The multi-adaptor proto-oncoprotein Cbl is a key regulator of Rac and actin assembly

Robin M. Scaife1,*, Sara A. Courtneidge2 and Wallace Y. Langdon1

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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The induction of protein tyrosine kinase signaling pathways is a principal mechanism for promoting cellular activation. Biochemical and genetic analyses have implicated the multi-adaptor proto-oncogene protein Cbl as a key negative regulator of activated protein tyrosine kinases. By inhibiting the function of Cbl as a multi-domain adaptor protein, through expression of a truncated form (480-Cbl), we demonstrate that Cbl is a potent negative regulator of actin assembly in response to receptor tyrosine kinase (RTK) activation. Expression of 480-Cbl dramatically enhances RTK-dependent induction of actin dorsal ruffles, which correlates with a pronounced increase in Rac activation. By contrast, mitogenic signaling by RTK targets, such as PI 3-kinase and MAP kinases, as well as RTK-mediated tyrosine phosphorylation do not appear to be affected by 480-Cbl expression. Further, we determined that Cbl undergoes a striking RTK-activation-dependent translocation to sites of active actin dorsal ruffle nucleation. Hence, the selective regulation of RTK signaling to the actin cytoskeleton appears to result from recruitment of signaling proteins on a Cbl template bound to the actin cytoskeleton.

Key words: Actin, Cbl, Rac, RTK, Src


    Introduction
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 Introduction
 Materials and Methods
 Results
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The actin cytoskeleton is centrally involved in many normal cellular functions associated with cell adhesion and migration, and its assembly into distinct networks plays important roles in pathologies such as neoplastic transformation and bacterial infection. Cell-substrate adhesion requires the assembly of actin microfilaments into focal-adhesion-associated stress fibers that span the cytoplasm, whereas in cell-cell adhesions cadherin-containing actin filaments are organized into filopodia at the cell periphery (Amano et al., 1997Go; Vasioukhin et al., 2000Go). Cell migration similarly requires localized assembly of actin fiber aggregates at the leading-edge lamellipodia (Nobes and Hall, 1999Go). The spatial and temporal organization of these actin-filament-based structures is regulated by signaling pathways involving the Rho-like GTPases, which include Rho, Rac and Cdc42 (Hall, 1998Go). The transient induction of actin dorsal ruffles in response to receptor protein tyrosine kinase (RTK) activation requires signaling by Rac, whereas stress fiber formation can be induced by serum components that lead to Rho activation (Ridley and Hall, 1992Go; Ridley et al., 1992Go). The Rho-like GTPases permit the coordinated assembly of these actin networks by signal transduction through kinases such as PAK, LIMK and ROCK that regulate the assembly and organization of actin fibers (Kaibuchi et al., 1999Go; Ridley, 1999Go; Schmidt and Hall, 1998Go).

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, 2001Go), 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., 1999Go; Levkowitz et al., 1999Go; Levkowitz et al., 1998Go; Miyake et al., 1998Go; Yokouchi et al., 1999Go). Although studies of Drosophila oogenesis and vulval induction in C. elegans unambiguously demonstrate that Cbl can attenuate RTK signaling (Pai et al., 2000Go; Yoon et al., 1995Go), 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., 1999Go), effects of Cbl overexpression on mitogenic signaling (Broome et al., 1999Go; Dolfi et al., 1998Go; Waterman et al., 1999Go) do not consistently correlate with Cbl-mediated influences on cell proliferation (Bonita et al., 1997Go; Miyake et al., 1999Go). Notably, in several instances Cbl has been reported to potentiate signaling by tyrosine kinases (Garcia-Guzman et al., 2000Go; Zhang et al., 1999Go).

Several reports indicate that Cbl proteins can affect the actin cytoskeleton (Feshchenko et al., 1999Go; Lee et al., 1999Go; Ribon et al., 1998Go). We have found that the expression of a dominant-negative Cbl construct (Petrelli et al., 2002Go) 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, 2000Go). In analogy with a recent study of Cbl-b regulation of Cdc42 and filopodia formation in T cells (Krawczyk et al., 2000Go), 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.


    Materials and Methods
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 Materials and Methods
 Results
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Cell culture and transfections
NIH 3T3 fibroblasts were obtained from ATCC and cultured in DMEM (Trace Biochemicals) containing 10% FCS (Gibco/BRL) and 2 mM L-glutamine (Trace Biochemicals) at 37°C and 5% CO2. Dominant-negative Rac, Src and the p85 subunit of PI 3-kinase were expressed by transient co-transfection (using FuGENE 6 transfection reagent, Boehringer) of column-purified pRK5, pUSEamp or pSG plasmid containing the cDNA coding for either N17 Rac1 (provided by A. Hall, University College London), K296R/Y528F Src (UBI), or p85{Delta}iSH2N (provided by S. Wennstrom, ICRF, London), with column-purified pcDNA3 plasmid coding for Enhanced Green Fluorescent Protein tagged with a nuclear localization signal (NLS-GFP, kindly provided by J. Borst NKI, Amsterdam). pJZenNeo vectors encoding HA-epitope-tagged c-Cbl cDNAs have been described previously (Andoniou et al., 1994Go). EGFP-ß—actin was subcloned into pBabePuro. For stable expression, pJZenNeo and pBabePuro constructs were electroporated into {psi}2 packaging cells to generate virus particles for infection of NIH 3T3 cells, which were selected with 400 µg/ml active G418 (Gibco/BRL) or 2 µg/ml puromycin.

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-{alpha} 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-{alpha} 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.


    Results
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Effect of dominant-negative Cbl expression on RTK-induced cytoskeletal dynamics
The activation of RTKs transiently induces cytoskeletal rearrangements that lead to the formation of circular actin dorsal ruffles (Ridley, 1999Go). To determine whether Cbl coordinates signaling from RTKs to the actin cytoskeleton we assayed the effect of a truncated dominant-negative c-Cbl construct [480-Cbl, (Petrelli et al., 2002Go)] on RTK-induced actin dorsal ruffle formation. This form of Cbl retains its TKB and RING finger domains but lacks C-terminal sequences involved in recruiting SH2- and SH3-domain-containing proteins (Fig. 1A). Both full-length c-Cbl and dominant-negative Cbl (truncated at amino-acid residue 480) were stably expressed in NIH 3T3 fibroblasts by retroviral infection of HA-tagged cDNA constructs. These constructs stably produced HA-tagged proteins at markedly higher levels than endogenous c-Cbl (Fig. 1B). In wild-type NIH 3T3 fibroblasts, activation by platelet-derived growth factor (PDGF) resulted in stress fiber disassembly concomitant with the formation of punctate semi-circular actin aggregates (red signal in Fig. 1C, panels 1 and 2). Similar results to this were also seen with c-Cbl-overexpressing NIH 3T3 fibroblasts (data not shown; Fig. 2A, panel 5). By contrast, PDGF treatment of cells stably overexpressing 480-Cbl caused the actin to reassemble within minutes to form extensive nearly circular bundles of F-actin (red signal in Fig. 1C, panels 4 and 5). These PDGF-induced actin bundles are restricted to the apical section of the cells (Fig. 1C, panel 5) and are hence referred to as actin dorsal ruffles. Staining with anti-tubulin antibodies revealed that the assembly of microtubules was not affected following 5 minutes of PDGF activation of either wild-type or 480-Cbl-expressing cells (green signal, Fig. 1C, panels 1 and 4, 3 and 6).



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Fig. 1. Effect of dominant-negative Cbl expression on RTK-induced cytoskeletal dynamics. (A) Truncation of c-Cbl at amino-acid residue 480 uncouples the N-terminal TKB and RING domains from the numerous C-terminal docking sites. (B) Wild-type, HA-c-Cbl-and HA-480-Cbl-expressing NIH 3T3 cells were lysed and aliquots of the lysates were subjected to SDS-PAGE and western blotting with anti-c-Cbl and anti-HA antibodies as indicated. (C) Serum-starved wild-type (panels 1, 2 and 3) and 480-Cbl-expressing NIH 3T3 cells (panels 4, 5 and 6) were treated with PDGF for 5 minutes. Merged confocal fluorescence microscopy Z-section stacks of phalloidin (red) and anti-tubulin (green) staining are shown in panels 1 and 3, whereas 1.5 µm thick apical sections are shown in panels 2 and 5 for phalloidin staining and panels 3 and 6 for anti-tubulin staining. A PDGF-induced actin aggregate in wild-type cells is indicated by the narrow arrowhead in panel 1. An actin dorsal ruffle is indicated by the broad arrowhead in panel 4. The scale bar represents 20 µm.

 


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Fig. 2. Cbl-mediated regulation of PDGF-induced actin dorsal ruffle assembly. (A) Serum-starved wild-type (panels 1 and 3), c-Cbl (panel 5), 480-Cbl (panels 2 and 4) and G306E 480-Cbl- (panel 6) expressing NIH 3T3 cells were either left untreated (panels 1 and 2) or treated with PDGF for 5 minutes (panels 3-6). Microfilaments were visualized by confocal fluorescence microscopy (the scale bar represents 100 µm), and (B) the percentage of cells that form actin dorsal ruffles at 0, 5 and 15 minutes of PDGF treatment was quantified (see Materials and Methods).

 

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).



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Fig. 3. Expression of 480-Cbl does not affect PDGF receptor activation or signaling to MAP kinase and PI 3-kinase. Wild-type, c-Cbl- and 480-Cbl expressing NIH 3T3 cells were lysed following serum starvation or after 1 and 5 minutes of PDGF activation as indicated. Aliquots of the lysates were subjected to SDS-PAGE and western blotting with anti-phosphotyrosine (A), anti-phospho-Shc (Tyr317) and anti-Shc (B), anti-phospho-MAP kinase (Thr202/Tyr204) and anti-MAPK (C) and anti-phospho-Akt (Ser473) and anti-Akt (D), as indicated.

 

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., 2002Go). 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.



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Fig. 4. Expression of 480-Cbl does not affect PDGF receptor phosphorylation and polyubiquitylation. Wild-type, c-Cbl- and 480-Cbl-expressing NIH 3T3 cells were lysed following serum starvation or after 3 and 10 minutes of PDGF activation, as indicated. Aliquots of the lysates were immunoprecipitated with anti-PDGF receptor antibodies and probed by western blot with anti-phosphotyrosine (A), anti-ubiquitin (B) or anti-PDGF receptor (C) following SDS-PAGE.

 

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., 1997Go), Src (SU6656 and PP2) (Blake et al., 2000Go) 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).



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Fig. 5. Enhancement of actin dorsal ruffle formation by 480-Cbl requires Src, PI 3-kinase and Rac. (A) Following serum starvation, 480-Cbl-expressing NIH 3T3 cells were treated for 20 minutes with either DMSO carrier, PKC inhibitor BIM I (5 µM), Rac inhibitor SCH 51344 (75 µM), Src inhibitors SU6656 (5 µM) or PP2 (5 µM), PI 3-kinase inhibitors Wortmannin (0.5 µM) or LY294002 (30 µM), or left untreated, as indicated. Cells were fixed and stained with TRITC phalloidin to quantify actin dorsal ruffle formation after 5 minutes of activation by FCS (10%), LPA (2.5 µM) or PDGF (10 ng/ml) as indicated. (B) 480-Cbl expressing cells were transiently transfected with NLS-GFP (panel 1), NLS-GFP and R296/F528-Src (panel 2), NLS-GFP and N17-Rac1 (panel 3) and NLS-GFP and p85{Delta}iSH2N (panel 4). The cells were treated with PDGF for 5 minutes following serum starvation and the NLS-GFP and TRITC phalloidin staining of representative samples were visualized by confocal fluorescence microscopy. The number of NLS-GFP-positive cells forming distinct actin dorsal ruffles is indicated in each panel. Bar, 30 µm.

 

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 p85{Delta}iSH2 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., 2000Go), 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., 2000Go). 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.



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Fig. 6. Effect of 480-Cbl expression on PDGF-mediated activation of Src. Wild-type, c-Cbl- and 480-Cbl-expressing NIH 3T3 cells were lysed following serum starvation or after 1 and 5 minutes of treatment with PDGF. Aliquots of the lysates were directly subjected to SDS-PAGE and probed by western blotting with anti-phospho-Shc (Y239) and anti-Shc (A) or anti-phospho-Stat3 (Y704) and anti-Stat3 (B) as indicated.

 

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., 1992Go), 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.



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Fig. 7. Effect of 480-Cbl expression on PDGF-mediated activation of Rac. Wild-type, c-Cbl- and 480-Cbl-expressing NIH 3T3 cells were lysed following serum starvation or after 1 and 5 minutes of treatment with PDGF. (A) Aliquots of the lysates were either directly subjected to SDS-PAGE and western blotting with anti-Rac antibodies or incubated with sepharose-bound GST-PAK-CD and bound proteins subjected to SDS-PAGE and western blotting with anti-Rac antibodies as indicated. (B) Densitometric scans for anti-Rac blots of GST-PAK-CD bound proteins obtained from five experiments were averaged and plotted. Error bars represent the standard deviations. (C) Aliquots of serum starved and PDGF treated wild-type, c-Cbl and 480-Cbl expressing NIH 3T3 cells were subjected to SDS-PAGE and Western blotting with anti-phospho-p38 MAPK (Thr180/Tyr182) and anti-p38 MAPK specific antibodies, as indicated.

 

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., 2000Go).

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, 2001Go; Meng and Lowell, 1998Go; Sanjay et al., 2001Go), 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.



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Fig. 8. Src and PI 3-kinase mediate the increased Rac-activation by 480-Cbl expression. (A) Following serum starvation, 480-Cbl-expressing NIH 3T3 cells were treated for 20 minutes with either DMSO carrier, PKC inhibitor BIM I (3 µM), Src inhibitors SU6656 (5 µM) or PP2 (10 µM) or PI 3-kinase inhibitor LY294002 (30 µM), as indicated. Cells were lysed either after no further treatment or after 1 minute of activation by PDGF (10 ng/ml). Aliquots of the lysates were either directly subjected to SDS-PAGE and probed by western blotting with anti-Rac antibodies or were incubated with sepharose-bound GST-PAK-CD and bound proteins subjected to SDS-PAGE and western blotting with anti-Rac antibodies. (B) Serum-starved NIH 3T3 expressing either full-length c-Cbl or truncations at amino acids 655, 563, 528, 480 (with and without the TKB domain inactivating G306E mutation) and 388 were treated with PDGF for 5 minutes. Microfilaments were visualized by confocal fluorescence microscopy following Tritc-phalloidin staining, and the percentage of cells that form actin dorsal ruffles was quantified (see Materials and Methods).

 

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., 2001Go; Soubeyran et al., 2002Go), 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.



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Fig. 9. RTK-induced actin dorsal ruffles are formed from c-Cbl containing actin-rich nucleation sites. (A) Confocal fluorescence images of serum-starved NIH 3T3 cells expressing EGFP-actin were recorded every minute following addition of PDGF to the culture medium. The arrowhead indicates a site of actin dorsal ruffle assembly in selected images. Bar, 30 µm. An animated compilation of the individual images is represented in Movie 1 (available at jcs.biologists.org/supplemental). (B) Serum-starved HA-c-Cbl-expressing NIH 3T3 cells were treated with PDGF for 2, 5 or 10 minutes. Single 1 µm apical sections of TRITC-phalloidin (red) and anti-HA staining (green) were visualized individually and as merged images by immunofluorescence confocal microscopy. The arrowheads indicate putative actin nucleation sites. Bar, 30 µm.

 

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.


    Discussion
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In agreement with several other reports indicating involvement of Cbl in actin cytoskeletal dynamics (Krawczyk et al., 2000Go; Levkowitz et al., 1999Go; Ribon et al., 1998Go), we previously demonstrated that expression of the dominant-negative 480-Cbl construct suppresses lamellipodia formation in migrating NIH 3T3 fibroblasts (Scaife and Langdon, 2000Go). We have now demonstrated unequivocally that the actin cytoskeleton is one of the principal targets of signaling pathways that are regulated by Cbl. Although inhibition of Cbl, by expression of 480-Cbl, had no discernible effect on the microtubule network, we found that actin dorsal ruffle formation in response to PDGF is greatly enhanced. A degree of actin rearrangement into punctate perinuclear rings or semi-circular dorsal ruffles occurs following RTK activation of wild-type cells. Expression of 480-Cbl greatly increases this response, resulting in the transient induction of very prominent actin dorsal ruffles in nearly all cells.

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., 2000Go; Kirsch et al., 2001Go; Meng and Lowell, 1998Go; Soubeyran et al., 2002Go; Zell et al., 1998Go). 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, 2001Go). 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., 2000Go; Chiang et al., 2000Go). Interestingly, the reported Cbl-b-depletion-dependent effects on the guanine nucleotide exchange factor Vav-1 (Bachmaier et al., 2000Go; Chiang et al., 2000Go) 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., 2000Go; Yoon et al., 2000Go).

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., 2000Go). 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., 2000Go). 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., 2000Go; Fang and Liu, 2001Go; Schmitt and Stork, 2002Go; Thien et al., 2001Go; Yoon et al., 2000Go). 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., 2000Go; Schmitt and Stork, 2002Go). 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., 2000Go; Chiang et al., 2000Go; Fang and Liu, 2001Go; Petrelli et al., 2002Go; Soubeyran et al., 2002Go; Thien et al., 2001Go). 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., 2000Go; Villalba et al., 2001Go).


    Acknowledgments
 
We thank Christine Thien and Hiroshi Maruta for helpful suggestions, Alan Hall for providing the N17-Rac cDNA construct, John Collard and Nathalie Morin for the GST-PAK-CD constructs, Jannie Borst for the NLS-GFP vector, Chandra Kumar for compound SCH51344. We also acknowledge Paul Rigby for assistance with the WA Lotteries Commission confocal microscope. This work was funded by the National Health and Medical Research Council (Canberra), MHRIF (Perth) and a grant from the Association pour la Recherche sur le Cancer (Paris).


    Footnotes
 
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    References
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 Materials and Methods
 Results
 Discussion
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