4228 Research Article

IntroductionGrowth factor receptors such as the PDGFR regulate essentialphysiological processes in metazoans, including tissuemorphogenesis, maintenance and repair. Dysregulated growth-factor-dependent signaling plays a role in a number of diseases suchas cancer (Aaronson, 1991; Heldin et al., 1998; Wells, 2000).Cellular migration, which is regulated in part by growth factors,relies on the coordinated assembly and turnover of cellularstructures. In particular, localized actin polymerization drivesmembrane protrusion, and the formation of focal adhesions (FAs)provides a physical link between the actin cytoskeleton and theunderlying extracellular matrix that enables productive cellmigration (Heldin et al., 1998; Ridley et al., 2003; Wells, 2000).

Activation of PDGFR by ligand binding leads to theautophosphorylation of tyrosine residues in the cytosolic portion ofthe receptor. These phosphorylated sites bind to SH2 domains, thusrecruiting a number of SH2-domain-containing proteins to thereceptor. Among the specific downstream consequences isreorganization of the actin cytoskeleton. One of essential events inthis pathway is thought to be activation of the small guanosinetriphosphatase (GTPase) Rac1, which promotes Arp2/3-dependentactin polymerization by increasing the actin-nucleating activity ofWAVE family proteins (Miki et al., 2000; Ridley et al., 1992). Atpresent, the mechanisms by which the recruitment of SH2-domain-containing proteins to the activated PDGFR influences theactivities of actin cytoskeleton regulators remain largely unknown.

The Crk family of adaptor proteins consists of CrkI, CrkII andCrkL (Fig. 1A) (Matsuda et al., 1992; Mayer et al., 1988; Reichmanet al., 1992; ten Hoeve et al., 1993). They are ubiquitously expressedin most tissues and are required for animal development (Guris et

al., 2001; Park et al., 2006). CrkII and CrkL consist of one SH2domain followed by two SH3 domains (designated hereafter asnSH3 and cSH3). In general, SH2 domains bind tyrosine-phosphorylated peptides whereas SH3 domains bind proline-richsequences (Pawson and Nash, 2003). The SH2 and SH3 domainsof CrkII and CrkL are highly similar in sequence (Reichman et al.,1992; ten Hoeve et al., 1993), and their interaction specificities arealmost identical (Feller, 2001). This suggests that CrkII and CrkLhave overlapping functions. Whereas the SH2 and nSH3 domainsare known to function as interaction domains, no partners otherthan the nuclear exporter Crm1 have been reported to bind to thecSH3 domain of either CrkII or CrkL (Smith et al., 2002). Thissuggests that the cSH3 domain might not function as a typical SH3domain.

In addition to these domains, CrkII and CrkL each have aregulatory tyrosine phosphorylation site (Y221 and Y207,respectively) located between the two SH3 domains.Phosphorylation of this site induces intramolecular binding betweenthe phosphorylated tyrosine and the SH2 domain. This masks thebinding surfaces of the SH2 and nSH3 domains, lowering theiraccessibility to other binding partners and shutting off Crk signaling(Kain and Klemke, 2001; Kobashigawa et al., 2007). CrkI is analternately spliced form of CrkII that lacks the cSH3 domain andregulatory tyrosine phosphorylation site (Matsuda et al., 1992).Thus, unlike CrkII and CrkL, CrkI signaling cannot bedownregulated by tyrosine phosphorylation (Kobashigawa et al.,2007), suggesting that the role of CrkI might be distinct from thatof CrkII and CrkL.

Crk family adaptors have been implicated in the regulation ofcellular adhesion, migration and proliferation (Feller, 2001). They

Crk family adaptors, consisting of Src homology 2 (SH2) andSH3 protein-binding domains, mediate assembly of proteincomplexes in signaling. CrkI, an alternately spliced form of Crk,lacks the regulatory phosphorylation site and C-terminal SH3domain present in CrkII and CrkL. We used gene silencingcombined with mutational analysis to probe the role of Crkadaptors in platelet-derived growth-factor receptor (PDGFR)signaling. We demonstrate that Crk adaptors are required forformation of focal adhesions, and for PDGF-stimulatedremodeling of the actin cytoskeleton and cell migration. Crk-dependent signaling is crucial during the early stages ofPDGFR activation, whereas its termination by Abl familytyrosine kinases is important for turnover of focal adhesionsand progression of dorsal-membrane ruffles. CrkII and CrkL

preferentially activate the small GTPase Rac1, whereas variantslacking a functional C-terminal SH3 domain, including CrkI,preferentially activate Rap1. Thus, differences in the activityof Crk isoforms, including their effectors and their ability tobe downregulated by phosphorylation, are important forcoordinating dynamic changes in the actin cytoskeleton inresponse to extracellular signals.

Supplementary material available online athttp://jcs.biologists.org/cgi/content/full/122/22/4228/DC1

Key words: SH2 domain, SH3 domain, Focal adhesion dynamics,PDGF, Small GTPase

Summary

Distinct roles for Crk adaptor isoforms in actinreorganization induced by extracellular signalsSusumu Antoku* and Bruce J. Mayer‡

Raymond and Beverly Sackler Laboratory of Genetics and Molecular Medicine, Department of Genetics and Developmental Biology, University of Connecticut Health Center, Farmington, CT 06030-3301, USA*Present address: Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA‡Author for correspondence (bmayer@neuron.uchc.edu)

Accepted 15 September 2009Journal of Cell Science 122, 4228-4238 Published by The Company of Biologists 2009doi:10.1242/jcs.054627

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JCS ePress online publication date 27 October 2009

4229Role of Crk adaptor isoforms in PDGF signaling

exert their biological effects by interacting with various proteinsthrough their SH2 and nSH3 domains. Key partners include thep130 Crk-associated substrate (p130Cas) and paxillin, both ofwhich are FA proteins that bind the SH2 domains of Crk adaptorswhen phosphorylated (Birge et al., 1993; Sakai et al., 1994). Crkadaptors also bind via their nSH3 domains and activate severalguanine nucleotide exchange factors (GEFs) for small GTPases,including Dock180 and C3G/GRF2 (Hasegawa et al., 1996;Knudsen et al., 1994; Tanaka et al., 1994). In addition, Crk familyadaptors associate via their nSH3 domains with Abl family non-receptor tyrosine kinases, which in turn can phosphorylate CrkIIand CrkL on the regulatory site (Knudsen et al., 1994). Many ofthese Crk family binding partners have been implicated inPDGFR signaling (Cote et al., 2005; Plattner et al., 1999; Riveraet al., 2006; Voss et al., 2003), and CrkII and CrkL are rapidlytyrosine phosphorylated after PDGF stimulation of cells(Matsumoto et al., 2000; Sorokin et al., 1998). Despite theseobservations, the functional roles of Crk family proteins duringPDGFR signaling remain obscure.

Here, we show that Crk family adaptors are required for FAformation, and for PDGF-stimulated rearrangement of the actincytoskeleton and cell migration. We also show that inactivation ofCrk signaling following PDGF stimulation is important for propercoordination of FA disassembly and to prevent an extended periodof actin-cytoskeleton rearrangement. Our results also reveal a novelrole for the Crk cSH3 domain in promoting Rac1 activation, whereasRap1 activation is favored in the absence of cSH3. These resultshighlight essential roles for Crk family adaptors in PDGFRsignaling, and differences in the biological activity of CrkI comparedto CrkII and CrkL.

ResultsCrk family adaptors are required for PDGF-stimulatedformation of circular dorsal membrane ruffles and cell motilityTo determine whether Crk family adaptors play an essential role inPDGFR signaling, CrkL, CrkI/II or both Crk family adaptorproteins were knocked down by vector-based siRNA in NIH-3T3fibroblasts, and the responses to PDGF-bb (PDGF) stimulationexamined. In these experiments, protein expression was decreasedby approximately 90% for CrkI, 85% for CrkII and 99% for CrkL,without affecting the levels of PDGFR (Fig. 1B).

We first examined rearrangements of the actin cytoskeleton,specifically the formation of circular dorsal ruffles. These are ring-shaped actin structures on the dorsal surface of the cell, whichoriginate at the cell periphery and propagate in a wave-like fashioninwards, ultimately leading to membrane internalization (Fig. 1C)(Heldin et al., 1998; Orth and McNiven, 2006). When stimulatedwith PDGF for 10 minutes, CrkI/II- or CrkL-knockdown cellsformed circular dorsal membrane ruffles to a similar extent as wild-type (wt) cells (Fig. 1C,D). By contrast, the percentage of CrkI/II/L-knockdown cells forming PDGF-induced circular dorsal ruffles wasreduced by ~65% (Fig. 1B,C). These results indicate that PDGF-stimulated formation of dorsal membrane ruffles is dependent onCrk family proteins, and that CrkI/II and CrkL have overlappingfunctions.

We next examined the role of Crk family adaptors in PDGF-stimulated cell migration, which is driven by reorganization of theactin cytoskeleton. Utilizing a wound-healing assay, we observedthat the PDGF-stimulated increase in migration was significantlylower in CrkI/II- or CrkL-knockdown cells than in wt cells, despiteno apparent reduction in basal motility (Fig. 1E,F). CrkI/II/L-

knockdown cells displayed a complete loss of PDGF-enhanced cellmigration as well as diminished basal motility. These resultsindicate that Crk family adaptors play a crucial role, not only inPDGF-stimulated cell motility, but also in regulation of cellmovement in general. Taken together, our results indicate that Crkfamily adaptors are required for linking PDGFR signaling to theactin cytoskeleton, and that in this role CrkI/II and CrkL are for themost part redundant.

PDGF stimulation induces downregulation of Crk adaptors byAbl family tyrosine kinasesPDGF stimulation of fibroblasts has been shown to induce tyrosinephosphorylation of CrkII and CrkL (Matsumoto et al., 2000;Sorokin et al., 1998). We next looked at which tyrosine kinase isresponsible for this. The Abl family of tyrosine kinases, consistingof c-Abl and Arg, are known to phosphorylate CrkII and CrkL invarious biological situations (Chodniewicz and Klemke, 2004). Wetherefore used RNA interference to test whether Abl kinasesphosphorylate CrkII and CrkL upon PDGF stimulation.Simultaneous knockdown of Abl and Arg (each to 10-15% ofcontrols) resulted in a significant decrease in PDGF-inducedphosphorylation of CrkII and CrkL, whereas the knockdown of Ablor Arg alone had no apparent effect (Fig. 2A,B). Thus,phosphorylation of CrkII and CrkL induced by PDGF stimulationis chiefly controlled by Abl family kinases.

Previously, SH2 domains of Crk family adaptors have beenshown to bind to the tyrosine-phosphorylated PDGFR(Matsumoto et al., 2000). We re-examined this using an SH2-domain overlay assay (Nollau and Mayer, 2001; Rivera et al.,2006). The SH2 domains of the p85 PI-3 kinase subunit, whichis known to interact with the activated PDGFR (McGlade et al.,1992), bound strongly to the PDGFR band co-migrating withthe 182-kDa marker supplementary material Fig. S1A). Bycontrast, the SH2 domains of CrkI/II and CrkL boundpreferentially to bands of approximately 120 and 70 kDa, probablycorresponding to p130Cas and paxillin, respectively (Birge et al.,1993; Machida et al., 2007; Sakai et al., 1994). These interactionsshowed a biphasic pattern, characterized by a small increase inbinding soon after PDGF stimulation followed by a rapid decreaseto below the level of binding in unstimulated cells (supplementarymaterial Fig. S1A).

Co-immunoprecipitation experiments also demonstrated a stronginteraction of Crk with p130Cas but not with PDGFR (Fig. 2C).We found that co-immunoprecipitation of p130Cas had decreasedby more than 95% at 12 minutes after PDGF stimulation in bothCrkII and CrkL immunoprecipitates (Fig. 2C, supplementarymaterial Fig. S1B). This decrease is due to tyrosine phosphorylationof CrkII and CrkL, because when complex formation with p130Caswas examined for an unphosphorylatable form of CrkII (CrkIIY221F), no decrease in the interaction with p130Cas was observedafter PDGF stimulation (supplementary material Fig. S1C). We alsoobserved that the tyrosine phosphorylation of p130Cas decreasedin concert with the dissociation of CrkII and CrkL after PDGFstimulation (Fig. 2C, supplementary material Fig. S1B). Presumably,p130Cas phosphotyrosine sites become exposed to phosphataseswhen the Crk SH2 domain disengages. Consistent with this, wehave observed that overexpression of the Crk SH2 domain issufficient to increase the tyrosine phosphorylation of p130Cas (ourunpublished results).

Together, these results indicate that PDGF stimulation promotesthe tyrosine phosphorylation of CrkII and CrkL, mediated by Abl

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family tyrosine kinases, leading to disruption of the Crk-p130Cascomplex and decreased p130Cas phosphorylation. These results areconsistent with a functional role for Crk family adaptors in PDGFRsignaling.

Crk family adaptors are required for FA formation and forPDGF-stimulated FA disassemblyDisassembly of FAs is important for cell motility (Ridley et al.,2003), and is observed after PDGFR activation, in concert withthe loss of actin stress fibers (Heldin et al., 1998; Ruusala et al.,2008). Crk family adaptors have been strongly implicated in FAformation (Antoku et al., 2008; Lamorte et al., 2003; Nievers etal., 1997). Therefore, we next examined the role of Crk in FAdynamics in PDGF-stimulated cells. FAs were visualized byimmunofluorescent staining with antibodies to vinculin, and weobserved that the prominent FAs seen in NIH-3T3 cells under serumstarvation quickly disassembled following PDGF stimulation (Fig.3A-C). We also consistently observed a decrease in pY397 Fak andto a lesser extent in pY118 paxillin, which are indirect biochemical

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indicators of FA formation, following PDGF stimulation (Fig. 2C,supplementary material Fig. S1B).

In contrast to wt cells, in CrkI/II/L-knockdown cells the numberof FAs was drastically reduced in unstimulated cells (Fig. 3A). Thisindicates that Crk family adaptors are important for FA formationor maintenance in fibroblasts. Despite the much lower number ofFAs present in CrkI/II/L-knockdown cells, quantitative imageanalysis (described in Materials and Methods) indicated that thoseFAs still disassembled in response to PDGF stimulation (Fig. 3C).

To explore the function of individual Crk domains and motifs inFA dynamics, we expressed RNAi-resistant Crk variants (fused toEYFP) in CrkI/II/L-knockdown cells (supplementary material Fig.S2A). In agreement with our result showing redundancy betweenendogenous CrkII and CrkL (Fig. 1), re-expression of either wt CrkIIor CrkL was sufficient to rescue FA formation in CrkI/II/L-knockdown cells under serum starvation (Fig. 3A). When these cellswere stimulated with PDGF, FAs disassembled (Fig. 3A,C) and thelocalization of CrkII and CrkL to FAs was lost (Fig. 3B,supplementary material Movies 1-4), as seen in wt cells. We also

Fig. 1. Crk family adaptors are required forformation of circular dorsal-membraneruffles and increased cell migration afterPDGF stimulation. (A)The domainstructure of Crk family adaptors. CrkI andCrkII have a PxxP motif in the SH2 domainthat is absent in CrkL. Y represents theregulatory tyrosine phosphorylation sitelocated at position 221 for human CrkII andposition 207 for human CrkL. Numbersbelow domains show percent identitybetween CrkII and CrkL for each domain,and numbers in parentheses show similarity.(B)Expression levels of Crk family proteinsdetermined by immunoblotting for NIH-3T3 cells infected with retrovirus carryingpSUPER vector only, or one with shRNAtargeting CrkL, CrkI/II or all Crk familygenes. Numbers to the left of blots indicaterelative molecular mass in kDa. (C)Serum-starved cells were fixed before or 10minutes after stimulation with 24 ng/mlPDGF. Fixed cells were stained withHoechst 33342 and phalloidin–Texas-redfor DNA (blue) and F-actin (red),respectively. Scale bar: 50mm. (D)Numberof cells forming circular dorsal membraneruffle(s) and total cells and were counted formultiple fields. Graph represents mean ±s.e.m. of percentage of cells with circulardorsal-membrane ruffle(s). ‡P<0.05 (n15).(E)Wounds were introduced on the surfaceof confluent dishes of serum-starved cells.After washing, 0.1% CS-containingmedium with or without 24 ng/ml PDGFwas added to the dish. Images were takenimmediately or 12 hours after wounds wereintroduced. Scale bar: 200mm. (F)Cellsmigrating more than 200mm from the initialedge of wound were counted 12 hours afterwound introduction. The number wasnormalized relative to control cells underserum starvation. This value is referred to asthe migration index. Graph represents mean± s.e.m. of migration index. ‡P<0.05 (n9).

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4231Role of Crk adaptor isoforms in PDGF signaling

Fig. 2. Tyrosine phosphorylation ofCrkII and CrkL by Abl familyproteins disrupts interactions withp130Cas after PDGF stimulation.(A)NIH-3T3 cells were infectedwith retrovirus carrying vector onlyor expressing shRNA targeting Abl,Arg or both. Cells were harvestedbefore and at various time pointsafter PDGF stimulation. Whole-celllysates (WCL) were immunoblottedto detect tyrosine phosphorylatedCrkII and CrkL, and expression ofc-Abl and Arg. (B)Graphs representmean ± s.e.m. of CrkII and CrkLtyrosine phosphorylation in wt, andin Abl-, Arg- or Abl/Arg-knockdown cells before and atvarious time points after PDGFstimulation. For CrkII, pY-CrkIIwas divided by total CrkII. ForCrkL, pY-CrkL was normalized bypY-CrkL values in wt cells underserum starvation. ‡P<0.05 (n3).(C)NIH-3T3 cells were harvestedbefore and at various time pointsafter PDGF stimulation. WCL weresubjected to immunoprecipitation(IP) with antibodies against CrkII,CrkL and p130Cas. WCL andimmunoprecipitates wereimmunoblotted with variousantibodies as indicated.

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observed the disappearance ofother FA proteins includingpaxillin, p130Cas and Nck2from adhesion sites after PDGFstimulation (supplementarymaterial Fig. S2C). We foundthat the SH2 and nSH3 domainsof CrkII were required forrestoring FA formation inunstimulated CrkI/II/L-knockdown cells (see CrkIISH2* or CrkII nSH3*, Fig.3A,B). By contrast, the cSH3-domain mutant CrkII (CrkIIcSH3*) rescued FA formation(Fig. 3A,B), indicating that thecSH3 domain is not required forthis activity.

We previously showed thatCrkII and CrkL are highlytyrosine-phosphorylated afterPDGF stimulation, resulting ininactivation. Therefore, we nextaddressed the importance of Crkphosphorylation in PDGF-stimulated FA disassembly. Theunphosphorylatable form ofCrkII (CrkII Y221F), whichcannot adopt the inactiveconformation, rescued FAformation under serumstarvation. However, in CrkII-Y221F-expressing cells, theseFAs following PDGFstimulation persistedsignificantly longer than theones from wt cells or CrkI/II/L-knockdown cells re-expressingwt CrkII (Fig. 3A-C). CrkI, thesplicing variant of CrkII, cannotbe regulated by tyrosine phosphorylation either and is unable toadopt the inactive conformation. Cells expressing CrkI showed asimilar phenotype to those expressing CrkII Y221F, because FAswere formed under serum starvation and these FAs persisted longerafter PDGF stimulation (Fig. 3A-C). Furthermore, EYFP fusionproteins of both CrkII Y221F and CrkI persisted in FAs longer thandid wt CrkII after PDGF stimulation (Fig. 3B, supplementarymaterial Movies 3, 5, 6). This is much more obvious in the moviesof living cells than in fixed cells. Thus, the phosphorylation-dependent inactivation of CrkII and CrkL is important for thecoordinated FA disassembly after PDGF stimulation

Tyrosine phosphorylation of CrkII and CrkL is required for‘closure’ of dorsal membrane ruffles induced by PDGFstimulationNext, we extended the strategy of gene silencing combined withmutational analysis to study the Crk-dependent regulation of dorsal-membrane ruffle dynamics. Consistent with the functionalcompensation between endogenous CrkII and CrkL (Fig. 1), re-expression of either CrkII or CrkL rescued PDGF-induced formationof circular dorsal-membrane ruffles in CrkI/II/L-knockdown cells

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(Fig. 3A,D). In addition, re-expression of CrkI and CrkII cSH3*rescued circular dorsal-membrane ruffles after PDGF stimulation,indicating that the cSH3 domain is not required for this activity;however, neither SH2- or nSH3-domain mutants of CrkII couldrescue formation of dorsal-membrane ruffles (Fig. 3A,D). Thus,the SH2 and nSH3 domains of Crk adaptors are strictly requiredfor PDGF-stimulated actin rearrangements.

Cells expressing the unphosphorylatable form of CrkII (CrkIIY221F) exhibited cytoskeletal rearrangements in response to PDGF.However, actin puncta at the cell periphery (resembling the earlystages of dorsal-ruffle formation) persisted for an extended periodwithout progressing to the wave-like contraction and closure typicalof circular dorsal ruffles (Fig. 3A,B, supplementary material Movie6). This suggests that phosphorylation of CrkII and CrkL isimportant for the normal maturation of circular dorsal-membraneruffles. Although CrkI cannot adopt the inactive conformation, cellsre-expressing CrkI were similar to those expressing wt CrkII withrespect to PDGF-stimulated formation of circular dorsal-membraneruffles. The difference between CrkI and CrkII Y221F is thepresence of the cSH3 domain. Therefore, we introduced a mutationin the cSH3 domain of CrkII Y221F, and its effects on the formation

Fig. 3. See next page for legend.

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4233Role of Crk adaptor isoforms in PDGF signaling

Fig. 3. Role of domains and regulatorytyrosine of Crk adaptors in FA formation andPDGF-stimulated actin rearrangement. NIH-3T3 cells were infected with retroviruscarrying pSUPER vector only or oneexpressing shRNA targeting all Crk familygenes. Cells were super-infected with MSCV-puro-derived retrovirus carrying EYFP orEYFP fused with wt or mutant Crk insensitiveto shRNA. (A,B) Serum-starved cells werefixed before or 7.5 minutes after stimulationwith 18 ng/ml PDGF. Fixed cells were stainedwith anti-vinculin–AlexaFluor 647,phalloidin–Texas-red, and Hoechst 33342 forvinculin, F-actin, and DNA, respectively.(A)Cell images are shown with vinculin(green), F-actin (red) and DNA (blue). Scalebar: 50mm. (B)These images were enlargedfrom A. EYFP or EYFP fusion proteins aredepicted in green and vinculin in red. Scalebar: 20mm. (C)The area occupied by FAs(discrete spots visualized by vinculinstaining) and total number of cells werequantified from fluorescent cell images.Graph represents mean ± s.e.m. of the relativechange of area occupied by FAs in cellsbefore and after PDGF stimulation. ‡P>0.05(n3). (D)The numbers of total cells and cellsforming circular dorsal-membrane ruffle(s)were counted from fluorescent cell images.Graph represents mean ± s.e.m. ofpercentages of cells with circular dorsal-membrane ruffle(s). ‡P<0.05 (n12).

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and structure of circular dorsal-membrane ruffles were examined.In CrkI/II/L-knockdown cells expressing this new mutant, CrkIIY221F cSH3*, circular dorsal ruffles with normal dynamics wereobserved (Fig. 3A,D). These results imply that although the cSH3domain is not required for dorsal-membrane-ruffle formation, itspresence in active (uninhibited) Crk prolongs actin cytoskeletonrearrangements and prevents normal maturation of circular dorsalruffles.

We also examined the importance of Crk for biochemicalsignaling pathways downstream of PDGF stimulation. We foundthat in CrkI/II/L-knockdown cells, PDGF-dependent activation ofthe MAP kinases Erk1/2 was blunted, particularly at low levels ofPDGF stimulation (supplementary material Fig. S3). By contrastthere was no apparent effect of knockdown on PDGF-dependentactivation of Akt. Thus, in addition to its crucial role in regulatingFAs and actin rearrangements, Crk adaptors play a supporting rolein regulating some pro-proliferative responses in PDGFRsignaling.

The cSH3 domain directs CrkII to activate Rac1 instead ofRap1The nSH3 domain of Crk interacts with Dock180, a Rac1 GEF, andthus can promote an increase in active (GTP-bound) Rac1(Hasegawa et al., 1996; Kiyokawa et al., 1998). Activation of Rac1is thought to be required for PDGF-induced formation of dorsalruffles (Ridley et al., 1992). Thus, we hypothesized that Crk familyadaptors induce actin-cytoskeleton rearrangement through Rac1activation in PDGFR signaling. Indeed, using a pulldown assay,we could demonstrate that activation of Rac1 after PDGFstimulation was severely impaired in CrkI/II/L-knockdown cells(Fig. 4, supplementary material Fig. S3A). This indicates that Crkfamily adaptors are important for Rac1 activation in PDGFRsignaling.

To further analyze how Crk adaptors regulate Rac1 activity duringPDGF stimulation, Rac1 activation was examined in CrkI/II/L-knockdown cells expressing wt or mutant Crk adaptors. In cells re-expressing either wt CrkII or CrkL, Rac1 was strongly activated 3minutes after PDGF stimulation, and then returned nearly to basallevels at 15 minutes (Fig. 4). In cells expressing the CrkII SH2* ornSH3*mutants, which failed to rescue formation of PDGF-induceddorsal ruffles, Rac1 was only modestly activated 3 minutes afterPDGF stimulation. Thus, CrkII appears to activate Rac1 bytransducing signals through its SH2 and nSH3 domains to induceactin cytoskeleton rearrangements during PDGFR signaling. TheCrkII Y221F mutant, which induced ‘persistent’ membrane ruffles(Fig. 3A, supplementary material Movie 6), also induced persistentRac1 activation (Fig. 4). This suggests that inactivation of CrkIIby tyrosine phosphorylation is required for termination of Rac1signaling shortly after PDGF stimulation.

To our surprise, Rac1 activation was severely impaired inCrkI/II/L-knockdown cells expressing CrkI or CrkII cSH3*, bothof which lack a functional cSH3 domain, although formation ofdorsal ruffles proceeded normally (Fig. 4). As an alternative to Rac1,Crk adaptors can activate the Rap1 small GTPase through anothernSH3-binding partner, C3G (Gotoh et al., 1995; Knudsen et al.,1994; Tanaka et al., 1994). Moreover, Rap1 is known to activatevarious downstream effectors including ARAP3, whose activationhas been shown to promote membrane ruffles (Krugmann et al.,2006; Krugmann et al., 2004). Thus, we tested the hypothesis thatCrkI and CrkII cSH3* induce membrane ruffles through Rap1activation in a Rac1-independent manner. Strikingly, in knockdown

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cells expressing CrkI or CrkII cSH3*, Rap1 activation was four-to fivefold higher at 3 minutes after PDGF stimulation comparedwith approximately twofold in wt cells or in cells expressing CrkIImutants with a functional cSH3 domain (Fig. 4). These resultssuggest that the cSH3 domain enables Crk adaptors to selectivelyactivate Rac1 instead of Rap1. Moreover, these results imply thatin PDGFR signaling, CrkI induces functional dorsal-membraneruffles via Rap1 activation in the apparent absence of Rac1activation.

DiscussionIn this study, the role of Crk family adaptors in PDGFR signalingwas investigated by a combination of gene silencing and mutationalanalysis. Our results demonstrate that Crk family adaptors play afunda mental role in FA formation and turnover in fibroblasts. Wealso demonstrated that Crk family adaptors are required forformation of circular dorsal-membrane ruffles and for promotingcell migration downstream of the PDGFR. In this pathway, CrkIIand CrkL showed overlapping functions and therefore dosagedependency. This redundancy among Crk family adaptors has beenalso observed in the integrin and reelin signaling pathways (Antokuet al., 2008; Matsuki et al., 2008; Park and Curran, 2008). In additionto positive roles for the Crk adaptors in the initiation of PDGFRsignaling, phosphorylation-mediated inhibition of CrkII and CrkLby Abl family tyrosine kinases is important for disassembly of FAsand for termination of reorganization of the actin cytoskeleton afterPDGF stimulation. Furthermore, we uncovered a novel role of the

Fig. 4. cSH3 domain and tyrosine phosphorylation of Crk are crucial forcoordinated activation of Rac1 and Rap1 after PDGF stimulation. NIH-3T3cells infected with retrovirus carrying pSUPER vector only or one withshRNA targeting all Crk family genes were super-infected with MSCV-puro-derived retrovirus expressing EYFP or EYFP fused with various types of Crk.Cells were harvested before and at 3 and 15 minutes after PDGF stimulation.WCL were subjected to GTPase assays. The amounts of Rac1 and Rap1 inpulldown fractions and their corresponding WCL were detected byimmunoblotting. The amounts of GTP-bound relative to total GTPase werenormalized to levels in wt cells under starvation. These values were used toobtain relative GTPase activities, and the means ± s.e.m. are represented byheat map graph. 1‡ vs 2‡ and 1¶ vs 2¶ in each column represent P<0.05 andP0.065, respectively (n3).

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4235Role of Crk adaptor isoforms in PDGF signaling

cSH3 domain in directing Crk adaptors to activate Rac1 at theexpense of Rap1 activation.

Regulation of FA dynamics by Crk adaptorsWe have shown that Crk family adaptors are essential for normalFAs (Fig. 3A,B); the few FAs seen in Crk family knockdown cellsare probably due to incomplete silencing of Crk expression. Thisobservation is consistent with previous studies showing thatoverexpression of v-Crk or Crk adaptors promotes FA formation(Antoku et al., 2008; Lamorte et al., 2003; Nievers et al., 1997).We also previously showed that Crk family adaptors are requiredfor FA formation by fibroblasts in the early stages of cell spreadingonto fibronectin-coated surfaces (Antoku et al., 2008). After PDGFstimulation, however, FAs rapidly disassemble (Fig. 3A-C), inconcert with the phosphorylation and functional inactivation of CrkIIand CrkL by Abl family kinases (Fig. 2). By contrast, expressionof an unphosphorylatable form of CrkII in CrkI/II/L-knockdowncells significantly slowed the disassembly of FAs stimulated byPDGF (Fig. 3A-C). Crk adaptors have been considered enhancersof FA formation (Feller, 2001; Nievers et al., 1997). Now, wereconsider this idea and propose that, rather than being merelyenhancers, Crk adaptors are essential components of FAs and arecrucial regulators of their assembly and disassembly. This modelis supported by studies by us and others, showing that changes intyrosine phosphorylation of Crk caused by modulating Abl activitytightly correlate with the number and size of FAs formed in cells(Antoku et al., 2008; Peacock et al., 2007).

Several lines of evidence suggest mechanisms whereby Crkadaptors could promote FA formation by mediating the assemblyof protein complexes. Fibroblasts lacking the major Crk SH2-binding partner p130Cas exhibit slow assembly of FAs (Antoku etal., 2008; Honda et al., 1998). Fibroblasts lacking the Crk nSH3-binding partner C3G show severely reduced FAs (Voss et al., 2003).Moreover, formation of the p130Cas-Crk-C3G complex has beensuggested to promote FA formation (Li et al., 2002). We show herethat dissociation of p130Cas from CrkII and CrkL coincides withthe disassembly of FAs that is induced by PDGF (Fig. 2C, Fig.3A,C, supplementary material Fig. S1B,C). Alternatively, Crkadaptors interact with the paxillin-GIT2--PIX complex, whichenhances formation of FAs (Lamorte et al., 2003).

One possible downstream effector of these complexes forregulation of FAs is the small GTPase RhoA. RhoA is a crucialregulator of FA dynamics, and its activity increases after PDGFstimulation, followed by a rapid decrease (Ruusala et al., 2008).ARAP3 is a GTPase activating protein (GAP) for RhoA and Arf6(Krugmann et al., 2002). This GAP is regulated by C3G throughanother small GTPase, Rap1 (Gotoh et al., 1995; Krugmann et al.,2004). Thus, Crk complexes might ultimately regulate FA dynamicsthrough a pathway that includes Rap1 and RhoA. However, we hadtechnical difficulties quantifying RhoA activity by GTPase pulldownassay, and thus were unable to assess the involvement of RhoA inCrk-mediated FA dynamics. This remains to be clarified by futurestudies.

Regulation of actin reorganization by Crk adaptorsWe showed that Crk adaptors are essential for the Rac1-dependentreorganization of the actin cytoskeleton that is induced by PDGFstimulation (Fig. 1B,C; Figs 3,4; supplementary material Fig.S3A). Interestingly, expression of CrkII Y221F in CrkI/II/L-knockdown cells resulted in prolonged dorsal ruffling and Rac1activation (Fig. 3A,B; Fig. 4, supplementary material Movie 6).

These results indicate that the functional inactivation of Crkadaptors (and thus downregulation of Rac1 activity) also plays animportant role in coordinating rearrangement of the actincytoskeleton. Furthermore, our data suggest that the activity of Crkadaptors is required only in the early period of PDGFR signaling.

Previously, the SH2 domains of Crk adaptors have been shownto bind to tyrosine-phosphorylated PDGFR (Matsumoto et al.,2000). However, in this study we did not detect specific interactionbetween Crk adaptors and PDGFR using SH2-domain overlayassay and co-immunoprecipitation (Fig. 2C, supplementarymaterial Fig. S1A). Instead, we observed strong binding of Crkadaptors to p130Cas (Fig. 2C, supplementary material Fig. S1B).p130Cas has 15 YxxP motifs, nine of which are YDxP sequencesfor which the Crk SH2 domain has strong affinity whenphosphorylated (Shin et al., 2004). Thus, it is probable that Crkadaptors are recruited to the membrane after PDGF stimulationthrough binding of their SH2 domains to p130Cas. We havepreviously shown that the interaction of Nck family adaptors, viatheir SH2 domains, with p130Cas is essential for efficient dorsal-membrane ruffle induction by PDGF (Rivera et al., 2006). LikeCrk SH2 domains, Nck SH2 domains have high affinity for YDxPmotifs (Frese et al., 2006). Thus, p130Cas is likely to play a centralrole in PDGF-mediated actin reorganization by recruiting both Crkand Nck to the plasma membrane, where active cytoskeletonrearrangement takes place. During this step, downstream effectorproteins bound to the Crk nSH3 domain are recruited to thecomplex. This is the key function for adaptor proteins, and explainswhy both SH2 and nSH3 domains are required for Rac1 activationand for rearrangement of the actin cytoskeleton during PDGFstimulation (Figs 3, 4). Nck is also required for the activation ofRac1 during PDGF signaling (Rivera et al., 2006), though its keydownstream effectors are unlikely to be identical to those of Crk;possible candidates for Nck effectors include Abl kinases andWASP family promoters of actin nucleation (Antoku et al., 2008;Rivera et al., 2004).

A possible downstream effector for Crk adaptors is the Rac1 GEFDock180, which is recruited to the plasma membrane after PDGFstimulation (Cote et al., 2005). In addition to the p130Cas-CrkII/L-Dock180 complex, the paxillin-CrkII-Dock180 complex has beenshown to enhance cell migration through Rac1 activation (Valleset al., 2004). Thus, this complex might partly contribute to Rac1activation in Crk-mediated PDGFR signaling. Crk adaptors alsobind another GEF, Sos, through their nSH3 domains (Feller et al.,1995). It is widely accepted that Sos is regulated by Grb2 andfunctions as a Ras GEF during growth-factor receptor signaling(Chardin et al., 1993; Nimnual and Bar-Sagi, 2002; Skolnik et al.,1993). However, Sos also has GEF activity toward Rac1 (Innocentiet al., 2002; Nimnual and Bar-Sagi, 2002), and therefore we cannotrule out the possibility that Sos contributes to Rac1 activation byCrk adaptors.

Unexpectedly, our mutational analysis also revealed that Crkadaptors lacking a functional cSH3 domain were unable to activateRac1, but could still rescue formation of dorsal ruffles in responseto PDGF (Figs 3, 4). We found that these forms of Crk stronglyactivated Rap1 instead of Rac1 during PDGF stimulation (Fig. 4).By contrast, Crk adaptors carrying a functional cSH3 domain didnot dramatically increase Rap1 activity (Fig. 4). A previous studysuggested that Rap1 activation itself results in Rac1 activation(Arthur et al., 2004). We did not detect Rap1-mediated Rac1activation during PDGFR signaling, though we cannot rule outlocalized increases in Rap1 activity in vivo. Fibroblasts lacking

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the Rap1 GEF C3G are deficient in formation of dorsal-membraneruffles after PDGF stimulation (Voss et al., 2003), consistent withan important role for Rap1 in this process. Overexpression orknockdown of ARAP3, a downstream effector of Rap1, was shownto induce cytoskeletal changes including altered peripheral ruffles(Krugmann et al., 2006), suggesting it might play a role downstreamof Rap1 in PDGF stimulated cells. It is interesting to note thatsustained Rap1 activation (as induced by CrkI) is compatible withnormal maturation of circular dorsal ruffles, whereas sustainedRac1 activation (as induced by CrkIIY221F) is not.

How might cSH3 direct activation of Rap1 instead of Rac1 in vivo?Biochemical and structural studies suggest two possible functions forthe cSH3 domain of Crk adaptors. First, the cSH3 domain reducesoverall affinity of the nSH3 domain towards its binding partners bylowering the accessibility of the binding pocket in the nSH3 domain(Kobashigawa et al., 2007; Sarkar et al., 2007). It is possible that thisconformation favors binding of certain effectors, such as C3G, at theexpense of others. Second, the cSH3 domain has been proposed topromote the homo-dimerization of Crk adaptors (Harkiolaki et al.,2006) and to promote nuclear export (Smith et al., 2002). Furtherstudies will be required to resolve this issue.

The selective activation of Rap1 by CrkI raises an interestingpoint about the distinct functional role of CrkI relative to CrkII andCrkL. In cells, the expression level of CrkI is generally much lowerthan that of CrkII (Matsuda et al., 1992). However, unlike CrkII,CrkI signaling cannot be turned off and, as we have shown, CrkIpreferentially activates Rap1 rather than Rac1. Thus, in resting cellsCrkII will initially dominate signaling; however, when CrkII islocally inhibited by signal-induced phosphorylation, CrkI signalingwill start to predominate at that site, albeit at lower levels. Thisprobably serves as a mechanism for cells to switch from Rac1 toRap1 signaling at specific sites, and might also play a role inshielding Crk SH2-binding proteins such as p130Cas and paxillinfrom total dephosphorylation.

ConclusionDynamic cell behaviors such as such as FA turnover and actin-cytoskeleton remodeling must be regulated precisely in time andspace. For example, in order for a cell to move, adhesions mustform at the leading edge, persist for a time and then disassemble.For a dorsal-membrane ruffle to form, contract and close, the zoneof active actin polymerization must move in a wave-like fashionfrom the cell periphery towards the center. Crk adaptors are wellsuited to play a central role in such behaviors, because they canmediate the assembly of protein complexes in response to tyrosinephosphorylation and then be rapidly downregulated by their ownphosphorylation. Presumably, engagement of the SH2 domain byphosphorylated targets not only recruits key downstream effectors,such as Dock180 and C3G, but also recruits the Abl family kinasesthat will locally shut down signaling after a short delay. Such delayedfeedback loops are typical of biological systems with wave-like oroscillatory behavior (Tyson et al., 2003). Additional adaptors, suchas those of the Nck family, can also recruit Abl kinases as well asdifferent downstream effectors to these sites, and might further playa role both in downregulating the Crk signal and in switchingeffectors over time (Antoku et al., 2008; Rivera et al., 2006). Thepresence of low levels of unphosphorylatable CrkI further enrichesthe dynamic behavior of the system by providing a lower limit toactivity, even after the most of the Crk signaling has beendownregulated. Multiple species of Crk family adaptors, whichdiffer in their activity and effectors, allow the cell to orchestrate

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precise spatiotemporal responses downstream of PDGFRactivation.

Materials and MethodsAntibodiesAntibodies directed against Abl (8E9, BD Pharmingen), CrkI/II (BD TransductionLaboratories), pY207-CrkL (Cell Signaling), CrkL (C-20, Santa Cruz Biotechnology),phosphotyrosine (P-Tyr-100, Cell Signaling Technology), vinculin (VIN-11-5, Sigma-Aldrich), pY397-Fak (44-624G, Invitrogen), Fak (C-20, Santa Cruz Biotechnology),pY118-paxillin (Cell Signaling), paxillin (BD Transduction Laboratories), p130Cas(BD Transduction Laboratories), Rac1 (23A8, Millipore), Rap1 (121, Santa CruzBiotechnology), p-Erk1/2, pT202/pY204 (Cell Signaling), Erk2 (D-2, Santa CruzBiotechnology), pS473-Akt (Cell Signaling), PDGFR (958, Santa CruzBiotechnology) EGFP/EYFP (FL, Santa Cruz Biotechnology) and actin (I-17, SantaCruz Biotechnology) were purchased from commercial sources. Antibodiesrecognizing Arg (AR11) and pY221-CrkII were generous gifts from Peter Davies(Albert Einstein College of Medicine, Bronx, NY) and Michiyuki Matsuda (KyotoUniversity, Kyoto, Japan), respectively.

PlasmidsThe short hairpin RNA (shRNA)-knockdown construct for CrkI/II/L was previouslydescribed (Antoku et al., 2008). The sequences for shRNA knockdown constructstargeting mouse CrkL (generous gifts from Nathaniel Allen and Jonathan Cooper,Fred Hutchinson Cancer Research Center, Seattle, WA), CrkI/II, Abl, Arg or Abl/Argwere; 5�-GGGCGAGCTTCTAGTGATAAT-3� (CrkL); 5�-GTGGAGTG ATT CT -CAGGCA-3� (CrkI/II); 5�-TGAGCTATGTGGACTCTAT-3� (Abl); 5�-GGAGC -CAAATTTCCTATTA-3� (Arg); or 5�-GAGTACTTGGAGAAGAAGA-3� (Abl/Arg),respectively. These shRNA sequences were inserted into the pSUPER.retro vector(Oligoengine). The cDNAs encoding human CrkI, CrkII, CrkL and Nck2; mousepaxillin; and rat p130Cas were previously described (Antoku et al., 2008; Rivera etal., 2006; Sharma et al., 2003). The cDNAs encoding EYFP and -actin were frompEYFP-C1 actin (Clontech). The cDNAs encoding human CrkII Y221F; mCherrythat was used for making pMSCV-puro (Clontech) mCherry-actin; and cerulean thatwas used for making pSUPER.retro.cerulean were generous gifts from MichiyukiMatsuda, Roger Tsien (UCSD, La Jolla, CA), and Dave Piston (Vanderbilt University,Nashville, TN) respectively. The shRNA-insensitive cDNAs were generated bychanging nucleotides G381C, C387G, G540T, C549A for CrkI and CrkII; and G513T,T522A, C525G and T528G for CrkL. CrkII SH2*, CrkII nSH3* and CrkII cSH3*were generated by changing the amino acid sequence of shRNA-insensitive CrkII toR38L, W169L and W275L, respectively. All these mutations were generated bypolymerase chain-reaction-based mutagenesis using Pfu DNA polymerase(Stratagene). All cDNAs were inserted into pMSCV-puro vectors containing N-terminal EYFP or pMIGR-1 vector containing N-terminal FLAG epitope (Antoku etal., 2008; Pear et al., 1998). pGEX4T-3 Ral-GDS GBD plasmid was obtained fromMichiyuki Matsuda. pET-Pak1-PBD, pGEX-6P-1 Crk-SH2, pGEX-6P-1 CrkL-SH2and pGEX-6P-1 PI3K-SH2 plasmids were previously described (Antoku et al., 2008;Machida et al., 2007).

Cell culture, transfection, viral infection and PDGF stimulation ofcellsMaintenance of HEK293T and NIH-3T3 cells, virus production and viral infectionwere previously described (Antoku et al., 2008). For re-expression of Crk familyadaptors in CrkI/II/L-knockdown cells, NIH-3T3 cells were first infected withpSUPER.retro.puro CrkI/II/L-derived virus. At 48 hours post-infection, the cells werereplated. The next day, cells were super-infected with MSCV-puro-derived virus. At24 hours post-super-infection, the cells were replated either on cell culture dishes oron coverslips coated with 0.1% (w/v) gelatin (Sigma-Aldrich) in cell-culture dishes,and serum-starved for further experiments. At 18-24 hours after serum starvation,the cells were stimulated with PDGF-bb (Millipore) for various times, and eitherharvested with lysis buffer or fixed for further experiments.

BiochemistryCell lysis, immunoprecipitation, western blotting and far-western blotting werepreviously described (Antoku et al., 2008). Briefly, for immunoprecipitation, 0.35mg anti-CrkI/II or anti-p130Cas, or 0.6 mg anti-CrkL antibodies were immobilizedon Protein G- or Protein A-Sepharose (Pierce or GE Healthcare, respectively). TheCrkI/II antibody was further chemically cross-linked to beads using disuccinimidylsuberate (DSS) (Pierce), following the manufacturer’s instructions. The GTPasepulldown assay was performed as previously described (Azim et al., 2000).Immunoblot band intensities were quantified using ImageJ software (NIH, Bethesda,MD).

Immunofluorescent staining, assay of dorsal-membrane-ruffleformation and live-cell imagingPDGF-stimulated cells were fixed on coverslips by adding one-tenth total volume of10� PEM buffer (1 M PIPES pH 6.9, 10 mM MgCl2, 10 mM EGTA pH 8.0) and37% (w/v) paraformaldehyde (Sigma-Aldrich) for 5 minutes, and permeabilized with

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phosphate-buffered saline (PBS) containing 0.5% (v/v) NP-40 for 5 minutes. Thecoverslips were blocked with PBS containing 1% (w/v) BSA for 1 hour at roomtemperature. Fixed Crk-knockdown cells were stained with phalloidin conjugatedwith Texas-red (Invitrogen), and Hoechst 33342 (Sigma-Aldrich). Alternatively, fixedCrk-knockdown cells expressing EYFP or EYFP fusion proteins were stained withanti-vinculin followed by goat-anti-mouse secondary antibody conjugated withAlexa Fluor 647 (Invitrogen), phalloidin conjugated with Texas-red, and Hoechst33342. Photomicrographs were obtained on a Zeiss 510 laser-scanning invertedconfocal microscope with 1.3 NA oil immersion objective lens. For assay of dorsal-membrane-ruffle formation the images of stained cells were examined for the numberof cells exhibiting circular actin ring structures on the dorsal side and the total numberof cells in each field. Each field contained approximately 15-25 cells. Percentagesof cells with dorsal-membrane ruffles were calculated. For each sample, four to fivefields were examined and the percentages were averaged. The quantification of FAswas calculated from images using NIH ImageJ software as the area occupied byvinculin staining divided by the total number of cells visualized by DNA and F-actinstaining. For live-cell imaging, a coverslip was placed into an Attofluor chamber(Invitrogen) prior to PDGF stimulation. Before and after stimulation, cell imageswere taken using a Nikon TE2000 inverted spinning disc microscope using a NikonPlan Fluor 40� 1.3 NA oil immersion objective lens.

Wound-healing assayAfter serum starvation, vertical and horizontal wounds were introduced using a P200micropipet tip (USA Scientific) on a 35-mm tissue-culture dish whose surface wasfully occupied by cells. The plate was then washed three times with PBS, andDulbecco’s modified Eagle’s medium (DMEM) containing 0.1% (v/v) calf serumwith or without PDGF was added. Immediately or 12 hours after placing in themedium, images for three locations of a marked wound were taken by a Nikon D50digital SLR camera mounted on a Nikon Eclipse TS100 inverted phase contrastmicroscope with Nikon Plan Fluor 10� 0.3 NA objective lens. From the images, thenumber of cells that had migrated more than 200 mm into the wound were countedfor each sample.

Statistical analysisStatistical significance among samples for immunoblot quantification, wound healingassay and assay of dorsal-membrane-ruffle formation were determined by ANOVAfollowed by Tukey’s test. Statistical significance of FA formation before and afterPDGF stimulation was determined using the Student’s t-test. All statistical valueswere obtained from three independent experiments.

We thank Gonzalo Rivera for critical reading of the manuscript;Nathaniel Allen and Jonathan Cooper for generously providing anunpublished reagent, the CrkL shRNA plasmid; Roger Tsien and DavePiston for cDNAs of mCherry and Cerulean, respectively; Peter Daviesfor Arg antibody; and Michiyuki Matsuda for pY221-CrkII antibody,CrkII Y221F cDNA, and pGEX4T-3 Ral-GDS GBD plasmid. Thesestudies were supported by grant CA82258 from the National Institutesof Health (to B.J.M.). Deposited in PMC for release after 12 months.This paper is dedicated by B.J.M. to the memory of HidesaburoHanafusa, deeply respected scientist and beloved mentor.

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