1 Department of Pathology, University of Western Australia, QE II Medical
Centre, Crawley WA 6009, Australia
2 Van Andel Research Institute, Grand Rapids, MI 49503, USA
* Author for correspondence (e-mail: rscaife{at}cyllene.uwa.edu.au)
Accepted 28 October 2002
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Summary |
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Key words: Actin, Cbl, Rac, RTK, Src
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Introduction |
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Genetic and biochemical evidence indicates that the Cbl family of
proto-oncogenes regulate signaling by both receptor and non-receptor tyrosine
kinases (Thien and Langdon,
2001), and Cbl has been shown to function as an E3 RING-type
ubiquitin protein ligase that can direct the polyubiquitylation and
downregulation of activated RTKs (Joazeiro
et al., 1999
; Levkowitz et
al., 1999
; Levkowitz et al.,
1998
; Miyake et al.,
1998
; Yokouchi et al.,
1999
). Although studies of Drosophila oogenesis and
vulval induction in C. elegans unambiguously demonstrate that Cbl can
attenuate RTK signaling (Pai et al.,
2000
; Yoon et al.,
1995
), evidence supporting a role for Cbl in regulating
proliferation of mammalian cells in response to growth factors is less clear.
Although depletion of c-Cbl has clearly been shown to enhance the
proliferation of macrophages (Lee et al.,
1999
), effects of Cbl overexpression on mitogenic signaling
(Broome et al., 1999
;
Dolfi et al., 1998
;
Waterman et al., 1999
) do not
consistently correlate with Cbl-mediated influences on cell proliferation
(Bonita et al., 1997
;
Miyake et al., 1999
). Notably,
in several instances Cbl has been reported to potentiate signaling by tyrosine
kinases (Garcia-Guzman et al.,
2000
; Zhang et al.,
1999
).
Several reports indicate that Cbl proteins can affect the actin
cytoskeleton (Feshchenko et al.,
1999; Lee et al.,
1999
; Ribon et al.,
1998
). We have found that the expression of a dominant-negative
Cbl construct (Petrelli et al.,
2002
) that only retains its tyrosine-kinase-binding (TKB) and RING
finger domains (480-Cbl) can perturb cell morphology and Rac-dependent actin
organization (Scaife and Langdon,
2000
). In analogy with a recent study of Cbl-b regulation of Cdc42
and filopodia formation in T cells
(Krawczyk et al., 2000
), we
report a potent negative regulatory role for Cbl in RTK-mediated signaling to
the actin cytoskeleton. We demonstrate that perturbation of Cbl function in
response to RTK activation dramatically increases actin dorsal ruffle
assembly. This cytoskeletal response corresponds with a potent activation of
Rac, whereas other RTK targets remain unaffected. The selective activation of
signaling to the actin cytoskeleton appears to be due RTK-induced
translocation of Cbl to sites of actin dorsal ruffle nucleation where it can
attenuate Rac-activation and actin assembly.
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Materials and Methods |
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Cells were grown to approximately 50% confluency prior to serum starvation for 24 hours in DME + 0.5% FCS. Serum-starved cells were activated at 37°C with 10 ng/ml PDGF (human recombinant AA homodimer, UBI or Gibco/BRL), 2.5 µM LPA or 10% FCS following a 20 minute incubation with pharmacological inhibitors of PKC (Bisindolylmaleimide I, CalBiochem), Src (SU6656, SUGEN corporation), PI 3-kinase (Wortmannin or LY294002, Sigma) or Rac (SCH 51344, provided by C. Kumar, Schering-Plough Research Institute) as indicated.
Immunofluorescence microscopy
Cells were seeded onto coverslips coated with polylysine (0.1 mg/ml) and
cultured at least 24-48 hours prior to fixation in 4% p-formaldehyde/PBS. The
fixed cells were then permeabilized for 2 minutes with 0.2% Triton X-100 in
PBS. Coverslips were rinsed with PBS and incubated for 30 minutes with 0.5
µg/ml TRITC-phalloidin (Sigma) or primary antibodies (7.5 µg/ml
anti-Tubulin antibodies, Sigma mAb, #T-4026 or 0.1 µg/ml anti-HA
antibodies, Boehringer mAb 3F10) at 37°C for 60 minutes in PBS containing
2.5 mg/ml BSA. Following a PBS wash the coverslips were incubated with 5
µg/ml biotin-SP-conjugated goat anti-mouse, biotin-SP-conjugated anti-rat
(Jackson Laboratories, catalog numbers 115-065-003, 112-065-003) at 37°C
for 60 minutes in PBS containing 2.5 mg/ml BSA. Biotin-labeled
antigen-antibody complexes were then visualized by incubation for 60 minutes
with PBS containing 2.5 mg/ml BSA and 2 µg/ml Alexa-488-conjugated
Streptavidin (Molecular Probes). Following a PBS rinse coverslips were mounted
with SlowFade Light Antifade reagent (Molecular Probes). Images of
representative fields were obtained with Comos and Confocal Assistant software
(BioRad) following capture on a Nikon Diaphot 300 microscope equipped for UV
laser scanning confocal microscopy (BioRad MRC 1000/1024). The 543 nm
excitation signal from TRITC and the 488 nm excitation signals from NLS-GFP
and Alexa 488 were collected sequentially with 580/32 and 522/35 nm emission
filters, respectively. Actin dorsal ruffle formation was quantified for each
sample by determination of the number of cells that contained visually
distinct actin dorsal ruffles by immunofluorescence microscopy (20x
objective). Triplicate counts of 150 cells each were scored for the presence
of distinct actin staining.
Cell lysis, immunoprecipitation, Rac-GTP precipitation and western
blotting
Cells were washed with PBS and lysed in ice-cold 50 mM HEPES buffer (pH
7.4) containing 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1%
sodium dodecyl sulphate, 10% glycerol, 1.5 mM MgCl2, 1 mM EDTA, 1
mM Na3VO4, 10 mM NaF and protease inhibitors. Following
centrifugation at 14,000 g for 10 minutes, aliquots of cell
lysates were directly subjected to SDS-PAGE and transferred onto
nitrocellulose membranes (Amersham). Membranes were blocked with 10% non-fat
milk (Carnation), 5% BSA (Boehringer) in TBS containing 0.5% Tween-20 and
probed with antibodies directed against either phospho-tyrosine (mAb 4G10,
kindly provided by Brian Druker, OHS Portland), c-Cbl (BD Transduction Labs
mAb 610441), HA (Boehringer mAb 3F10), PDGF- receptor (Santa Cruz
SC-431), phospho-Shc (Tyr317) (Cell Signaling # 2431), phospho-Shc (Tyr239)
(UBI # 07-209), Shc (mAb Santa Cruz SC-967), phospho-MAP kinase (NEB mAb, #
9106), MAP kinase (Santa Cruz # 094), phospho-Akt (NEB # 9271 and # 9275), Akt
(NEB # 9272), phospho-Stat3 (UBI mAb, # 05-485), Stat3 (Santa Cruz mAb #
8019), phospho-p38 MAP kinase (Thr180/Tyr182) (Cell Signaling #9211) or p38
MAP kinase (Cell signaling #9212), followed by an HRP-conjugated secondary
antibody (Silenus). Antigens were then visualized by chemiluminescence (ECL,
Amersham) using Hyperfilm MP (Amersham). Rac-GTP was precipitated from cells
lysed in 50 mM Tris pH 7.4, 100 mM NaCl, 1% NP40, 2 mM MgCl2, 10%
glycerol and protease inhibitors by incubation for 30 minutes at 4°C with
glutathione-sepharose (Pharmacia) -bound GST-PAK-CD (PAK-CRIB domain amino
acid residues 56-141), followed by three washes with lysis buffer. Rac was
detected by western blotting with anti-Rac antibodies (Transduction Labs mAb #
R55620). PDGF-
receptor was immunoprecipitated using polyclonal
anti-PDGF receptor antibodies (Santa Cruz SC-431) following lysis of cells in
50 mM Tris pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM Na3VO4,
10 mM NaF, 1% NP40 containing protease inhibitors and centrifugation at 1500
g for 4 minutes. Immune complexes were subjected to SDS-PAGE
and probed by western blot with 1/400 diluted anti-ubiquitin antibodies
(NovoCastra NCL-UBIQ) following an 8 minute 120°C autoclave of the
membrane.
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Results |
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Assembly of actin dorsal ruffles in response to PDGF is rapid and transient. Maximal actin dorsal ruffle formation occurred within 5 minutes of PDGF activation, and these structures fully disassembled after 30 minutes of PDGF treatment (Fig. 2A, panels 3 and 4; data not shown). Not only was actin ruffle formation more distinctive in 480-Cbl-expressing cells, but the number of cells capable of assembling these structures was enhanced by nearly 10-fold by the expression of this Cb1 construct (Fig. 2B). Mutational inactivation (G306E) of the TKB domain abolished the ability of 480-Cbl to increase PDGF-induced actin dorsal ruffle formation (Fig. 2B, panel 6), indicating that the enhanced cytoskeletal response is due to competition for Cbl TKB domain targets.
Expression of 480-Cbl does not affect PDGF receptor regulation or
signaling to MAP kinase and PI 3-kinase
We wished to determine whether 480-Cbl expression has a general effect on
RTK signal output or whether it specifically enhances RTK signaling to the
actin cytoskeleton. The PDGF receptor and several intracellular targets
undergo dose-dependent tyrosine phosphorylation in response to PDGF activation
of serum-starved cells. By assaying total cellular tyrosine phosphorylation
levels prior to and immediately following PDGF addition to the culture medium,
we found that PDGF treatment induced comparable protein tyrosine
phosphorylation in c-Cbl overexpressing, 480-Cbl-expressing and wild-type NIH
3T3 cells (Fig. 3A). This
indicates that 480-Cbl expression does not affect the tyrosine phosphorylation
cascades induced by activation of the PDGF receptor. In order to confirm that
480-Cbl expression does not alter the kinase activity of the PDGF receptor,
phosphorylation of tyrosine residue 317 of the PDGF receptor substrate Shc was
assayed. We found that phosphorylation of Shc tyrosine residue 317 was
comparable in c-Cbl-overexpressing, 480-Cbl-expressing and wild-type NIH 3T3
cells (Fig. 3B). These results
suggest that 480-Cbl expression does not enhance RTK-mediated tyrosine
phosphorylation. We therefore examined PDGF-mediated mitogenic signaling
pathways to determine whether, in addition to the effect on the actin
cytoskeleton, 480-Cbl expression can also affect these responses. Activation
of the PDGF receptor induces a MAPK-phosphorylation-dependent mitogenic
signaling pathway that was assayed using Thr202/Tyr204 phospho-p44/42 MAP
kinase antibodies. Surprisingly, the kinetics and magnitude of the transient
induction of Thr202/Tyr204 MAPK phosphorylation in wild-type, c-Cbl and
480-Cbl-expressing cells were comparable
(Fig. 3C). Similarly, we
indirectly probed for PDGF-induced PI 3-kinase activity by assaying
activation-dependent phosphorylation of the PI 3-kinase effector Akt using
Ser-473 phospho-Akt antibodies. Expression of 480-Cbl had no discernible
effect on PDGF-induced Ser-473 phospho-Akt levels
(Fig. 3D).
|
Although truncated Cbl constructs, such as 480-Cbl, have E3 ligase activity
in vitro, ubiquitylation-mediated downregulation of the EGF receptor requires
C-terminal Cbl sequences in addition to the N-terminal TKB and RING domains
(Waterman et al., 2002). We
therefore examined whether the effects of 480-Cbl on actin cytoskeletal
dynamics could reflect effects on PDGF receptor polyubiquitylation owing to
inhibition of the ability of endogenous Cbl to function as an E3 ubiquitin
ligase. Although enhanced stimulation-dependent polyubiquitylation of the PDGF
receptor occurred upon c-Cbl overexpression, polyubiquitylation of the PDGF
receptor was comparable in wild-type and 480-Cbl-expressing cells
(Fig. 4). These results
therefore indicate that the profound cytoskeletal response of PDGF-stimulated
480-Cbl cells is not due to a block in endogenous Cbl E3 ligase activity. It
does appear, however, that 480-Cbl has a role in reducing the level of
tyrosine-phosphorylated PDGFR after 10 minutes compared with wild-type
cells.
|
PDGF-induced enhancement of actin dorsal ruffle formation by 480-Cbl
requires signaling by Src, PI 3-kinase and Rac
In order to identify the signaling pathways from RTKs to the actin
cytoskeleton that may be regulated by Cbl, we assayed the effect of several
pharmacological inhibitors of key signal-transducing molecules. We found that
treatment with inhibitors of Rac (SCH51344)
(Walsh et al., 1997), Src
(SU6656 and PP2) (Blake et al.,
2000
) or PI 3-kinase (Wortmannin and LY294002) fully abolished the
PDGF-induced actin dorsal ruffle formation in 480-Cbl-expressing cells
(Fig. 5A). These inhibitors had
no discernable effects on the morphology and cytoskeleton of serum-starved
cells prior to treatment with PDGF, indicating that their effect on
PDGF-induced actin dorsal ruffle formation is not due to unrelated secondary
effects on the cells. The induction of actin dorsal ruffles is specific to RTK
activation since we found that stimulation with FCS or LPA, which primarily
activates G-protein-coupled seven-transmembrane domain receptors, did not
result in actin dorsal ruffle formation
(Fig. 5A).
|
Interestingly, we found that actin dorsal ruffle formation is mechanistically distinct from actin lamellipodium formation. Indeed, in contrast to the induction of actin dorsal ruffles by RTK activation, the formation of peripheral actin lamellipodia by spreading NIH 3T3 fibroblasts is not blocked by the inhibition of Src with SU6656 or PP2 (data not shown).
The requirement of Rac, Src and PI 3-kinase for PDGF-induced actin dorsal
ruffle formation in 480-Cbl-expressing cells was confirmed using
dominant-negative constructs. Although actin dorsal ruffles were still formed
in control nuclear localization signal (NLS)-tagged GFP-transfected
480-Cbl-expressing cells, co-transfection of NLS-GFP with N17-Rac,
R296/F528-Src or p85iSH2 essentially abolished actin dorsal ruffle
formation (Fig. 5B). The effect
of 480-Cbl on actin dorsal ruffle assembly is therefore dependent on signaling
events involving Src, PI 3-kinase and Rac.
Although we observed no pronounced effect of 480-Cbl overexpression on the
tyrosine phosphorylation of cellular substrates or the
phosphorylation-dependent activation of MAP kinase and Akt, the requirement
for Src, PI 3-kinase and Rac for actin dorsal ruffles formation suggests that
480-Cbl exerts its effect on the actin cytoskeleton by specifically affecting
one, or more, of these signaling components. Since enhanced signaling by Src
can induce similar circular actin structures
(Blake et al., 2000), we wished
to determine whether 480-Cbl expression leads to increased Src activation by
PDGF. Src activation was determined by assaying tyrosine phosphorylation
levels of Shc (at residue 239) and Stat3 (at residue 704); two principal Src
substrates in NIH 3T3 cells (Goi et al.,
2000
). We found that, relative to control NIH 3T3 cells and
c-Cbl-overexpressing cells, the PDGF-induced tyrosine phosphorylation by Src
of both Shc and Stat3 were not affected by 480-Cbl expression
(Fig. 6). These results
indicate that 480-Cbl expression does not alter the total cellular activity of
Src towards two well defined substrates; however, a localized effect on a
subpopulation of Src remains a possibility.
|
Increased PDGF-mediated activation of Rac in 480-Cbl-expressing
cells
Since Rac activation results in actin dorsal ruffle formation in response
to RTK-induced signaling (Ridley et al.,
1992), we wished to determine whether 480-Cbl expression enhances
the PDGF-induced activation of Rac. PDGF-induced Rac activation was assayed by
the binding of Rac-GTP to a GST-PAK-CD fusion protein. Expression of 480-Cbl
resulted in a pronounced increase in Rac-GTP levels relative to levels in
PDGF-treated wild-type and c-Cbl-overexpressing cells
(Fig. 7A). PDGF treatment of
480-Cbl-expressing cells consistently resulted in a rapid and pronounced
activation of Rac, and the averaged results from five separate Rac-activation
experiments are shown in Fig.
7B.
|
In light of the pronounced effect of 480-Cbl expression on PDGF-induced
Rac-GTP levels, we examined the activation of p38 MAPK, a downstream target of
Rac. Thr180/Tyr182 phospho-p38 MAPK-specific antibodies revealed no effect of
480-Cbl expression on the phosphorylation of p38 MAPK
(Fig. 7C). This suggests that
the enhanced activation of Rac may be specific for signaling events that
directly regulate actin assembly. This is analogous to results obtained with
Cbl-b-depleted T-cells. In this case, increased filopodia formation and Cdc42
activation in response to T-cell receptor stimulation did not result in
increased levels of SAPK or p38 MAPK activity
(Krawczyk et al., 2000).
Requirements of Src and PI 3-kinase for the increased Rac-activation
by 480-Cbl expression
As shown in Fig. 5, our data
clearly demonstrate that Src and PI 3-kinase are required for the induction of
actin dorsal ruffles in PDGF-treated 480-Cbl-expressing cells. Since Cbl has
been implicated in the regulation of both Src and PI 3-kinase
(Fang and Liu, 2001;
Meng and Lowell, 1998
;
Sanjay et al., 2001
), we
wished to determine whether these kinases are also involved in the enhanced
activation of Rac in PDGF-treated 480-Cbl-expressing cells. PDGF-induced
activation of Rac in 480-Cbl expressing cells was assayed following incubation
with the synthetic Src inhibitors SU6656 and PP2. Similar to the effect of
these inhibitors on actin dorsal ruffle assembly
(Fig. 5), SU6656 and PP2
pre-incubation of 480-Cbl-expressing cells greatly reduced the subsequent
PDGF-induced activation of Rac (Fig.
8A). Similarly, inhibition of PI 3-kinase by pre-incubation of the
cells with LY294002 also blocked the subsequent PDGF-induced activation of Rac
in 480-Cbl cells (Fig. 8A). By
contrast, the synthetic PKC inhibitor BIM 1, which was ineffective at
inhibiting actin dorsal ruffle formation
(Fig. 5), failed to reduce Rac
activation following PDGF treatment of 480-Cbl-expressing cells
(Fig. 8A). These data indicate
that although Src and PI 3-kinase do not appear to exhibit enhanced activity
in 480-Cbl cells they are essential components in the pathway leading from the
PDGF receptor to Rac.
|
The C-terminal portion of Cbl can form associations with many signaling
proteins, which include Src family kinases, p85, Crk and CIN85
(Fig. 1A). To better define the
critical region in c-Cbl that needs to be deleted to reveal the
dominant-negative effect on actin ruffle formation we examined the effect of
overexpressing a range of C-terminal truncations
(Fig. 8B). We found that
expression of Cbl truncated at amino-acid residue 655 had no effect on
PDGF-induced actin dorsal ruffle formation, whereas a Cbl construct bearing a
deletion of the sequences between amino acids 655 and 563 produced an actin
response equivalent to that of 480-Cbl. This was a significant result as it
ruled out any involvement of Cbl with CIN85/CMS, which associates with a
proline motif in this region (Kirsch et
al., 2001; Soubeyran et al.,
2002
), with Crk, which associates with phosphorylated tyrosine
residues 700 and 774, and with p85, which associates with tyrosine 731
(Fig. 1A). Thus the key
requirement for producing an interfering form of Cbl that enhances signaling
to the actin cytoskeleton involves proteins that interact with residues
between 528 and 655. Although this region of c-Cbl contains 4 PxxP motifs,
which could potentially function as SH3-binding sites, this region has not
been extensively studied and to our knowledge no interacting proteins have
been identified. The study of C-terminal truncations also revealed that a form
of Cbl that has a disrupted RING finger (i.e. 388-Cbl) also functioned as a
dominant-negative protein that could enhance actin ruffle formation. Thus
competition for RING finger binding proteins is not a factor in this
response.
RTK-induced actin dorsal ruffles are formed from c-Cbl containing
actin-rich nucleation sites
In order to address how c-Cbl can selectively affect Rac-mediated signaling
by RTKs to the actin cytoskeleton we directly examined the assembly of actin
dorsal ruffles in response to PDGF activation. Using NIH 3T3 cells that stably
express GFP-actin we were able to visualize PDGF-induced actin dynamics by
fluorescence microscopy. Within 4 minutes of PDGF addition distinct small
cytoplasmic actin aggregates appeared that transiently coalesced and arranged
into a highly dynamic actin ring (Fig.
9A; Movie 1, available at
jcs.biologists.org/supplemental).
The PDGF-induced formation of actin dorsal ruffles therefore appears to occur
by assembly and spatial arrangement of actin-rich precursor aggregates.
|
In order to determine the involvement of c-Cbl in the formation of actin dorsal ruffles we stimulated NIH 3T3 cells stably expressing HA-c-Cbl with PDGF and examined the cells by double-label immunofluorescence microscopy. We found an extensive degree of c-Cbl colocalization with the fully formed circular actin dorsal ruffles after 10 minutes of PDGF activation (Fig. 9B, right-hand panels). At an earlier time point following RTK activation (e.g. 5 minutes after PDGF addition), we found a striking colocalization of c-Cbl with the perinuclear actin aggregates (i.e. the actin-rich dorsal ruffle precursors) (Fig. 9B, central panels). Further, a degree of c-Cbl colocalization with actin ruffle nucleation sites was also clearly discernable very soon after RTK activation (e.g. 2 minutes after PDGF addition to the cells) (Fig. 9B, left-hand panels). These results demonstrate that c-Cbl, unlike 480-Cbl (data not shown), is recruited to sites of actin dorsal ruffle assembly and indicate that c-Cbl may play a role in regulating signaling required for actin dorsal ruffle nucleation. The greatly enhanced assembly of actin dorsal ruffles in RTK-activated 480-Cbl-expressing cells may therefore be due to a loss of signal attenuation by endogenous Cbl at these specific subcellular actin nucleation sites.
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Discussion |
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The induction of dorsal ruffles in PDGF-treated cells is clearly quite
different from the inhibition of leading-edge lamellipodia formation in
unstimulated 480-Cbl expressing cells. This effect of 480-Cbl on unstimulated
cells may reflect the involvement of Cbl in a number of signaling pathways
that could affect cell morphology and the actin cytoskeleton
(Baumann et al., 2000;
Kirsch et al., 2001
;
Meng and Lowell, 1998
;
Soubeyran et al., 2002
;
Zell et al., 1998
). Further,
PDGF-induced actin dorsal ruffle formation is mechanistically distinct from
actin assembly at the leading-edge of spreading cells since this latter
process is not dependent on Src (R.M.S., unpublished).
In order to identify the signaling proteins involved in enhanced
reorganization of the actin cytoskeleton of 480-Cbl-expressing cells we used
synthetic inhibitors and dominant-negative forms of Src, PI 3-kinase and Rac.
We found that PDGF induction of actin dorsal ruffles in both wild-type and
480-Cbl-expressing cells is totally dependent on these proteins. However, the
cytoskeletal effects of 480-Cbl expression were not mirrored by parallel
changes in the total cellular activities of the PDGF receptor, Src, and PI-3
kinase: three proteins that have been extensively studied as key targets for
attenuation by Cbl of RTK signaling (Thien
and Langdon, 2001). Rather, we found that 480-Cbl leads to greatly
increased Rac-GTP levels following activation of the PDGF receptor, and this
activation of Rac correlates with the induction of actin dorsal ruffles. These
results indicate that Cbl can function as a negative regulator of Rac and
actin dorsal ruffle formation following RTK activation. The effect of 480-Cbl
expression on the Rac-mediated actin cytoskeletal rearrangements of NIH3T3
fibroblasts is hence similar to the effect of Cbl-b depletion on Cdc42
activation and filopodia formation in T-cells
(Bachmaier et al., 2000
;
Chiang et al., 2000
).
Interestingly, the reported Cbl-b-depletion-dependent effects on the guanine
nucleotide exchange factor Vav-1 (Bachmaier
et al., 2000
; Chiang et al.,
2000
) are also in agreement with genetic analyses of the C.
elegans Cbl orthologue SLI-1. These analyses have implicated Cbl/SLI-1 in
the regulation of nucleotide exchange factors for low molecular weight GTPases
(Chang et al., 2000
;
Yoon et al., 2000
).
The results presented here suggest that the association of Cbl with the
actin cytoskeleton permits its direct participation in regulating actin
cytoskeletal dynamics. Significantly, we have demonstrated that c-Cbl is
targeted to PDGF-induced actin dorsal ruffles, where it is involved in the
regulation of Rac-mediated nucleation of actin assembly. Regulation of actin
cytoskeleton dynamics therefore appears to involve spatially restricted
recruitment of signaling proteins by Cbl, permitting attenuation of selected
Rac-mediated signaling targets. Indeed a study of changes in T-cell receptor
clustering and filopodia formation in Cbl-b-deficient T-cells has similarly
revealed no effect of Cbl-b depletion on SAPK/JNK and MAPK/ERK activation
(Krawczyk et al., 2000).
Spatially restricted changes in actin dynamics by targeted assembly of
multi-protein scaffolds, involving PKA and Abl, have also been described by
others (Westphal et al.,
2000
). Hence the selective effects of Cbl on actin assembly,
without concomitant effects on p38 and JNK, following Rac activation, may
similarly be due to its ability to target the assembly of multi-protein
signaling complexes at sites of actin nucleation. Although 480-Cbl is not
localized to the actin dorsal ruffles (data not shown), its selective binding
to TKB substrates presumably precludes their recruitment to these
multi-protein signaling complexes.
In light of the E3-ligase activity of Cbl, its involvement in RTK signaling
has generally been interpreted in terms of ubiquitylation-dependent
degradation of signaling complexes. However, the inadequacy of a single
mechanism to explain Cbl activity is readily apparent, and this limitation has
been emphasized by several recent studies
(Baumann et al., 2000;
Fang and Liu, 2001
;
Schmitt and Stork, 2002
;
Thien et al., 2001
;
Yoon et al., 2000
). Of
particular relevance is the recently identified Cbl-dependant regulation of
TC-10 and Rap-1. These involve tyrosine-kinase-dependent recruitment of a
Crk-C3G complex by Cbl in what appears to be a ubiquitylation-independent
signaling event. (Baumann et al.,
2000
; Schmitt and Stork,
2002
). Interestingly, this multi-adaptor function of Cbl permits
regulation of signaling to these GTPases without affecting the activity of
`upstream' signaling molecules such as Src kinases.
The true value of multi-adaptor proteins such as Cbl may therefore be
realized by their selective recruitment and targeting, permitting orchestrated
assembly of signaling complexes at specific subcellular sites. It is becoming
increasingly clear that the role of Cbl in the selective assembly of signaling
complexes is paramount for regulation of extracellular signals
(Bachmaier et al., 2000;
Chiang et al., 2000
;
Fang and Liu, 2001
;
Petrelli et al., 2002
;
Soubeyran et al., 2002
;
Thien et al., 2001
). Indeed
the regulation of Rac-dependent actin cytoskeletal rearrangements by Cbl is
likely to be of considerable relevance for many important cellular and
physiological functions (Baumann et al.,
2000
; Villalba et al.,
2001
).
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Acknowledgments |
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Footnotes |
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