Cofilin phosphatases and regulation of actin dynamics
Timothy Y Huang, Céline DerMardirossian and Gary M Bokoch1
Cofilin is a ubiquitous actin-binding factor required for the
reorganization of actin filaments in eukaryotes. The
dephosphorylation of cofilin enables its actin severing and
depolymerizing activity and drives directional cell motility, thus
providing a simple phosphoregulatory mechanism for actin
reorganization. To date, two cofilin-specific phosphatases
have been identified: Slingshot and Chronophin. These cofilin
phosphatases are unrelated in sequence and regulatory
properties, each potentially providing a unique mechanism for
cofilin activation under varying biological circumstances.
Addresses
The Scripps Research Institute, Departments of Immunology and
Cell Biology, 10550 N. Torrey Pines Road, La Jolla, California, 92037,
USA
Corresponding author: Bokoch, Gary M (bokoch@scripps.edu)
1
T Huang and C DerMardirossian contributed equally to this review.
Current Opinion in Cell Biology 2006, 18:26–31
This review comes from a themed issue on
Cell structure and dynamics
Edited by J Victor Small and Michael Glotzer
Available online 7th December 2005
0955-0674/$ – see front matter
# 2005 Elsevier Ltd. All rights reserved.
DOI 10.1016/j.ceb.2005.11.005
Introduction
The reorganization of actin at the leading edge of eukaryotic cells is a fundamental aspect of cell motility, and
requires the coordinated function of both actin-polymerizing and actin-depolymerizing/severing factors. To drive
the cell front forward, branched actin filaments are generated at the leading edge through the action of the Arp2/
3 complex and/or filamin A [1,2]. Actin depolymerizing
components, including proteins of the cofilin/ADF (actin
depolymerizing factor) family, disassemble F-actin from
the rear of the actin network to recycle actin monomers to
the leading edge for further rounds of polymerization.
There is also growing evidence that cofilin-dependent Factin severing activity is important for actin polymerization, as cofilin-severed F-actin fragments act as preferred
substrates from which Arp2/3 builds actin networks [3].
Precise spatial regulation of cofilin-dependent actindepolymerizing/severing activity appears to be crucial
to cell motility, since site-specific activation of caged
cofilin can determine the direction of cell movement
[4].
Current Opinion in Cell Biology 2006, 18:26–31
We discuss here current knowledge of the regulation and
biological roles of the cofilin phosphatases in actin
dynamics.
Single-site phosphoregulation of cofilin
Cofilin and related ADF family proteins (henceforth
generically referred to as cofilin) are phosphorylated at
a conserved N-terminal serine, Ser3 [5]. Early studies
revealed that in its phosphorylated form cofilin is unable
to bind actin, and that dephosphorylation of this site
reactivated the actin-depolymerizing activity of cofilin
[6]. From these studies, it became clear that cofilin
phosphorylation/dephosphorylation at Ser3 acts as a simple switch for actin assembly and disassembly/severing
(Figure 1).
Two kinase families have been shown to phosphorylate
and deactivate cofilin: the LIM (Lin-11/Isl-1/Mec-3)
kinases and the TES (testicular protein) kinases. Interestingly, no other substrates have been identified for
these kinases. The LIM kinases, LIMK1 and LIMK2,
are ubiquitous in their tissue distribution [7,8], whereas
TESK1 is expressed most abundantly in testicular tissue
[9,10]. LIM kinases are activated by phosphorylation of
Thr508/505 (LIMK1/2) within the kinase activation loop
through divergent Rho GTPase pathways: Rac/Cdc42
acting through p21-activated kinase (PAK)1 and PAK4
[11,12], Cdc42 acting through MRCK (myotonic-dystrophy-related cdc42-binding kinase [13], and RhoA through
ROCK (Rho-associated coiled-coil-containing kinase)
[14]. By contrast, TESK activation is dependent on
integrin engagement upon cell attachment, and occurs
independently of PAK1/ROCK activation [15]). Functionally, the LIM and TES kinases promote F-actin
stability through cofilin dephosphorylation and deactivation, as their overexpression in cell lines promotes F-actin
accumulation [15].
Clearly, LIMK regulation is a key factor in the cofilin
phospho-regulatory switch mechanism, but it was not
certain whether lack of LIM kinase activity is sufficient
to cause the decreases in phosphorylation that result in
cofilin activation, or whether phosphatase activation is
also necessary. In some cell types, cell stimulation
induced changes in net phosphocofilin levels, while in
other cells (e.g. 3T3 and A431) a significant increase in
phosphocofilin turnover was observed with no significant
change in the total phosphocofilin pool [16]. The latter
observations suggested that the activity of both cofilin
kinases and cofilin phosphatases was being regulated by
common upstream stimulatory signals. As LIMK and
TESK evolved the specific task of phosphorylating cofiwww.sciencedirect.com
Cofilin phosphatases and regulation of actin dynamics Huang, DerMardirossian and Bokoch 27
Figure 1
Phosphoregulatory cycle of cofilin. Cofilin phosphorylation at serine 3 by LIMK and TESK inhibits cofilin binding to F-actin, leading to F-actin
stabilization. Cofilin dephosphorylation by SSH and CIN stimulates F-actin depolymerization and severing. The severing function can promote actin
nucleation (via Arp 2/3), initiating additional actin assembly and directional motility.
lin, the possibility was raised that cofilin-specific phosphatases might also exist.
motility, neurodegenerative stress responses, and even
apoptosis.
Although earlier studies implicated the involvement of
phosphatases with broad substrate specificities, such as
PP1, PP2A and PP2B, inhibitors active against these
phosphatases largely fail to block cofilin dephosphorylation [5]. Such observations suggested that these general
phosphatases may not account for the majority of cofilin
phosphatase activity observed in response to cellular
stimuli. Recent observations also indicate that cofilin
dephosphorylation can be spatially restricted within specific cellular subcompartments [17,18] and that directed
cofilin phosphatase activity within these locations may be
sufficient to drive processes such as cell protrusion and
Thus far, two types of cofilin-selective phosphatases have
been identified: the Slingshot family of phosphatases, and
Chronophin.
www.sciencedirect.com
Slingshot, a conserved family of cofilin
phosphatases
Slingshot was initially identified as a dedicated cofilin
phosphatase through genetic studies in Drosophila, where
its dysfunction was noted to cause disorganized epidermal
cell morphogenesis, including splintered hair bristles
(hence the name Slingshot) [19]. A single gene (D-ssh)
in Drosophila codes for the 125-kDa Slingshot (dSSH)
Current Opinion in Cell Biology 2006, 18:26–31
28 Cell structure and dynamics
Table 1
Comparison of SSH and CIN phosphatases.
Slingshot
Chronophin
Isoforms
hSSH1 (115.5 kDa)
hSSH2 (158.2 kDa)
hSSH3 (73.0 kDa)
31.7 kDa
Tissue distribution
(based on mouse
tissues)
Widespread; braina,
thymusa, hearta, livera,
spleena, kidneya, small
intestine, skeletal muscle
and testis examined
Widespread; braina,
thymusa, hearta, livera,
spleen, kidney, small
intestine, skeletal muscle
and testisa examined
Widespread; braina,
thymus, heart, liver,
spleen, kidneya, small
intestinea, skeletal muscle
and testisa examined
Widespread; abundant in brain, heart,
skeletal muscle, liver and kidney
Regulatory motifs
F-actin binding region (except for SSH3)
14-3-3 binding motifs
p85 SH3 binding motif,
Akt, PKC consensus phosphorylation motifs
p85 SH3 binding motif,
PLCg binding motif
Localization
F-actin-rich lamellipodia
Membrane protrusions
Mitosisb: metaphase — cortical actin; anaphase/telophase — cleavage furrow;
late telophase/cytokinesis — cleavage furrow and midbody
F-actin-rich lamellipodia
Membrane protrusions
Mitosisb: prophase — cytoplasm;
metaphase — cytoplasm;
anaphase/telophase — cell poles;
late telophase/cytokinesis — cleavage
furrow, acto-myosin contractile ring
and midbody
Regulatory proteins 14-3-3 proteins
Calcium-regulated protein phosphatase (Calcineurin)
PtdIns 3-kinase
PAK4
PtdIns 3-kinase?
PLCg?
Substrates
Cofilin, ADF, LIMK
Cofilin, ADF, Pyridoxal 50 -phosphate c
Implicated cell
functions
Mitosis, growth cone motility/morphology, neurite extension,
regulation of membrane protrusion
Mitosis, membrane protrusion?,
vitamin B6 metabolism c
a
Most abundant. bAs shown in HeLa cells. cNon-protein substrate.
protein. In both human and mouse, the Slingshot phosphatases are represented by three genes (SSH-1, -2, and 3), each with long and short variants with distinct tissue
expression patterns (Table 1). SSH seems to be widely
expressed in various organisms, but is notably absent in
Saccharomyces cerevisiae, Caenorhabditis elegans and Arabidopsis thaliana. In mammalian cells SSH-1L, along with
SSH-2L and SSH-3L, dephosphorylates both phosphoADF and phospho-cofilin at the critical Ser3 residue,
thereby suppressing actin filament assembly induced
by LIMK1 or TESK1 [19]. Notably, SSH-3L was less
effective in dephosphorylating these substrates in comparison with the two other isoforms. SSH phosphatases
contain a protein phosphatase domain (PTP) with a
canonical catalytic HCxxGxxR sequence found within
other dual-specificity and protein tyrosine phosphatases.
Although SSH-1L and SSH-2L both co-localize with
actin filaments in mammalian cells through a C-terminal
F-actin binding region, the three SSH family phosphatases differ in their subcellular localization, F-actin binding activity, specific activity and tissue expression
patterns (Table 1) [20].
process. Indeed, SSH-1L was found to localize at the
cleavage furrow and midbody during cytokinesis [21].
Localization to these sites correlated with elevated
SSH-1L activity during telophase and cytokinesis, and
coincided with the temporal pattern of cofilin dephosphorylation during these stages [21]. Overexpression
of phosphatase-inactive SSH1 led to aberrant accumulation of F-actin and phosphocofilin during late cytokinesis,
accompanied by frequent regression of the cleavage furrow and formation of multinucleated cells, further supporting a role for SSH in cell division.
SSH has also been implicated in the control of the motility
and morphology of the growth cone and in neurite extension through cofilin regulation. SSH induced highly motile
growth cones and enhanced neurite extension rates, while
antagonizing the repressive effects on growth cone behavior of LIMK1 [22]. SSH may regulate actin dynamics
during membrane protrusion, as SSH-1L is activated in
insulin-stimulated MCF7 cells and accumulates in protrusions where active cofilin, but not inactive phosphocofilin,
is concentrated [23,24]. These studies reflect multiple
roles for SSH in various cell types.
Biological activities
Localized cofilin function was previously shown to be of
importance during cell division, which suggested that
cofilin regulators may also be involved in the cell division
Current Opinion in Cell Biology 2006, 18:26–31
Localization
As site-specific cofilin activation was previously shown to
be critical for the regulation of various biological processes,
www.sciencedirect.com
Cofilin phosphatases and regulation of actin dynamics Huang, DerMardirossian and Bokoch 29
the localization of SSH family phosphatases to specific
subcellular sites is also likely to be required for their role in
regulating actin dynamics. During cell motility, cofilin is an
important catalyst of actin filament disassembly required to
maintain lamellipodial protrusion. To facilitate actin reorganization at the leading edge, it has been reported that
SSH becomes activated through its release from 14-3-3 in
the cytoplasm, consequently translocating to the F-actinrich lamellipodia during cellular stimulation with neuregulin-1 [24] or insulin [23]. The subcellular distribution of
SSH-1L varies in mitotic HeLa cells, ranging from a uniform colocalization with cortical F-actin throughout prometaphase and metaphase to a distribution coincident with
F-actin staining at the cleavage furrow during telophase
[21].
The extent to which SSH localization is determined by its
F-actin binding activity or through its association with
other cellular components is still not entirely clear. For
example, SSH localization to insulin-induced membrane
protrusions was found to be PtdIns-3-kinase-dependent
[23]. Whether this was due to a direct regulatory effect of
PtdIns-3-kinase activity on SSH localization as opposed
to an effect on SSH binding to F-actin remains unknown.
Moreover, recent evidence indicates that 14-3-3 proteins
associate with cofilin and SSH-1L in a phosphorylationdependent manner, thereby preventing cofilin dephosphorylation and translocation of SSH to F-actin-rich
cortical regions, respectively [24,25,26]. 14-3-3 proteins
may therefore play a role in the formation of localized
regulatory complexes, which include cofilin, LIMK and
F-actin as well as SSH [25,26,27].
Regulation
Multiple signaling pathways, including those involving
Ca2+, cAMP and PtdIns 3-kinase, have previously been
proposed to modulate stimulus-induced cofilin dephosphorylation in many different cell types. Accordingly,
several of these cofilin-dephosphorylating pathways have
now been linked to SSH. Recent studies have shown that
the Ca2+ ionophore A23187 and Ca2+-mobilizing agonists,
such as ATP and histamine, induced SSH-1L activation,
correlating with cofilin dephosphorylation in vivo [28].
Calcineurin, a calcium-regulated protein phosphatase,
was observed to dephosphorylate and activate SSH-1L,
supporting the idea that SSH-1L is negatively regulated
by phosphorylation. Indeed, SSH activity was inhibited
by PAK4-mediated phosphorylation, and dephosphorylation of SSH by l-phosphatase was shown to boost its
phosphatase activity [26]. In most systems, there is also
evidence for calcium-independent regulatory pathways.
The PtdIns 3-kinase inhibitor wortmannin has been
shown to antagonize cofilin dephosphorylation induced
by several receptor stimuli [5]. Nishita et al. (2004)
reported that insulin-dependent actin reorganization
occurs through PtdIns 3-kinase and SSH, since insulinstimulated MCF7 cells exhibit SSH activation and cofilin
www.sciencedirect.com
dephosphorylation that is abrogated by PtdIns 3-kinase
inhibition [23]. Additionally, SSH phosphatase activity
appears to be directly stimulated up to 10-fold by F-actin
binding, indicating that existing actin filaments may
participate in promoting additional actin reorganization
through SSH/cofilin activation [24,26].
Interestingly, recent evidence indicates that SSH phosphatase activity may not be limited to cofilin, as SSH
appears to dephosphorylate and inactivate LIMK1 [26].
SSH-1L and LIMK1 form a complex, resulting in dephosphorylation of both autophosphorylated LIMK1 sites
and the critical Thr508 residue in the activation loop of
LIMK1, thereby inactivating its ability to phosphorylate
cofilin. This finding probably explains the robust cofilin
dephosphorylation observed in vivo upon overexpression
of SSH-1L. These observations complicate our understanding of cofilin regulation, as it is difficult to demonstrate whether changes in phosphocofilin levels are due to
direct SSH-mediated cofilin phosphatase activity, SSHmediated LIMK inactivation, or a combination of both.
Furthermore, it is unclear whether certain signals
upstream of SSH can affect its specificity towards LIMK
or cofilin, and whether different signals can affect the
association of SSH with LIMK. These issues will have to
be addressed in any future studies involving the biological
effects of SSH-1L.
Chronophin, a unique HAD-family cofilin
phosphatase
Chronophin (CIN) has been identified as a second potential cofilin phosphatase through a biochemical screen
based on in vitro dephosphorylation of cofilin [29].
CIN belongs to the haloacid dehalogenase (HAD) family
of phosphatases, whose involvement in mammalian signal
transduction pathways is poorly characterized. CIN contains a highly conserved catalytic domain and three conserved sequence motifs characteristic of the HAD
hydrolases. HAD phosphatases use an unconventional
catalytic mechanism whereby a transition state phospho-aspartate intermediate forms as a result of nucleophilic attack on the substrate phosphate group [30].
Mutation of the nucleophilic aspartate (Asp25 in CIN)
markedly compromises CIN activity in vitro. Interestingly, CIN has also been shown to exhibit pyridoxal
(vitamin B6) phosphatase activity [31,32].
In addition to its biological activity (described below),
CIN exhibits several unique characteristics that suggest it
may represent a novel means of regulating cofilin-dependent actin dynamics. First, HAD family members bearing
similarity to CIN can be found in many organisms,
including several that lack SSH orthologs (e.g. yeast
and bacteria), and is widely distributed in human tissues,
being particularly abundant in brain. Second, CIN cofilin
phosphatase activity is insensitive to classical thiol-based
serine/threonine phosphatase inhibitors, as reported for
Current Opinion in Cell Biology 2006, 18:26–31
30 Cell structure and dynamics
physiological cofilin phosphatase activity [5,33]. Third,
CIN exhibits several predicted interaction motifs potentially linking CIN to regulation by both PtdIns 3-kinase
and PLCg, both of which have been suggested to be
involved in signaling to cofilin dephosphorylation in vivo
[5].
Biological activities
CIN dephosphorylates a limited number of protein substrates in vivo and in vitro (see supplementary data to
[29]), and is active against both cofilin and ADF in vivo
[29]. As opposed to SSH, CIN fails to exhibit significant
activity towards LIMK, perhaps accounting for its less
dramatic effect on phosphocofilin levels when overexpressed in cells. However, CIN overexpression does
decrease basal phospho-cofilin levels, and effectively
antagonizes the enhanced cofilin phosphorylation stimulated by LIMK. By contrast, catalytically inactive CIN or
CIN depletion by siRNA induces enhanced cellular
phospho-cofilin pools. High CIN activity is consistently
associated with decreased cellular F-actin content, while
decreased CIN activity promotes the accumulation of
polymerized actin. CIN localizes to actin-rich ruffles
and membrane protrusions, and our preliminary studies
indicate that CIN function is directly linked to actin
dynamics at the leading edge of motile cells (C DerMardirossian, V Delorme and GM Bokoch, unpublished).
Manipulation of CIN activity produces pronounced
effects on cell division. Specifically, the overexpression
of dominant negative forms of CIN (or downregulation by
CIN siRNA treatment) causes aberrant actin assembly,
frequent cleavage furrow regression, and a significant
population of multinucleated cells, correlating with
changes in phosphocofilin levels. Similar defects have
been reported when cofilin (Twinstar in Drosophila) function is disrupted genetically in the fruit fly, and upon the
injection of cofilin antibodies in dividing Xenopus blastomeres, suggesting that CIN and cofilin function are
linked during cell division [34,35]. The colocalization of
CIN with cofilin to the ingressing cleavage furrow and to
the actomyosin contractile ring at later mitotic stages
supports the observation that CIN function is critical
during cell division. Interestingly, CIN appeared to localize to membranous regions at the cell poles during
telophase, and seemed to have a more cytoplasmic distribution with limited localization to the cleavage furrow
at earlier stages of mitosis. This differs from SSH, which
strongly localizes to the ingressing furrow at early telophase, suggesting that these two cofilin phosphatases
may play distinct regulatory roles during cell division
(Table 1).
dantly or whether they activate cofilin in specific functional contexts. Superficially, both phosphatases associate
with actin (CIN through its colocalization with cellular Factin, and SSH through direct binding), and the overexpression or dysfunction of both phosphatases leads to
qualitatively similar cellular phenotypes. However, the
two phosphatases are not functionally redundant in the
context of mitosis, as siRNA-mediated depletion of either
protein in the same cell type caused distinct temporal
changes in phospho-cofilin levels in synchronized mitotic
cells, and depletion of both proteins caused enhanced
increases in phospho-cofilin levels [29]. Viewed from a
reductionist perspective, the lack of any sequence similarity between SSH and CIN suggests that the phosphatases may fundamentally differ in their biochemical
nature (for example, how they are activated and regulated), and in their regulatory function. Furthermore,
associated signaling proteins have been identified that
may form localized regulatory complexes and hence
modify the functional properties of these cofilin phosphatases. A challenge for future studies will be to investigate the activities of cofilin phosphatases in the context
of the normal biological, regulatory, spatial and temporal
constraints that control their function in actin dynamics.
Acknowledgement
The authors acknowledge support from the National Institutes of Health,
Grant GM44428 (to GMB).
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