotoxic effects of A␤. Although the reasons for the current discrepancies are unclear, it almost certainly reflects the complexity of AD and
the multiple and varied roles of SUMOylation in cellular
systems.
2. SUMO and tau
The role of SUMOylation in tau aggregation has been less
extensively investigated than its roles in A␤ generation and
toxicity. Tau can be SUMOylated in heterologous cells,
primarily at K340, located in the fourth microtubule binding site (54). Interestingly, evidence suggests that K340 is
also a ubiquitination site and that SUMOylation may antagonize ubiquitination of tau. Tau SUMOylation is also
regulated by phosphorylation, as levels are increased by
phosphatase inhibition (54). Phospho-tau aggregates in the
Tg2576 APP mouse model of AD also stain positively for
SUMO1 (264), implicating tau-SUMOylation in the pathology of AD. However, these results have not been recapitulated
by studies of tangles in the post-mortem human brain (214).
Thus it remains unclear what role, if any, SUMOylation of tau
may have in the pathology of AD.
3. SUMO regulation of synaptic protein trafficking
in AD
Away from the two major disease proteins, there are other
indications that SUMOylation has a role in AD pathology.
A study profiling the levels of SUMOylated and ubiquitinated proteins in the brains of Tg2576 AD model mice
found changes in the abundance of specific SUMOylated
proteins compared with wild-type mice (178). Although the
identity of many of these proteins is unclear, these results
indicate that, in addition to SUMOylation of APP and tau,
dysfunction of SUMOylation of multiple substrates may be
involved in AD progression.
Importantly, synaptic dysfunction and loss is the primary
cause of dementia (35, 238, 241). However, the precise
signaling cascades underlying synapse collapse and the resulting cognitive loss remain elusive. As outlined above,
SUMOylation of synaptic proteins is a key modulator of
synaptic kainate receptor trafficking and function (32, 113,
174), and is also necessary for AMPAR trafficking in synaptic scaling (45) and LTP (113). The fact that AMPAR
trafficking pathways are disrupted in AD, combined with
published reports of a strong association between SUMOylation and AD (55, 153, 178, 315), raise the possibility
that misregulation of SUMO-dependent AMPAR traffick-
ing may be a causative factor in the synaptic dysfunction
observed during AD, and we expect that this is likely to be
an active area of future investigation.
C. Parkinson’s Disease
Parkinson’s disease (PD) is a progressive neuropathy resulting from the specific loss of dopaminergic neurons projecting from the substantia nigra compacta to the striatum.
These neurons are associated with the initiation of voluntary movements, and as a result the symptoms of the disease
include muscular rigidity, resting tremor, postural instability, and difficulty initiating movements. The vast majority
of PD cases are idiopathic, although there are known genetic risk factors, and SUMOylation has been implicated in
PD disease pathology (63).
One of the characteristics of PD is the presence of inclusions
called Lewy bodies in the cytosol of affected neurons. These
inclusions stain strongly for ␣-synuclein, a natively unfolded protein of undetermined function, which is a known
SUMO substrate (54). In particular, SUMOylation of ␣-synuclein has been shown in the brains of transgenic mice
expressing His6-tagged SUMO2 (139), mainly at K96 and
K102. SUMOylation reduces aggregation and toxicity of
␣-synuclein, both in heterologous cells and in the dopaminergic neurons of the substantia nigra in a rat model of PD.
However, another study has suggested that SUMOylation
of ␣-synuclein increases its aggregation (193). Thus the exact function of ␣-synuclein SUMOylation in PD remains
uncertain.
About 1–2% of early-onset PD cases are caused by mutations in the transcriptional regulator DJ-1 (63). DJ-1 is a
multifunctional protein, but one of its major roles is in the
cellular response to oxidative stress (263). SUMOylation of
DJ-1 at K130 is enhanced by UV irradiation and is required
for full activity of the protein (249). Interestingly, the most
severe DJ-1 mutation associated with PD is L166P, which
leads to aberrant DJ-1 SUMOylation and a decrease in protein solubility (249). L166P DJ-1 expressing cells showed a
similar sensitivity to UV-induced apoptosis as DJ-1 knockdown cells, implying that the PD mutation and consequent
aberrant SUMOylation causes a loss of the oxidative stress
response.
The most common autosomal recessive cause of early-onset
PD is mutation of the PARK2 gene, encoding parkin, a
ubiquitin E3 ligase (63). Parkin is present in the majority of
Lewy bodies in both familial and sporadic PD, implicating
this protein in the pathology of the disease. Although it has
not been shown to be SUMOylated, parkin interacts noncovalently with SUMO1, and this interaction increases its
E3 ligase activity, including autoubiquitination (280). In
addition, interaction of SUMO1 with parkin increases its
nuclear transport. Therefore, noncovalent interaction of
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HENLEY ET AL.
SUMO-1 with parkin clearly plays an important regulatory
role, although whether this has any relation to the pathology of PD is unknown. It is also of note that one of the
targets of parkin ubiquitin ligase activity is RanBP2 (281),
which is itself a SUMO E3 ligase, thus demonstrating the
often complex interplay between these two modifications.
D. SUMOylation in Ischemia
preconditioning. Furthermore, transgenic mice overexpressing Ubc9 have elevated global SUMOylation levels,
and also exhibit increased protection against focal cerebral
ischemic damage (150). Therefore, the evidence points
strongly to a cytoprotective role of neuronal protein
SUMOylation during ischemic insult and preconditioning.
However, until very recently no SUMO targets were identified as critical to this protective response.
1. Drp1
Rapid global increases in neuronal protein SUMOylation
occur in response to excessive neuronal excitation, or other
forms of cellular stress such as heat shock, oxidative stress
and ischemia (147, 301), leading to the hypothesis that this
modification is cytoprotective during extreme cell stress.
Indeed, it is well established that protein SUMOylation levels are greatly increased in the brain, liver, and kidney during hibernation torpor in squirrels (149), when blood flow
is severely reduced, and that this SUMOylation represents a
critical cytoprotective mechanism. Another study has
shown that transient focal cerebral ischemia causes a massive increase in SUMO2/3 conjugation (312), with the largest increase seen in the parietal cortex (⬃20-fold). Similar
results were obtained using two different animal models of
stroke (41). In the rat model used in this study, a dramatic
increase in conjugation of SUMO1 and SUMO2/3 was observed in the infarct region at 6 and 24 h. Interestingly, there
was also an increase in SUMO2/3 conjugation in the hippocampus, which was not subject to ischemia, and in the
mouse model this was the only site of increased SUMO2/3
conjugation, whereas increases in SUMO1 conjugation
were found in the infarct region (41). Significantly, kainate
receptor levels were decreased in the infarct region, in line
with the observation that SUMOylation of GluK2 causes
decreased KAR EPSCs (174).
Furthermore, OGD increases SUMO1 and SUMO2/3 conjugation to substrate proteins in cultured hippocampal neurons (40). Surprisingly, levels of SENP1 were also increased
by OGD, suggesting that the neuronal response to OGD
involves a sophisticated interrelationship between SUMOylation and deSUMOylation (40). Broadly in line with these
results, blockade of neuronal SUMO2/3 translation in primary cortical neurons increased neuronal vulnerability following OGD, consistent with SUMO2/3 conjugation being
an endogenous neuroprotective stress response (49).
Taken together, these different studies support the concept
that increases in protein SUMOylation represent a cytoprotective neuronal response to ischemic insult. SUMOylation
also has a role in ischemic preconditioning, an intrinsic
process in which repeated short subtoxic episodes of ischemia protect against a subsequent major ischemic insult,
and overexpression of SUMO1 or SUMO2 in either cortical
neurons or SHSY5Y cells leads to increased survival following OGD (148). Consistent with this, RNAi depletion of
SUMO1 has the opposite effect and attenuated the effect of
1276
The GTPase Drp1 plays a major role in regulating mitochondrial morphology and integrity (220, 326). Under conditions of extreme cell stress, Drp1 is recruited to mitochondria and mediates, among other things, fragmentation and
cytochrome c release, which are potent signals for cell death
(34). Drp1 is SUMOylated by both SUMO1 and SUMO2/3,
and this modification appears to regulate its mitochondrial
partitioning (88, 89, 93, 291). Interestingly, SUMO2/3
modification of Drp1 has been shown to mediate its partitioning away from mitochondria, promoting cell survival
during OGD (89) (FIGURE 8). The deSUMOylating enzyme
SENP3 mediates SUMO2/3 removal from Drp1, and
SENP3 is rapidly degraded during ischemia (89). The OGDinduced loss of SENP3 is triggered by activation of the
endoplasmic reticulum (ER) unfolded protein response
(UPR), a cell stress pathway that is activated by the accumulation of unfolded proteins in the ER (98). The UPR
seeks to restore cellular homeostasis but, when overwhelmed, can initiate cell death. More specifically, OGD
activates the ER-resident UPR kinase PERK, resulting in
cathepsin B-mediated degradation of SENP3 (89). Downregulation of SENP3 during the insult stabilizes global
SUMO2/3 conjugation and prolongs SUMO2/3 modification of Drp1, sequestering it away from mitochondria, and
reducing Drp1-mediated cytochrome c release and consequent cell death (88, 89).
A notable feature of ischemic damage is that very few cells
die during the ischemic episode; rather, they undergo delayed cell death following the reintroduction of oxygen, a
phenomena termed “reperfusion injury.” Correlating with
this, SENP3 levels rapidly recover during reoxygenation,
thereby reducing SUMO2/3 modification of Drp1, and promoting cytochrome c release and cell death. A key observation is that knockdown of SENP3 blocks the cell death
associated with reoxygenation, suggesting that SENP3, acting as a novel arm of the UPR, mediates the delayed cell
death observed upon ischemic insult (88, 89).
This represents a novel adaptive pathway to extreme cell
stress in which dynamic changes in SENP3 stability and
regulation of Drp1 SUMOylation by SUMO2/3 are crucial
determinants of cell fate (89). Furthermore, these data highlight SENP3 as a potentially useful therapeutic target to
prevent reperfusion injury. It is highly likely that other ischemia-induced SUMO substrates will be identified in the
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NEURONAL SUMOYLATION
Acetylation
SU
SAE2
deSUMOylation
SIRT1
SAE2
SAE1
Ubc9
Drp1
S2
S2
?
S2
?
?
?
MO
SU
?
Apoptosis
MO
SAE1
O
Ubc9 SUM
Ac
Drp1
Drp1
?
S2
S2
S2 SUMO2/3
S2
Pro-survival
Cytochrome c
SENP3
CatB
PERK
ER
CatB
P
Nucleus
PERK
?
CatB
Lysosome
CatB
HYPOXIA
PERK
FIGURE 8. SUMOylation and ischemia. Hypoxia induces the accumulation of unfolded proteins in the ER,
resulting in ER stress, which is sensed by the ER-resident kinase PERK. Activation of PERK, through an
unknown mechanism, results in lysosomal rupture and release of the protease cathepsin B (CatB) into the
cytosol, which mediates degradation of the SUMO2/3-specific SUMO protease SENP3. Loss of SENP3 leads
to increased SUMOylation of multiple SUMO2/3 substrates, including the mitochondrial GTPase Drp1. Drp1
is a crucial mediator of mitochondrial fission and pro-apoptotic cytochrome c release. Enhanced SUMOylation
of Drp1 during ischemia favors its distribution to the cytosol, where it is unable to stimulate cytochrome c
release, promoting cell survival. In addition to stimulating degradation of SENP3, hypoxia also promotes
SUMOylation by inducing SIRT1-mediated deacetylation of Ubc9, which enhances its activity against NDSMcontaining substrates, and deSUMOylation of the E1 subunit SAE2, which promotes its activity.
coming years, and will ultimately lead to novel therapies for
ischemic insults such as stroke.
XX. SUMMARY
The field of posttranslational modification is undergoing a
renaissance with increased understanding of the multifaceted roles of ubiquitination and the emergence of multiple
UBL proteins. SUMO is currently the best characterized of
the UBL family, but there many other, as yet much less
studied, family members, and more are continually being
discovered (282). Overall, the effects of SUMOylation in
different neurological and neurodegenerative diseases are
complex, and it is clear a great deal of further work is
required. As highlighted throughout this review, despite the
many advances achieved, many questions remain. For ex-
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HENLEY ET AL.
ample, we still do not yet know in any detail which steps in
synaptic transmission, plasticity, and neuroprotection pathways are regulated by SUMOylation. Nor do we know how
the process of SUMOylation itself is regulated to control
synaptic function and cell survival in neurons. However, we
expect that progress in this field will continue to advance
rapidly and further define the molecular triggers, targets,
and functional outcomes of protein SUMOylation in neurons. On a larger scale, while much evidence supports a role
for SUMOylation in neuronal and synaptic function, no
studies have yet investigated the impact of perturbed
SUMOylation on learning and memory in vivo, or examined how SUMOylation of specific substrates regulates
brain function. Together with the progress already made,
these studies will reveal exciting and potentially clinically
important data on the targets, mechanisms, and consequences of SUMOylation, and we anticipate that components and substrates of the SUMO pathway will eventually
provide attractive and tractable therapeutic targets for a
wide range of neurological and neurodegenerative diseases.
ACKNOWLEDGMENTS
Address for reprint requests and other correspondence:
J. M. Henley, School of Biochemistry, Medical Sciences
Building, Univ. of Bristol, Bristol BS8 1TD, UK (e-mail:
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GRANTS
We are grateful to the European Research Council, the
Medical Research Council, and the Biotechnology and Biological Sciences Research Council for financial support.
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DISCLOSURES
No conflicts of interest, financial or otherwise, are declared
by the authors.
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