Journal of Neurochemistry, 2007, 100, 1169–1176
doi:10.1111/j.1471-4159.2006.04292.x
NGF augments the autophosphorylation of Ret via inhibition of
ubiquitin-dependent degradation
Brian A. Pierchala,*, Cynthia C. Tsui,* Jeffrey Milbrandtà and Eugene M. Johnson ,§
*Department of Biological Sciences, University at Buffalo, SUNY Buffalo, New York, USA
Department of Molecular Biology and Pharmacology, àDepartment of Immunology and Pathology, and
§Department of Neurology, Washington University School of Medicine St Louis, Missouri, USA
Abstract
Nerve growth factor (NGF) is required for the trophic maintenance of postnatal sympathetic neurons. A significant
portion of the growth-promoting activity of NGF is from NGFdependent phosphorylation of the heterologous receptor
tyrosine kinase, Ret. We found that NGF applied selectively to
distal axons of sympathetic neurons maintained in compartmentalized cultures activated Ret located in these distal
axons. Inhibition of either proteasomal or lysosomal degradation pathways mimicked the effect of NGF on Ret activation.
Likewise, NGF inhibited the degradation of Ret induced by
glial cell line-derived neurotrophic factor-dependent activation,
a process that requires ubiquitination and proteasomal deg-
radation. NGF induced the accumulation of autophosphorylated Ret predominantly in the plasma membrane, in contrast
to GDNF, which promoted the internalization of activated Ret.
An accretion of monoubiquitinated, but not polyubiquitinated,
Ret occurred in NGF-treated neurons, in contrast to glial cell
line-derived neurotrophic factor that promoted the robust
polyubiquitination of Ret. Thus, NGF stimulates Ret activity in
mature sympathetic neurons by inhibiting the ongoing ubiquitin-mediated degradation of Ret before its internalization and
polyubiquitination.
Keywords: degradation, glial cell line-derived neurotrophic
factor, nerve growth factor, Ret, sympathetic neuron, ubiquitin.
J. Neurochem. (2007) 100, 1169–1176.
Neurotrophic factors control many aspects of neural development and their contribution to the establishment of the
sympathetic nervous system is well documented. Nerve
growth factor (NGF) controls the target-dependent survival
of late prenatal and early postnatal sympathetic neurons
(Levi-Montalcini and Booker 1960; Levi-Montalcini 1987;
Barde 1989), and NGF is required for the trophic maintenance of adult neurons (Angeletti et al. 1971; Goedert et al.
1978; Gorin and Johnson 1980; Ruit et al. 1990). Survival
and growth promotion by neurotrophins are conveyed via
activation of receptor tyrosine kinases (RTKs) known as
Trks, and the RTK mediating NGF actions is TrkA (Bothwell
1995; Segal et al. 1996; Kaplan and Miller 2000).
Another family of neurotrophic factors critical for neural
development, the glial cell line-derived neurotrophic factor
(GDNF) family ligands (GFLs), function via activation of the
RTK Ret, but unlike the neurotrophins, GFLs do not bind
directly to Ret to activate it. The high-affinity binding
components of GFL receptors are GPI-anchored coreceptors
termed GFRas, which as a GFL-GFRa complex, bind to and
activate Ret (Baloh et al. 2000; Airaksinen and Saarma
2002). Two isoforms of Ret are generated by alternative
splicing that have different C-termini encoding either 9
amino acids (Ret9) or 51 different amino acids (Ret51)
(Tahira et al. 1990). This seemingly modest change leads to
striking differences both in the signaling capacities of Ret9
and Ret51 and in their functional characteristics (de Graaff
et al. 2001; Tsui-Pierchala et al. 2002b). It has recently been
appreciated that 20–30% of the trophic activity of NGF on
Received July 1, 2006; revised manuscript received August 16, 2006;
accepted August 16, 2006.
Address correspondence and reprint requests to Brian A. Pierchala
Department of Biological Sciences, University at Buffalo, SUNY,
Buffalo, NY 14260, USA. E-mail: bap7@buffalo.edu
Abbreviations used: GDNF, glial cell line-derived neurotrophic factor;
GPCR, G-protein coupled receptor; IP, immunoprecipitation; NGF, nerve
growth factor; RTK, receptor tyrosine kinases; TGN, trans-golgi network.
Ó 2007 The Authors
Journal Compilation Ó 2007 International Society for Neurochemistry, J. Neurochem. (2007) 100, 1169–1176
1169
1170 B. A. Pierchala et al.
mature sympathetic neurons is conveyed by Ret51 (TsuiPierchala et al. 2002a). NGF does not enhance Ret phosphorylation by altering the availability of GFLs, but instead
NGF regulates Ret through a GFL-independent mechanism
that has slow kinetics, requiring hours to induce Ret
phosphorylation maximally (Tsui-Pierchala et al. 2002a).
Several populations of neurons respond synergistically to
multiple neurotrophic factors, or require several growth
factors simultaneously during development. Cells must
respond appropriately to the presence of multiple extracellular signals simultaneously, and mechanisms of cross-talk
between signaling cascades remain largely unknown. For
example, the pathway responsible for the NGF-dependent,
inter-RTK activation of Ret has remained uncharacterized.
Here we identify the mechanism by which NGF modulates
Ret activation, namely through the inhibition of ubiquitindependent degradation pathways that clear activated Ret
from sympathetic neurons, thus increasing the basal level of
Ret phosphorylation and enhancing growth.
Experimental procedures
Sympathetic neuronal cultures
Primary sympathetic neurons from the superior cervical ganglion
were generated and maintained in vitro in the presence of NGF as
described previously (Tsui-Pierchala et al. 2002a). These neurons
were maintained in vitro for 17–19 days. Before their treatment with
either NGF or GDNF, NGF was removed from the dissociated
neurons, they were washed once, and then maintained without NGF
for 48 h before stimulation. Compartmentalized cultures of mature
sympathetic neurons were produced as described previously (TsuiPierchala and Ginty 1999). In compartmentalized cultures, neurons
were maintained for 21 days before stimulation; during the 10–
12 days before the experimental treatments these neurons were
maintained with NGF only on the distal axons and terminals.
Immunoprecipitations
Ret51 and TrkA immunoprecipitations were performed identically
to our previous study (Tsui-Pierchala et al. 2002a). Briefly, after the
treatments described in the Figure legends, the neurons were washed
twice with ice-cold phosphate-buffered saline (PBS) and then
detergent extracted using an immunoprecipitation (IP) buffer (Tris,
pH 7.4, 10% glycerol, 1% Nonidet P-40, protease inhibitors, and
500 lmol/L sodium vanadate). To these cleared extracts anti-Ret51
antibodies or anti-Trk antibodies were added (10 lL; Santa Cruz,
Inc., Santa Cruz, CA, USA) along with protein A and protein G
(Invitrogen Corp., Carlsbad, CA, USA). After 3 h of gentle agitation
at 4°C, the immunoprecipitates were then washed three times with
IP buffer and the complexes were prepared for sodium dodecyl
sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) by adding
2X sample buffer and heating this for 5 min in a boiling water bath.
For the immunoprecipitations using mono-Ub and poly-Ub antibodies (Chemicon, Temecula, CA, USA; and Stressgen Bioreagents,
Victoria, BC, Canada, respectively), the same IP procedure was
followed except that only 5 lL of each antibody was used.
Biochemical experiments with compartmentalized cultures were as
follows: the distal axon and cell body compartments were lysed
separately by gently applying 100 and 60 lL of IP buffer,
respectively, without agitation at 4°C. The compartments were
gently scraped and the extracts were collected with microcapillary
pipette tips. These extracts were then cleared by centrifugation and
then subjected to Ret followed by Trk immunoprecipitation as
described above. Immunoprecipitations from compartmentalized
cultures required that the extracts from 4 to 5 Campenot chambers
were pooled for each experimental condition.
Cell surface biotinylation
Cell surface proteins were distinguished from internalized proteins
by labeling them with biotin. Mature sympathetic neurons were
treated as described in the Figure legends and then cooled to 4°C to
stop membrane traffic. The neurons were washed with ice-cold PBS
and then surface proteins were biotinylated for 20 min at 4°C using
2 mmol/L sulfo-NHS-LC-biotin (in PBS; Pierce, Inc., Rockville, IL,
USA). The neurons were subjected to a second 20-min incubation
with NHS-biotin, washed once with PBS alone, and next incubated
with lysine (10 mmol/L) for 15 min on ice. The neurons were
washed again with PBS and then detergent extracted with IP buffer.
Biotinylated proteins were precipitated with immobilized Neutravidin (Pierce, Inc.) in an identical fashion as the immunoprecipitations, and after this Neutravidin precipitation, internalized Ret was
isolated by performing a standard Ret immunoprecipitation from the
supernatants of the Neutravidin precipitation. These precipitates
were then prepared for SDS-PAGE as described above.
Immunoblotting
Immunoblotting experiments were conducted as reported previously
(Tsui-Pierchala et al. 2002a). The antibodies for immunoblotting
were as follows: anti-phosphotyrosine was used at a 1:3000 dilution
of the stock (4G10; Upstate Biotechnology, Inc., Lake Placid, NY,
USA), anti-Ret51 and anti-PYRet antibodies are rabbit polyclonal
antibodies described previously (Tsui-Pierchala et al. 2002b), and
anti-actin was used at a 1:1000 dilution (Santa Cruz). In some cases,
the immunoblots were quantified using the UN-SCAN-IT software
(Silk Scientific, Orem, UT, USA). All immunoblots that were
quantified were within the linear range of the film and within the
parameters of the software. Graphs are shown as the mean ± SD.
Values reported in the Results for Fig. 2a are the mean ± range, and
the mean ± SD for Figs 2b and 4b.
Results
Ret levels and Ret autophosphorylation are
concomitantly regulated by nerve growth factor
The discovery that NGF induces Ret activation independently of GFLs as a means of augmenting cellular growth and
metabolism led to an investigation of the mechanism
responsible for this cross-talk between TrkA and Ret. We
speculated that the mechanism responsible could be (1) the
inhibition of phosphatases that down-regulate Ret phosphorylation, (2) the activation of kinases that phosphorylate
Ret, or (3) the up-regulation of an adaptor molecule that
dimerizes and, thus, activates Ret (Tsui-Pierchala et al.
2002a). Biochemical approaches in mature sympathetic
Ó 2007 The Authors
Journal Compilation Ó 2007 International Society for Neurochemistry, J. Neurochem. (2007) 100, 1169–1176
NGF inhibits ubiquitin-dependent Ret degradation 1171
neurons were undertaken to determine whether any of these
three hypotheses were correct. The use of multiple techniques and experimental conditions in mature sympathetic
neurons has not yielded data to corroborate any of these
possibilities (data not shown). Thus, no available evidence
supports the hypotheses that NGF modulates Ret activity by
regulating phosphatases, kinases, or adaptor molecules.
The initial examination of NGF-dependent Ret phosphorylation did not resolve which tyrosine residues in Ret
become phosphorylated upon NGF treatment (Tsui-Pierchala
et al. 2002a). This question was addressed by using four
antibodies that each recognizes a specific tyrosine in Ret
when autophosphorylated (Tsui-Pierchala et al. 2002b).
After NGF treatment for 48 h, Y905, Y1015, Y1062 and
Y1096 were all more highly phosphorylated in Ret when
compared with treatment with medium alone (Fig. 1a). The
protein level of Ret51, the predominant Ret isoform regulated by NGF, increased with NGF treatment as well,
compared with other proteins expressed in mature sympathetic neurons such as actin (Fig. 1a). When the increase in
phosphorylation of these four tyrosines was compared with
the increase in Ret51, this quantification revealed that only
Y905 and Y1062 phosphorylation were greater than the
increase in Ret protein (Fig. 1b). The differences we
observed in the relative level of phosphorylation of tyrosines
in Ret between neurons treated with GFLs and NGF suggest
that Ret activated by NGF will have different signaling
capacities than Ret activated by GFLs. Because Y1015 is a
docking site for phospholipase C-c (Borrello et al. 1996), it
is likely that phospholipase C-c, and downstream PKC
activity, will not be as prominent upon NGF-activation of Ret
when compared with GFL-activation of Ret. Y905 is an
autocatalytic tyrosine in Ret and, therefore, NGF treatment
likely increases Ret kinase activity and does not simply
augment Ret phosphorylation. Therefore, NGF coordinately
regulated Ret levels and GFL-independent Ret autophosphorylation, with the phosphorylation of tyrosines 905 and
1062 being the most pronounced.
Nerve growth factor regulates Ret phosphorylation in
distal axons independently of local protein synthesis
To better understand the mechanism responsible for NGF
regulation of Ret, compartmentalized cultures of mature
sympathetic neurons were established. This culture system
allows the direct examination of signaling events occurring
in distal axons and terminals separately from events occurring at the cell bodies and proximal axons (Campenot 1977,
1979). To determine whether NGF applied only to the distal
axons of sympathetic neurons induces Ret autophosphorylation in the distal axons or in the cell bodies, sympathetic
neurons were maintained with NGF applied only to the distal
axons for 48 h, and the phosphorylation status of Ret was
monitored in both the distal axons and cell bodies. Surprisingly, NGF locally regulated Ret levels and Ret auto-
(a)
(b)
Fig. 1 Ret phosphorylation and Ret levels are concomitantly regulated by nerve growth factor (NGF). (a) Mature sympathetic neurons
were treated with NGF (50 ng/mL) or medium alone for 48 h and then
detergent extracted. The extracts were immunoblotted with antibodies
specific for Ret when phosphorylated on tyrosine 905, 1015, 1062 or
1096 (labeled on right). The amount of Ret51 was examined with
immunoblotting (fifth panel) and equal loading of each condition was
confirmed with actin immunoblotting (bottom panel). (b) Three independent experiments as shown in (a) were quantified and graphed as
the amount of PY-Ret divided by the amount of Ret51 in each condition.
phosphorylation in distal axons (Fig. 2a, Ret increased 3.5fold ± 1.0 and P-Ret increased 5.5-fold ± 0.7). NGF acting
only on the terminals also increased Ret autophosphorylation
in the cell bodies (Fig. 2a, Ret increased 2.8-fold ± 0.8 and
P-Ret increased 4.2-fold ± 0.4), indicating that NGF was
able to act retrogradely. Consistent with previous reports
(Bhattacharyya et al. 1997; Riccio et al. 1997; Senger and
Campenot 1997), treatment of distal axons with NGF
induced TrkA activation in the distal axons as well as in
the cell bodies (Fig. 2a). Unlike its effect on Ret, NGF
treatment of the distal axons did not alter the levels of TrkA
in the distal axons (Fig. 2a). Because activated RTKs do not
move anterogradely in sympathetic neurons (Ye et al. 2003),
Ó 2007 The Authors
Journal Compilation Ó 2007 International Society for Neurochemistry, J. Neurochem. (2007) 100, 1169–1176
1172 B. A. Pierchala et al.
with NGF for 4 h, Ret levels and Ret autophosphorylation
increased in the terminals (Fig. 2b, Ret increased 2.8fold ± 0.6 and P-Ret increased 4.2-fold ± 0.9). Inhibition of
protein translation in the terminals with CHX did not alter the
ability of NGF to regulate Ret there (Fig. 2b, Ret increased
3.1-fold ± 0.9 and P-Ret increased 4.8-fold ± 1.2), indicating that axonal protein translation is not required for NGF
induction of Ret phosphorylation in distal axons. In conclusion, NGF locally regulated the protein level and autophosphorylation of Ret in the distal axons of mature
sympathetic neurons via a mechanism that did not require
protein translation in the distal axons.
(a)
(b)
Fig. 2 Nerve growth factor (NGF) regulates Ret phosphorylation and
levels locally in distal axons independently of protein synthesis. (a)
Mature sympathetic neurons maintained in compartmentalized cultures were treated with NGF or medium alone only on the distal axons
for 48 h. The cell body compartment contained anti-NGF. Detergent
extracts were produced from the cell body or distal axon compartment
and Ret activation (top panel) and TrkA activation (third panel) were
determined after immunoprecipitation of these receptors. The levels of
Ret and TrkA were deduced by reprobing these immunoblots with
Ret51 and Trk antibodies, respectively. (b) The distal axons of sympathetic neurons deprived of NGF were treated with NGF (50 ng/mL)
for 4 h or were treated with NGF in the presence of cycloheximide
(1 lmol/L) on only the distal axons. Ret activation in the distal axons
was examined as in (a). These experiments were performed two to
three times with similar results. Actin immunoblotting of the immunoprecipitate supernatants confirmed that equal amounts of protein were
analyzed.
these data suggest that NGF acting on distal axons regulated
Ret locally. The observation that NGF coordinately regulated
Ret phosphorylation and Ret levels in distal axons suggests
the possibility that the local translation of Ret mRNA in
distal axons accounts for this phenomenon. To test this
hypothesis, cycloheximide (CHX) was used to locally inhibit
protein translation. When the distal axons of mature sympathetic neurons in compartmentalized cultures were treated
Nerve growth factor promotes Ret autophosphorylation
by inhibiting Ret degradation
The proteasomal degradation pathway was recently demonstrated in axons and is involved in axon pruning and axon
degeneration (Watts et al. 2003; Zhai et al. 2003). The
ability of NGF to regulate Ret levels and Ret phosphorylation in axons led us to hypothesize that NGF regulates Ret by
inhibiting the degradation of autophosphorylated receptor.
This possibility was examined in mass cultures of mature
sympathetic neurons. As before, the treatment of sympathetic
neurons with NGF for 4 h induced Ret phosphorylation and
increased Ret protein and this effect of NGF was maximal
within 48 h (Fig. 3a) (Tsui-Pierchala et al. 2002a). Inhibition
of the proteasome with the selective proteasomal inhibitors
lactacystin or epoxomicin for 8 h increased both the
phosphorylation and the amount of Ret in sympathetic
neurons in the absence of NGF (Fig. 3a). Inhibition of
lysosomes with either concanamycin or ammonium chloride
increased Ret protein and Ret phosphorylation levels as well
(Fig. 3a), indicating that inhibition of either proteasome or
lysosome activity was sufficient to increase Ret levels and
autophosphorylation in mature sympathetic neurons.
The observation that proteasomal and lysosomal inhibitors
mimicked the effects of NGF suggested that the effects of
NGF on Ret involved inhibition of the degradation pathways
that down-regulate activated RTKs. Recent studies have
revealed that Ret9 and Ret51, after ligand activation, are
rapidly ubiquitinated and degraded (Scott et al. 2005;
Pierchala et al. 2006), and in sympathetic neurons this
occurs predominantly via the proteasome (Pierchala et al.
2006). Interestingly, the kinetics of degradation of Ret51 are
markedly faster than Ret9 and best conform to first order
kinetics, whereas Ret9 degradation is zero order (Pierchala
et al. 2006). If NGF increases the levels of Ret and Ret
phosphorylation by inhibiting the clearance of activated
receptors, then NGF should inhibit the ligand-dependent
degradation of Ret. Stimulation of mature sympathetic
neurons with GDNF induced the rapid degradation of
Ret51, and the majority of Ret51 was lost within 3 h of
GDNF treatment (Fig. 3b). NGF maintenance of sympathetic
neurons inhibited markedly the GDNF-dependent degrada-
Ó 2007 The Authors
Journal Compilation Ó 2007 International Society for Neurochemistry, J. Neurochem. (2007) 100, 1169–1176
NGF inhibits ubiquitin-dependent Ret degradation 1173
(a)
(b)
(c)
Fig. 3 Nerve growth factor (NGF) modulates the level of autophosphorylated Ret in sympathetic neurons via inhibition of activationdependent Ret degradation. (a) Mass cultures of sympathetic neurons
that were deprived of NGF for 48 h were then treated with medium
alone, with NGF (50 ng/mL) for 4 h or 48 h, or with lactacystin
(10 lmol/L), epoxomicin (5 lmol/L), concanamycin (200 nmol/L), or
ammonium chloride (2 mmol/L) for 8 h. An additional set of
NGF-deprived neurons were also treated with both epoxomicin and
ammonium chloride for 8 h. The neurons were then subjected to
phospho-Ret analysis as was done in Fig. 2. (b) Mass cultures of
sympathetic neurons that were maintained in NGF (50 ng/mL) or
without NGF for 48 h were stimulated with glial cell line-derived neurotrophic factor (50 ng/mL) for various lengths of time and subjected to
phospho-Ret analysis. Actin immunoblotting confirmed the analysis of
equal amounts of protein. These experiments were performed three to
four times with similar results. (c) The results of (b) were quantified and
graphed. Error bars represent standard deviation.
tion of Ret51 (Figs 3b and c). Quantification of these
experiments indicated that the stability of Ret51 after GDNFdependent activation was increased dramatically by NGF and
appeared to change from first order kinetics (r2 = 0.91) to
zero order kinetics (r2 = 0.96, Fig. 3c). NGF also increased,
over time, the amount of autophosphorylated Ret induced by
GDNF because of the slower kinetics of degradation (Fig.
3b). Therefore, NGF modulates the levels of activated Ret by
inhibition of the degradation machinery required for the
clearance of activated Ret in sympathetic neurons. These
results explain the previous observation that both Ret protein
and Ret autophosphorylation increase concomitantly (Fig. 1)
because the half-life of activated Ret is increased dramatically in NGF-maintained neurons, leading to the accumulation
of activated Ret in these neurons.
Nerve growth factor blocks the degradation of activated
Ret prior to its internalization and polyubiquitination
The inhibitory effect of NGF on the degradation of activated
Ret may occur at multiple steps in the degradation pathway.
For most RTKs, ligand-initiated degradation requires that the
receptor is internalized and transported to lysosomes and/or
proteasomes (Bonifacino and Traub 2003; Dikic and Giordano 2003). To determine whether NGF inhibited the
internalization of autophosphorylated Ret thereby leading
to its accumulation in the plasma membrane, cell surface
biotinylation experiments were conducted. Mature sympathetic neurons were treated with NGF for either 4 or 48 h, or
with GDNF for 30 s or 30 min. After these treatments, the
neurons were then rapidly cooled to 4°C to stop membrane
traffic and plasma membrane-spanning proteins were labeled
with biotin and purified with Neutravidin. NGF treatment
caused the robust accumulation of autophosphorylated Ret
on the cell surface and GDNF treatment also led to the rapid
appearance of phosphorylated Ret on the cell surface (Fig.
4a). Examination of Ret that escaped biotinylation and was in
internal membranes revealed that NGF did not promote the
accumulation of phosphorylated Ret in internal membranes,
in contrast to GDNF-treated neurons in which activated Ret
was internalized rapidly (Fig. 4a, third panel). Ret was not
purified with Neutravidin from GDNF-treated sympathetic
neurons that were not cell surface biotinylated, which
confirmed the specificity of Neutravidin for biotinylated
proteins (Fig. 4a). These observations indicate that NGF
induces the accumulation of activated Ret on the cell surface,
perhaps by inhibition of the internalization of activated Ret
that would normally lead to its degradation, thus, resulting in
the sustained accumulation of phosphorylated Ret on the cell
surface.
An event required for the internalization of many
autophosphorylated RTKs is ubiquitination on lysine
residues (Bonifacino and Weissman 1998; Hicke 2001).
Ubiquitin is added to proteins as a monomer or as chains of
ubiquitin, and these modifications initiate internalization or
Ó 2007 The Authors
Journal Compilation Ó 2007 International Society for Neurochemistry, J. Neurochem. (2007) 100, 1169–1176
1174 B. A. Pierchala et al.
autophosphorylated Ret accumulates on the plasma membrane.
(a)
Discussion
(b)
Fig. 4 Nerve growth factor (NGF) inhibits the ubiquitin-dependent
degradation of Ret before internalization and polyubiquitination. (a)
Mass cultures of mature sympathetic neurons were treated with
medium alone, NGF (50 ng/mL), or glial cell line-derived neurotrophic
factor (GDNF) (50 ng/mL) for various lengths of time. Ret located in
the plasma membrane was labeled with biotin (B) along with other cell
surface proteins and purified away from Ret located in internal membranes by Neutravidin precipitation (P: NA). As a negative control,
neurons treated with GDNF for 30 min were not cell surface labeled
(right-most lane). Both pools of Ret were subjected to phospho-Ret
analysis as in Fig. 2. (b) Sympathetic neurons were treated with
medium alone, NGF (50 ng/mL) for 48 h, or GDNF (50 ng/mL) for
15 min. Monoubiquitinated (left three lanes) or polyubiquitinated (right
three lanes) proteins were immunoprecipitated and subjected to Ret51
immunoblotting. Actin served as a control for the analysis of equal
amounts of protein; all of these experiments were performed two to
three times with similar results.
transport of proteins to the proteasome, respectively
(Bonifacino and Weissman 1998; Hicke 2001). To determine
whether Ret that accumulates upon NGF treatment was
ubiquitinated, antibodies that preferentially detect either
monomeric or polymeric ubiquitin were used. NGF treatment
of mature sympathetic neurons induced the accumulation of
monoubiquitinated Ret (increased 2.4-fold ± 0.5 when compared with actin), but not polyubiquitinated Ret (Fig. 4b). In
contrast, Ret activated by GDNF preferentially incorporated
polyubiquitin (Fig. 4b), presumably because monoubiquitination is followed rapidly by extension of these moieties into
chains of ubiquitin. Therefore, NGF inhibited Ret degradation before polyubiquitination and this monoubiquitinated,
Nerve growth factor is required for the trophic maintenance
of the adult sympathetic nervous system (Angeletti et al.
1971; Goedert et al. 1978; Gorin and Johnson 1980; Ruit
et al. 1990). A significant portion of the effects of NGF on
the metabolism of mature sympathetic neurons is conveyed
by NGF modulation of Ret (Tsui-Pierchala et al. 2002a).
NGF acting on distal axons and terminals can regulate Ret
autophosphorylation locally. Although NGF also coordinately increases the level of Ret protein, the effect of NGF on
Ret in axons does not require local protein synthesis. Rather,
NGF increases the level of activated Ret in sympathetic
neurons by inhibition of the phosphorylation-dependent
degradation of Ret. NGF acts proximal to Ret internalization
and polyubiquitination, leading to the accumulation of
monoubiquitinated, autophosphorylated Ret in the plasma
membrane.
Of the several examples of cross-talk pathways between
receptor signaling complexes, one of the most relevant to this
study is the cross-talk mechanism between G-protein coupled
receptors (GPCRs) and Trks (Lee and Chao 2001; Lee et al.
2002; Rajagopal et al. 2004). The adenosine and PACAP
receptors activate TrkA by a Src-dependent mechanism that
causes the accumulation of activated TrkA in the trans-golgi
network (TGN) (Lee et al. 2002; Rajagopal et al. 2004).
TrkA activated by GPCRs has distinct signaling properties,
possibly because of its activation in the TGN rather than at
the plasma membrane (Lee et al. 2002; Rajagopal et al.
2004). NGF regulation of Ret is similar in that Y905 and
Y1062 are more highly autophosphorylated when compared
with Ret activated by GFLs, suggesting that NGF-activated
Ret possesses different signaling capacities than Ret activated by GFLs (Coulpier et al. 2002; Tsui-Pierchala et al.
2002b). It will be interesting to determine to what extent
other autophosphorylation sites in Ret, such as Y981
(Encinas et al. 2004), are augmented by NGF and how they
alter downstream signaling events.
The modulation of Ret by TrkA represents a fundamentally different mechanism in which one receptor (TrkA)
augments the signaling of a second receptor (Ret) through
inhibition of the down-regulation pathway for the second
receptor (Fig. 5). NGF regulation of Ret could be considered
a passive mechanism of receptor activation because it
essentially ‘resets’ the basal level of autophosphorylation
that exists for RTKs such as Ret by attenuating the ongoing
clearance pathway for the low level of phosphorylated Ret
occurring in the absence of ligand (Schlessinger 2000). An
equilibrium exists between phosphorylated and unphosphorylated receptor. This equilibrium is affected both by
the amount of Ret expressed (which explains the observation
Ó 2007 The Authors
Journal Compilation Ó 2007 International Society for Neurochemistry, J. Neurochem. (2007) 100, 1169–1176
NGF inhibits ubiquitin-dependent Ret degradation 1175
Patricia Osborne for critical reading of the manuscript, and Mary
Bloomgren for secretarial assistance.
Ub
Ret
NGF
Ub
Ub
Ub
Ub
Internalization
Degradation
Fig. 5 Schematic representation of the effect of nerve growth factor
(NGF) on Ret. In mature sympathetic neurons there is a low basal
level of Ret51 autophosphorylation that occurs when Ret monomers
transiently associate with each other. When Ret becomes phosphorylated it is ubiquitinated and targeted for degradation. NGF inhibits this
process after monoubiquitination but before the internalization of Ret.
Thus, activated Ret accumulates in sympathetic neurons and augments their metabolic status.
that Ret overexpression leads to higher levels of Ret
phosphorylation) and by the rate of clearance of activated
receptor, the point where NGF acts. Importantly, these results
demonstrate that RTKs may function independently of ligand
through mechanisms that alter the stability of activated
RTKs, in some respects similar to oncogenic mutations in
RTKs that alter their stability (Peschard and Park 2003).
These data suggest that proteins degraded by Cbldependent mechanisms may accumulate in mature sympathetic neurons maintained in NGF. These data, therefore,
reveal a new paradigm by which growth factors augment the
metabolism and growth of cells: the selective inhibition of
protein catabolism. Inhibition of cellular catabolic events, in
addition to the well-known effect of growth factors on
cellular anabolism, may represent two ‘arms’ of the growth
response to neurotrophic factors, coordinately providing the
cellular substrates necessary for maintenance of the adult
nervous system. Likewise, this mechanism has important
implications for neurodegenerative disorders involving alterations in catabolic pathways, such as the proteasomal
pathway. Because NGF acted locally to regulate Ret, and
potentially other proteins degraded by the proteasome,
growth factor inhibition of catabolism may affect axonopathies and Wallerian degeneration that occur in many
neuropathologic conditions. Beyond disease, neurotrophic
factor regulation of catabolic processes, particularly at the
level of internalization and polyubiquitination, may have a
modulatory function in vesicular trafficking and retrograde
signaling pathways that are required for survival, growth, and
plasticity.
Acknowledgements
This research was supported by National Institutes of Health grants
R37AG-12947 and AG-13729 (E.M.J.), AG-13730 (J.M.), and K01
NS-045221 (B.A.P.). We thank Tim Fahrner for technical assistance,
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