1172 Research Article

IntroductionThe platelet-derived growth factor (PDGF) β receptor (PDGFβR;also known as PDGFRB) plays a key role in the proliferation andmotility of fibroblasts, vascular smooth muscle cells, capillaryendothelial cells and neurons. The binding of dimeric ligand, PDGF-BB, to the extracellular domain of the receptor induces dimerizationand activation of the receptor, resulting in autophosphorylation ofkey tyrosine residues in its cytoplasmic domain. Oncephosphorylated, these tyrosine residues become docking sites forimportant SH2 domain-containing substrates (reviewed by Heldinet al., 1998). These include the 85 kDa regulatory subunit (p85) ofphosphoinositide 3-kinase (PI3K), phospholipase Cγ (PLCγ), Srcfamily members, the protein tyrosine phosphatase SHP2, signaltransducers and activators of transcription (STATs), the Ras-GTPaseactivating protein (p120RasGAP), and several signaling adaptorssuch as Grb2, Shc and Nck. Once recruited to the receptor, thesesubstrates activate signaling pathways leading to cell proliferation,morphological changes and motility.

Sustained ligand-dependent activation of the PDGFβR hasoncogenic effects both in vitro and in vivo. Overexpression of thegene encoding the PDGF-B chain, c-sis, or its viral homolog, v-sis(Doolittle et al., 1983; Waterfield et al., 1983), promotestransformation of rodent fibroblast lines (Beckmann et al., 1988;Clarke et al., 1984). Similarly, uncontrolled expression of PDGF-B resulting in an autocrine loop for PDGFβR receptor activationis thought to play a role in the development of several humancancers, including glioblastomas, fibrosarcomas and menigiomas

(Betsholtz et al., 1989; Fleming et al., 1992; Heldin and Westermark,1999; Hermanson et al., 1992).

Sustained ligand-independent activation of the PDGFβR by theE5 protein of bovine papillomavirus (BPV) also results intransformation. E5 is a 44 amino acid transmembrane protein thatdimerizes via two cysteine residues (Cys37 and Cys39) located inits predicted extracellular domain (Burkhardt et al., 1989; Horwitzet al., 1988; Schlegel et al., 1986). E5 is capable of binding to andconstitutively activating the endogenous PDGFβR in rodent, bovineand human fibroblasts (Petti and DiMaio, 1992; Petti and DiMaio,1994; Petti et al., 1991; Petti and Ray, 2000). By itself, E5 cantumorigenically transform rodent fibroblast lines (Bergman et al.,1988; DiMaio et al., 1986; Schiller et al., 1986), and its transformingactivity depends on its ability to constitutively activate the PDGFβR(Nilson and DiMaio, 1993; Riese, 2nd and DiMaio, 1995). LikePDGF-BB, E5 binds to the PDGFβR as a dimer and therebypromotes receptor dimerization followed by receptorautophosphorylation and activation (Lai et al., 1998). However,accumulated genetic evidence indicates that E5 binds to thePDGFβR in a unique and ligand-independent manner, via multipleintermolecular transmembrane domain interactions (Klein et al.,1999; Klein et al., 1998; Nappi and Petti, 2002; Nappi et al., 2002;Nilson et al., 1995; Petti et al., 1997).

Previous studies reported on the requirement of particular PDGFβRsignaling substrates for transformation of immortal mouse fibroblastlines. Evidence from one study suggested that SHP2 and PLCγ weresufficient for loss of contact inhibition of NIH 3T3 fibroblasts induced

The platelet-derived growth factor β receptor (PDGFβR) playsan important role in proliferation and motility of fibroblasts.We have been investigating the effects of sustained PDGFβRactivation in mortal human diploid fibroblasts (HDFs), whichare typically difficult to transform. We have previously shownthat the bovine papillomavirus E5 protein, through its abilityto crosslink and constitutively activate the PDGFβR, inducesmorphological transformation, enhanced growth and loss ofcontact inhibition (focus formation) in HDFs. Here, wecharacterized two E5 mutants as being severely defective forfocus formation but still competent for enhanced growth,suggesting that proliferation is insufficient for loss of contactinhibition. These E5 mutants were then used in a comparativestudy to distinguish the PDGFβR signaling intermediatesrequired for the enhanced growth phenotype from those

required for focus formation. Our data suggested that a PI 3-kinase (PI3K)-AKT-cyclin D3 pathway, a Grb2-Gab1-SHP2complex and JNK played a role in the enhanced growthphenotype. However, a SHP2-p66Shc-p190BRhoGAP complexand ROCK were implicated exclusively in focus formation. Wespeculate that a SHP2-p66Shc-p190BRhoGAP signalingcomplex recruited to the activated PDGFβR promotes a distinctRho-dependent process required for focus formation but notgrowth of HDFs.

Supplementary material available online athttp://jcs.biologists.org/cgi/content/full/121/8/1172/DC1

Key words: PDGF receptor, Signaling, Transformation

Summary

Transforming signals resulting from sustainedactivation of the PDGFβ receptor in mortal humanfibroblastsLisa M. Petti*, Elizabeth C. Ricciardi, Heather J. Page and Kristen A. PorterCenter for Immunology and Microbial Disease, Albany Medical College, MC-151, 47 New Scotland Avenue, Albany, NY 12208, USA*Author for correspondence (e-mail: pettil@mail.amc.edu)

Accepted 14 January 2008Journal of Cell Science 121, 1172-1182 Published by The Company of Biologists 2008doi:10.1242/jcs.018713

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by continuous PDGF expression (Uren et al., 1996). Asecond study reported that either PLCγ or PI3K wasrequired for PDGF-induced anchorage independentgrowth (DeMali et al., 1997). A third report suggestedthat PLCγ, SHP2, Shc and Nck were required forPDGF-stimulated DNA synthesis in NIH 3T3 cells(Roche et al., 1996). One limitation of these studies isthat they used mouse fibroblast lines, which are proneto transformation and may not use the signalingpathways that play a role in human cancers. Thus, thePDGFβR signaling substrates identified as beingimportant for transformation in these studies may notbe the same as those required for transformation ofnormal human fibroblasts.

We have been investigating the effect of sustainedPDGFβR activation by BPV E5 in young mortalhuman diploid fibroblasts (HDFs). Compared withrodent fibroblast lines, HDFs represent a morephysiologically relevant system to study mechanismsof human cell transformation, as they have notaccumulated the genetic alterations that predisposeimmortal rodent fibroblast lines to transformation.Although HDFs are difficult to transform, we havepreviously demonstrated that E5, through its abilityto constitutively activate the PDGFβR, could inducethree transforming phenotypes in HDFs: focusformation (loss of contact inhibition), morphologicaltransformation and continued growth beyond thenormal saturation density (Petti and Ray, 2000).Expression of v-Sis in these cells induced a similarset of phenotypes. However, neither E5 nor v-Sis couldinduce anchorage-independent growth or substantialgrowth in low serum. Moreover, HDFs expressing E5or v-Sis eventually undergo apoptosis, which mayrepresent a negative feedback response that preventsfull-scale transformation (Petti and Ray, 2000; Zhanget al., 2002).

Here, we investigated the role of specific PDGFβRsignaling pathways in E5-induced growthenhancement and focus formation of HDFs. Usingpartially defective E5 mutants, we demonstrated thatenhanced growth could be dissociated from focusformation, suggesting that alleviation of growthcontrol is insufficient for loss of contact inhibition.Further evidence implicated PI3K- and Grb2-mediated pathways in the enhanced growth phenotypeand a SHP2-p66Shc-mediated pathway in focusformation.

ResultsCertain E5 mutants can dissociate focus formation of HDFsfrom enhanced growthThe previously described E5 mutants W32S and C39S (Fig. 1A)were found to be defective for transformation but still able to bindto and activate the PDGFβR in mouse C127 fibroblasts (Horwitzet al., 1988; Nilson et al., 1995). Here, we examined the effect ofthese E5 mutants in HDFs in an effort to define more clearly theirdefect for transformation.

HDFs were made to stably express wild-type or mutant E5 byretroviral mediated gene transfer, and the ability of an E5 proteinto bind to and activate the PDGFβR in these cells was first examined.

E5 immunoprecipitation followed by PDGFβR immunoblottingrevealed that the W32S and C39S mutants were fully competentfor forming a stable complex with the PDGFβR in HDFs (Fig. 1B).PDGFβR immunoprecipitation followed by anti-phosphotyrosineimmunoblotting indicated that the W32S and C39S mutants inducedsubstantial tyrosine phosphorylation of both mature andincompletely processed precursor forms of the PDGFβR, but to alesser extent compared with wild-type E5 (Fig. 1B). Therefore,although both of these E5 mutants were fully capable of bindingto the PDGFβR in HDFs, they were not fully competent for inducingreceptor tyrosine phosphorylation.

Fig. 1. The effect of the C39S and W32S E5 mutants in HDFs. (A) The amino acid sequenceof the BPV E5 protein showing the positions of the amino acid substitutions present in theW32S and C39S mutants. Boxed region indicates the predicted transmembrane (TM)domain. (B) Extracts of HDFs stably expressing wild-type (wt) E5, W32S, C39S or no viraloncogene (control) were immunoprecipitated (IP) with an anti-E5 or anti-PDGFβR (PR)antiserum. Immunoprecipitates then were subjected to immunoblotting for PDGFβR (PR),E5 or phosphotyrosine (PY) as indicated. Mature (m) and precursor (p) forms of thePDGFβR (PR) are indicated by the arrows on the left. (C) HDFs expressing wild-type (wt)E5, the W32S or C39S mutant, or no viral oncogene (control) were plated at 1.6�105 cellsper 60 mm dish and counted in triplicate at various times thereafter. The mean number ofcells with the standard error is plotted. (D,E) A focus-forming assay was performed byinfecting HDFs with recombinant retroviruses expressing wild-type (wt) E5, C39S, W32S orno viral oncogene (control) and then maintaining cells at confluence as described in theMaterials and Methods. In D, Crystal Violet-stained monolayers are shown for visualizationof foci. In E, the number of foci induced by E5, C39S and W32S were counted, averaged,corrected for virus titer and expressed as the mean percent (with standard error) relative tothe number of foci induced by wild-type E5.

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Next, the growth kinetics of HDFs expressing the different E5proteins was assessed. As shown in Fig. 1C, the wild type E5-,W32S- and C39S-expressing HDFs grew at a similar rate and to asimilar saturation density, which was approximately twofold greaterthan that of control cells not expressing a viral oncoprotein.Therefore, the W32S and C39S mutants were competent forenhancing the growth of HDFs.

Finally, we examined the focus-forming activity of the E5mutants in HDFs. Although wild-type E5 readily induced focusformation of HDFs, the C39S and W32S mutants were severely

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defective for this phenotype (Fig. 1D,E). Thus, the W32S and C39Smutants were fully competent for enhanced growth, but were almostcompletely defective for focus formation. This suggests that anotherprocess besides the ability of cells to outgrow their normal saturationdensity is required for focus formation of HDFs. As these E5mutants separated enhanced growth from focus formation, weproceeded to use them as tools for distinguishing the PDGFβRsignaling pathways involved in these two transforming phenotypes.

PDGFβR-substrate interactions in the presence of the differentE5 proteinsGiven the phenotype of the W32S and C39S mutants, it stands toreason that these mutants somehow disable the PDGFβR frominteracting with those substrates that are required for focus formationbut not for enhanced growth. Therefore, in an effort to ascertainthe PDGFβR substrates involved focus formation of HDFs, wecompared the ability of specific substrates to be recruited to thereceptor in wild-type E5-, mutant E5- and v-Sis-expressing HDFs.

The in vivo binding of specific substrates to the PDGFβR wasdetermined by co-immunoprecipitation analysis in whichPDGFβR immunoprecipitates were analyzed for the presence ofvarious substrates by immunoblotting. As shown in Fig. 2, asubstantial amount of PLCγ, p85-PI3K, SHP2, RasGAP and Grb2could be detected in PDGFβR immunoprecipitates from E5- andv-Sis-expressing HDFs but not from control cells. This indicatesthat when the PDGFβR is activated by wild-type E5 or v-Sis inHDFs it recruits as a minimum these five substrates. We wereunable to detect Nck, Src, Shc or STAT3 co-immunoprecipitatingwith the PDGFβR in any of the different HDF cell strains (datanot shown).

Substrate-receptor binding was reduced in the mutant E5-expressing HDFs, but the magnitude of reduction varied dependingon the substrate examined. As shown in Fig. 2, co-immunoprecipitation of p85-PI3K and Grb2 with the PDGFβR wasonly modestly reduced in the presence of the E5 mutants, while co-immunoprecipitation of PLCγ and RasGAP was reduced to a greaterextent. Notably, SHP2 co-immunoprecipitation with the receptorin the presence of the E5 mutants was reduced by the greatestmagnitude (by ~80%; Fig. 2). Quantitation of results from multipleexperiments was performed and revealed that this trend wasreproducible (Fig. 2B). Thus, p85 PI3K and Grb2 showed thegreatest amount of binding to the PDGFβR in the presence of theE5 mutants, while SHP2 showed the least amount of binding.Therefore, given the fact that these E5 mutants were fully competentfor enhanced growth and greatly defective for focus formation,recruitment of PI3K and Grb2 to the receptor correlated primarilywith enhanced growth, while recruitment of SHP2 correlatedspecifically with focus formation.

Status of downstream signaling intermediates in HDFsexpressing the different E5 proteinsNext, we examined the activity of signaling intermediatesdownstream of PI3K and Grb2 in proliferating HDFs expressingthe different E5 proteins. Specifically, we ascertained thephosphorylation status of AKT, JNK, c-Jun and ERK1/2 as anindication of their activity in these cells by immunoblot analysisusing phospho-specific antibodies (Fig. 3A). Surprisingly,phosphorylation of ERK1/2 in HDFs did not increase in responseto expression of wild-type E5, v-Sis or any of the E5 mutants. Bycontrast, phosphorylation of AKT and JNK was substantiallyincreased in the wild-type E5-, C39S-, W32S- and v-Sis-expressing

Fig. 2. Recruitment of substrates to the PDGFβR in HDFs expressing thedifferent E5 proteins. (A) HDFs stably expressing the indicated E5 protein,v-Sis or no viral oncogene (control) were lysed after growing to ~85-95%confluence, and cell extracts were immunoprecipitated (IP) with a PDGFβRantiserum. PDGFβR (PR) IPs were subjected to immunoblotting for p85-PI3K(PI3-K), SHP2, PLC-γ, p120RasGAP or Grb2. The uppermost portion of eachblot was probed for PDGFβR to determine the amount of total receptor thatwas immunoprecipitated. For each substrate, a representative blot from severaldifferent experiments is shown. (B) The relative band intensities of theindicated substrates that co-immunoprecipitated with the PDGFβR as depictedin A were quantitated using the Image J program and normalized for totalreceptor amounts. Each normalized value was converted to a percentagerelative to the normalized value for the wild-type E5-expressing HDFs, and themean percentage from several different experiments with standard error isshown. This illustrates the amount of substrate bound to the PDGFβR relativeto that in the wild-type E5-expressing HDFs.

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HDFs compared with the control cells. In addition, phosphorylationof c-Jun, a major substrate for JNK, was also increased in thesecells, confirming that the activity of JNK was augmented. Thus,increased phosphorylation/activation of JNK and AKT, but not ofERK1/2, correlated with the ability of an E5 protein or v-Sis toenhance the growth of HDFs.

We also examined cyclin D3 levels in proliferating HDFsexpressing the different E5 proteins. As shown in Fig. 3B (upperpanel), cyclin D3 expression was increased in response to E5, C39S,W32S or v-Sis expression. Moreover, enhanced cyclin D3expression in the E5-expressing HDFs was inhibited by the PI3Kinhibitors LY294002 and wortmannin (Fig. 3B, lower panel).Therefore, cyclin D3 expression increased in response to sustainedPDGFβR activation in HDFs, required a PI3K-dependent pathway,and correlated with enhanced growth.

Characterization of SHP2-containing signaling complexes inHDFs expressing the different E5 proteinsAs recruitment of SHP2 to the PDGFβR showed the strictestcorrelation with focus formation of HDFs, we attempted to identifypossible downstream effectors of SHP2 that are involved in apathway for focus formation but not growth. In some systems, SHP2has been proposed to function as a signaling adaptor because itcontains several protein-protein interaction motifs, including twoSH2 domains, two phosphotyrosine residues and a proline-rich SH3-binding domain (Poole and Jones, 2005). Therefore, to initiallycharacterize the SHP2-mediated pathway involved in focus

formation, we attempted to identify its associated proteins in ourdifferent HDF strains.

We first asked whether or not a well-established binding partnerfor SHP2, Gab1, associates with SHP2 in HDFs expressing thedifferent E5 proteins. Gab1 was originally characterized as adocking protein that binds to Grb2 and is abundantly tyrosinephosphorylated in response to growth factors (Holgado-Madrugaet al., 1996). In the context of the ligand-activated PDGFβR, it wasshown that once Gab1 is recruited indirectly to the receptor viaGrb2 it becomes tyrosine phosphorylated and binds SHP2 (Kallinet al., 2004). To detect a Gab1-SHP2 interaction in our system, SHP2immunoprecipitation followed by Gab1 immunoblotting wasperformed. Fig. 4A shows that an abundant amount of Gab1 wasin a complex with SHP2 in the E5- and v-Sis-expressing HDFs butnot in control cells. This suggests that sustained PDGFβR activation

Fig. 3. Status of downstream signaling intermediates in HDFs expressing thedifferent E5 proteins. (A, upper panel of B) Whole-cell extracts ofproliferating HDFs expressing the indicated E5 protein, v-Sis or no viraloncoprotein (control) were subjected to immunoblotting. In A, blots wereprobed with antibodies specific for phosphorylated (p) forms of JNK, AKT,ERK1/2 or c-Jun, and then stripped and reprobed with pan-specific antibodiesdetecting the total amount of each protein. JNK isoforms 1 and 2 are indicatedon the right. In B (upper panel) the blot was probed with a cyclin D3-specificantibody and then reprobed with actin antibodies. In B (lower panel) E5-expressing HDFs were either untreated (–) or treated with of 20 μM LY294002(LY) or 310 nM wortmannin (W) for 24 hours. Whole-cell lysates wereprepared and immunoblotted for cyclin D3 and β-tubulin.

Fig. 4. Gab1 tyrosine phosphorylation and association with SHP2 and Grb2correlate with the enhanced growth phenotype. SHP2 (A) or Gab1 (B) wasimmunoprecipitated (IP) from lysates of HDFs expressing no (control) or theindicated viral oncoprotein. Immunoprecipitates were subjected toimmunoblotting for Gab1, SHP2, Grb2 or phosphotyrosine (PY) as indicated.For the graphs shown below representative blots, the relative band intensitiesfor the Gab1 and Grb2 blots in A, and the PY and Grb2 blots in B werequantitated using the Image J program and normalized for SHP2 (A) or totalGab1 (B) amounts. These normalized values are expressed as a percentagerelative to the amount of co-immunoprecipitation or PY-Gab1 observed in thewild-type E5-expressing HDFs. The mean values from two differentexperiments with standard error are shown.

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in HDFs promotes the binding of SHP2 to Gab1. Tyrosinephosphorylation of Gab1 was also substantially increased in theE5- and v-Sis-expressing HDFs (Fig. 4B), suggesting that it is asubstrate for the activated PDGFβR or an associated tyrosine kinasein these cells. Importantly, a considerable amount of Gab1 associatedwith SHP2 and was tyrosine phosphorylated in the C39S- andW32S-expressing HDFs (Fig. 4). Thus, given the phenotype of thesemutants, Gab1 tyrosine phosphorylation and its association withSHP2 correlated with enhanced growth rather than focus formationof HDFs. HDFs expressing the different E5 proteins were alsoexamined for the presence of Grb2-Gab1 and Grb2-SHP2complexes by co-immunoprecipitation analysis. Grb2 interactionswith Gab1 and SHP2 increased substantially in response to mutantE5, wild-type E5 or v-Sis expression (Fig. 4). Therefore, PDGFβR-Grb2, Grb2-Gab1, Gab1-SHP2 and Grb2-SHP2 interactions allcorrelated with enhanced growth and not focus formation of HDFs.This is consistent with the notion that a Grb2-Gab1-SHP2 signalingcomplex is recruited to the activated PDGFβR in HDFs andpromotes a signal for enhanced growth.

We also tested the ability of the adaptor protein Shc to interactwith SHP2 in HDFs expressing the different E5 proteins. As shownin Fig. 5A, the 66 kDa isoform of Shc (p66Shc) co-immunoprecipitated with SHP2 in the E5- and v-Sis-expressingHDFs but not in control cells. This indicates that sustained PDGFβRactivation promotes a stable association between SHP2 and p66Shcin HDFs. Interestingly, unlike the SHP2-Gab1 interaction, the SHP2-p66Shc interaction was substantially reduced in the presence of the

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C39S and W32S mutants compared with wild-type E5 (Fig. 5A).Thus, although the presence of a SHP2-Gab1 complex in HDFscorrelated with enhanced growth, the presence of a SHP2-p66Shccomplex correlated strictly with focus formation and recruitmentof SHP2 to the activated PDGFβR.

The p66Shc isoform differs both structurally and functionallyfrom its 52 and 46 kDa splice variants (reviewed by Pellegrini etal., 2005). p66Shc possesses an additional N-terminal domain, andphosphorylation of Ser36 within this domain is required for itsdistinctive activities, which include inactivating the Ras/ERKpathway and mediating oxidative stress responses (Migliaccio etal., 1999; Smith et al., 2005; Tiberi et al., 2006). Therefore, weexamined the status of p66Shc-Ser36 phosphorylation in HDFsexpressing the different E5 proteins. Shc was immunoprecipitatedfrom cell extracts and then subjected to immunoblotting using aphospho-Ser36-p66Shc-specific antibody. As shown in Fig. 5B,p66Shc-Ser36 phosphorylation was increased substantially in theE5- and v-Sis-expressing HDFs compared with control cells,suggesting that p66Shc is phosphorylated at Ser36 in response tosustained PDGFβR activation in HDFs. However, no increase inSer36 phosphorylation was observed in HDFs expressing an E5mutant (Fig. 5B). Therefore, Ser36 phosphorylation of p66Shc inHDFs correlated with focus formation and SHP2 interactions withthe PDGFβR and p66Shc.

To determine whether SHP2 is required for Ser36phosphorylation of p66Shc in HDFs, E5-expressing HDFs weretransiently transfected with a control or SHP2-specific siRNA andthen examined for Ser36 phosphorylation of p66Shc. As shown inFig. 6A, the SHP2-specific siRNA reduced SHP2 expression asexpected, and also reduced Ser36 phosphorylation of p66Shc in theE5-expressing HDFs. This suggests that SHP2 plays a role inphosphorylation of p66Shc at Ser36 in response to PDGFβRactivation. Taken together, our results suggest that once bound tothe activated PDGFβR, SHP2 recruits p66Shc and facilitates itsphosphorylation at Ser36.

As it has previously been reported that a Gab1-SHP2 complexis required for downstream activation of JNK (Holgado-Madrugaand Wong, 2003), we assessed the requirement of SHP2 for JNKactivation in our system using RNA interference of SHP2expression. Specifically, phosphorylation of JNK was examined

Fig. 5. p66Shc association with SHP2 and Ser36 phosphorylation correlatewith the focus-formation phenotype. (A,B) SHP2 (A) or Shc (B) wereimmunoprecipitated (IP) from lysates of HDFs expressing no (control) or theindicated viral oncoprotein. IPs were subjected to immunoblotting for Shc(A,B), SHP2 (A) or Ser36 phosphorylated (PS36)-p66Shc (B), as indicated.For the graphs shown below representative blots, the relative band intensitiesfor the p66Shc blot in A and the phospho-Ser36 p66Shc blot in B werequantitated using the Image J program and normalized for total SHP2 andp66Shc amounts, respectively. These normalized values are expressed as apercentage relative to the level of p66Shc-SHP2 co-immunoprecipitation (A)or p66Shc Ser36 phosphorylation (B) observed in the wild-type E5-expressingHDFs. The mean values from two (A) or three (B) different experiments withthe standard error are shown.

Fig. 6. The effect of siRNA-mediated knockdown of SHP2 on phosphorylationof p66Shc and JNK. (A,B) Whole-cell lysates of E5-expressing HDFstransfected with a control siRNA (250 pmoles) or a SHP2-specific siRNA(250 or 500 pmoles) were subjected to immunoblotting for SHP2, Ser36phosphorylated p66Shc (PS36-p66Shc) or phosphorylated JNK (pJNK) asindicated. In A, the PS36-p66Shc blot was reprobed for total p66Shc and theSHP2 blot was reprobed for β-tubulin. In B, the pJNK blot was reprobed fortotal JNK and SHP2. JNK isoforms 1 and 2 are indicated.

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after transient siRNA-mediated knockdown of SHP2 in the E5-expressing HDFs. As shown in Fig. 6B, phosphorylation of JNK1was reduced when expression of SHP2 was inhibited. This suggeststhat SHP2 plays a role in activation of JNK in the E5-expressingHDFs.

To assess the requirement of p66Shc Ser36 phosphorylation intransformation of HDFs, the effect of expressing anonphosphorylatable p66Shc mutant, S36A, on E5- and v-Sis-induced growth and focus formation was determined. S36A or wild-type p66Shc was introduced and stably expressed in HDFs byretroviral-mediated gene transfer. Increased expression of p66Shcin cells harboring a transgene confirmed that exogenous wild-typep66Shc or S36A was expressed (Fig. 7B). Furthermore, the levelp66Shc phosphorylated at Ser36 increased when wild-type p66Shcbut not S36A was overexpressed, confirming the inability of S36Ato be phosphorylated at this position (Fig. 7B). First, we determinedthe effect of wild-type p66Shc or S36A overexpression on enhancedgrowth induced by E5 or v-Sis. As shown in Fig. 7A, E5 or v-Sisexpression resulted in a threefold increase in cell density regardlessof whether or not exogenous mutant or wild-type p66Shc wasexpressed. This suggests that p66Shc does not play a role in theenhanced growth phenotype induced by E5 or v-Sis. We also askedwhether overexpressing wild-type or S36A p66Shc could affect theability of E5 or v-Sis to increase AKT and JNK phosphorylation,which correlated with the enhanced growth phenotype. As shownin Fig. 7B, E5- and v-Sis-induced phosphorylation of AKT and JNKwas unaffected by the co-expression of either exogenous wild-typep66Shc or S36A, further confirming the notion that p66Shc doesnot play a role in signaling events associated with enhanced growthof HDFs. Finally, the focus-forming activity of E5 and v-Sis in HDFswas assessed in the absence or presence of overexpressingexogenous wild-type p66Shc or S36A. As shown in Fig. 7C,overexpression of wild-type p66Shc slightly enhanced focus

formation induced by E5 or v-Sis. However, S36A expressionreduced the focus-forming activity of these oncogenes by ~40%.Taken together, these results suggest that Ser36 phosphorylation ofp66Shc plays a role in focus formation but not in enhanced growthinduced by sustained PDGFβR activation in HDFs. It is importantto note that the amount of E5- and v-Sis-induced Ser36phosphorylation of p66Shc did not decrease upon S36A expression(compare S36A with LXSN lanes in Fig. 7B), suggesting that S36Adid not inhibit Ser36 phosphorylation of endogenous p66Shc. Thisimplies that inhibition of focus formation by S36A was not at thelevel of Ser36 phosphorylation.

To initially characterize the signaling events downstream ofp66Shc that play a role in focus formation of HDFs, we attemptedto identify signaling proteins that interact with p66Shc as a resultof sustained PDGFβR activation in these cells. Co-immunoprecipitation analysis revealed that p66Shc formed a stablecomplex with p190BRhoGAP in E5- and v-Sis-expressing HDFs,but not in control cells (Fig. 8). This indicates that sustainedPDGFβR activation in HDFs promotes an interaction betweenp66Shc and p190BRhoGAP. Furthermore, the p66Shc-p190BRhoGAP interaction was greatly reduced in the C39S- andW32S-expressing HDFs (Fig. 8A), correlating this interaction withfocus formation but not enhanced growth of HDFs. Given the roleof p190BRhoGAP in inactivating Rho-family GTPases (Vincent andSettleman, 1999), these results raise the possibility that a SHP2-p66Shc-p190B complex affects a Rho GTPase signaling pathwayinvolved in focus formation of HDFs.

To determine whether or not p66Shc required Ser36phosphorylation for its ability to bind to p190B or SHP2, the abilityof the S36A p66Shc mutant to interact with these proteins wasassessed. Briefly, p66Shc was immunoprecipitated from extracts ofHDFs overexpressing wild-type p66Shc or S36A in the presenceof E5 or v-Sis and then subjected to SHP2 or p190B

Fig. 7. The effect of overexpressing wild-type or S36A mutant p66Shc on focusformation and enhanced growth of HDFs.(A) HDFs stably co-expressing wild-type(WT) p66Shc, S36A or the correspondingempty retroviral expression vector(LXSN) with E5, v-Sis or no viraloncogene (Puro) were seeded at an equaldensity in a 24-well dish. At varioustimes thereafter, cells were trypsinizedand counted in triplicate. The mean cellnumber with standard error is plottedagainst the time after plating. (B) Whole-cell extracts of cells co-expressing theindicated transgenes were subjected toimmunoblot analysis for Ser36phosphorylated p66Shc (PS36-p66Shc), orphosphorylated AKT or JNK (pAKT orpJNK, respectively). Blots were reprobedfor total p66Shc, AKT or JNK. JNKisoforms 1 and 2 are indicated. (C) Afocus-forming assay was performed byinfecting HDFs expressing wild-typep66Shc, S36A or no transgene (LXSN)with E5- or v-Sis-containing retrovirus.Foci were counted and averaged fromseveral experiments. Focus formation isexpressed as the mean percent (withstandard error) relative to the number offoci formed by the LXSN-HDFs.

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immunoblotting. As expected, p190B-p66Shc and SHP2-p66Shccomplex formation increased when wild-type p66Shc wasoverexpressed in the presence of E5 or v-Sis (Fig. 8B). Similarincreases in these complexes were observed when S36A wasexpressed, suggesting that the S36A mutant could interact withp190B and SHP2. This indicates that the ability of p66Shc to interactwith SHP2 and p190B does not require Ser36 phosphorylation.

The effect of kinase inhibitors on growth and focus formationof HDFsThe data presented thus far implicate PI3K-AKT-, JNK- and Rho-mediated pathways in the transforming phenotypes induced bysustained PDGFβR activation in HDFs. To determine therequirement of these pathways for E5- and v-Sis-induced enhancedgrowth and focus formation of HDFs, we assessed the effect ofspecific kinase inhibitors on these phenotypes. For these experimentswe used LY294002 and wortmannin (inhibitors of PI3K), SP600125(a JNK inhibitor), and Y27632 (an inhibitor of the Rho effectorkinase ROCK) at concentrations predetermined to be effective inthe E5-expressing HDFs (see Fig. S1 in the supplementary material).First, the effect of these inhibitors on the focus forming activity ofE5 or v-Sis in HDFs was determined. As shown in Fig. 9A, allinhibitors reduced focus formation of HDFs induced by E5 or v-Sis. Complete inhibition was achieved with LY294002, whilewortmannin, SP600125 and Y27632 inhibited focus formation by~40-50%. Next, we determined the effect of each inhibitor on thegrowth kinetics of E5-expressing HDFs. As shown in Fig. 9B,C,LY294002 efficiently arrested the growth of the E5-expressingHDFs, while wortmannin and SP600125 substantially slowed thegrowth rate of these cells. This suggests that PI3K and JNK activitiesare required for proliferation of the E5-expressing HDFs. Bycontrast, the ROCK inhibitor Y27632 had little effect on enhancedgrowth of the E5-expressing HDFs (Fig. 9D). Thus, PI3K-AKTand JNK pathways are required for both enhanced growth and focusformation of HDFs, while a ROCK signaling pathway is requiredexclusively for focus formation and not enhanced growth.

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DiscussionThe work presented here relates specific PDGFβR signalingpathways in normal human fibroblasts to two different aspects ofoncogenic transformation, enhanced growth and focus formation.We first described certain partially defective BPV E5 mutants (C39Sand W32S) as being competent for enhanced growth but defective

Fig. 8. p66Shc forms a complex with p190BRhoGAP in E5- and v-Sis-expressing HDFs. Total Shc (A) or p66Shc (B) was immunoprecipitated (IP)from lysates of HDFs expressing no transgene (control or LXSN-Puro) or theindicated viral oncoprotein in the absence (A) or presence (B) of exogenouswild-type (WT) p66Shc, S36A or empty LXSN. Immunoprecipitates weresubjected to immunoblotting for p190B, SHP2 or Shc as indicated.

Fig. 9. The effect of specific kinase inhibitors on growth and focus formationof HDFs. (A) Focus formation of HDFs by E5 or v-Sis was assessed in theabsence (–) or presence (+) of LY294002 (LY), wortmannin (W), SP600125(SP) or Y27632. Foci were counted and averaged from two experiments.Focus formation is expressed as the mean percent (with standard error) relativeto the number of foci formed by the untreated cells. (B-D) E5-expressingHDFs were seeded at an equal density either into 60 mM dishes (B) or a 24-well dish (C,D). Two days after plating, cells were either left untreated (–) ortreated with the above inhibitors. At various times thereafter, cells weretrypsinized and counted in triplicate. The mean cell number with standarderror is plotted against the time after plating.

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for focus formation of HDFs. This implies that focus formation ofthese cells requires distinct PDGFβR signaling pathways that arenot required for proliferation. The C39S and W32S mutants wereless effective than the wild-type E5 protein at inducing PDGFβRtyrosine phosphorylation, even though they were fully capable ofinteracting with the receptor. We speculate that although the C39Sand W32S mutants bind to the PDGFβR, they promote an alteredreceptor dimer conformation that is suboptimal for receptorautophosphorylation. This altered conformation may also disablethe receptor from recruiting those signaling substrates that arerequired primarily for focus formation.

Accordingly, we performed a comparative analysis of the abilityof the PDGFβR to recruit its substrates and activate downstreamsignaling intermediates in the presence of the different E5 proteinsand then correlated specific pathways with focus formation orenhanced growth. First, PI3K- and Grb2-mediated pathwayscorrelated with enhanced growth. The C39S and W32S mutants,like wild-type E5, could promote (1) substantial recruitment ofPI3K- and Grb2 to the PDGFβR, (2) activation of AKT and JNK,which could serve as downstream effectors of these substrates, (3)a PI-3-kinase-dependent increase in cyclin D3 expression, (4) Grb2complex formation with Gab1 and SHP2, and (5) Gab1 tyrosinephosphorylation and complex formation with SHP2. Experimentsusing kinase inhibitors confirmed that PI3K and JNK were requiredfor both focus formation and enhanced growth (Fig. 9). As theactivity of these kinases correlated with enhanced growth ratherthan with focus formation, we propose that they indirectly contributeto focus formation by promoting growth.

It stands to reason that a PI3K-dependent increase in cyclin D3expression correlated with enhanced growth of HDFs (Fig. 3B).The PI3K-AKT pathway previously has been implicated inincreased expression of cyclin D3 and cell proliferation (Spoffordet al., 2006; Zhu et al., 2001), and in some systems upregulationof cyclin D3 occurred through AKT-induced activation of mTOR(Feng et al., 2000; Garcia-Morales et al., 2006). Thus, it isreasonable to conclude that PDGFβR stimulation of a PI3K-AKTpathway in HDFs leads to increased expression of cyclin D3, whichin turn allows these cells to grow beyond their normal saturationdensity.

As PDGFβR-Grb2, Grb2-Gab1, Gab1-SHP2 and SHP2-Grb2complexes were abundant in HDFs expressing the C39S or W32Smutant, we propose that a single complex containing Grb2, Gab1and SHP2 is recruited to the activated PDGFβR via Grb2 and playsa role in the enhanced growth phenotype. In support of this notion,a previous report indicated that a Grb2-Gab1-SHP2 complex wasrecruited to the Grb2-binding site on the activated PDGFβR (Kallinet al., 2004). If this is the case in our system, the amount of SHP2that remained associated with the receptor in the presence of theE5 mutants (~20% of the SHP2 bound to the wild-type E5-activatedreceptor; Fig. 2B) may represent the pool of SHP2 that is bound tothe receptor indirectly via a Grb2-Gab1 complex. Furthermore,several studies have linked Gab1 to downstream activation of JNK(Garcia-Guzman et al., 1999; Sun et al., 2004), and two reportsdemonstrated that an interaction between Gab1 or Gab2 and SHP2is required for JNK activation (Holgado-Madruga and Wong, 2003;Yu et al., 2006). These studies, together with our finding that SHP2was required for JNK1 activation (Fig. 6B), raise the possibilitythat a Grb2-Gab1-SHP2 complex recruited to the activated PDGFβRrelays a signal for activation of JNK rather than ERK1/2 in HDFs.This is consistent with our observation that ERK1/2 was notactivated in HDFs expressing E5 or v-Sis (Fig. 3). Taken together,

our data suggests that PI3K and a Grb2-Gab1-SHP2 signalingcomplex recruited to the activated PDGFβR stimulate AKT andJNK signaling pathways, leading to enhanced growth.

Notably, a link was established between recruitment of SHP2 tothe activated PDGFβR and focus formation, as receptor binding toSHP2 was greatly impaired in the presence of the W32S or C39Smutant (Fig. 2). When recruited to the activated PDGFβR, SHP2has been proposed to promote Ras/ERK1/2 signaling either byselectively dephosphorylating the p120RasGAP-binding site on thereceptor or by binding to Grb2 (DeMali et al., 1999; Li et al., 1994).However, SHP2 must play a different role in HDFs expressing E5or v-Sis, as activation of ERK1/2 was not observed in these cells(Fig. 3) and recruitment of SHP2 to the PDGFβR did not inverselycorrelate with recruitment of p120RasGAP (Fig. 2). Because SHP2contains several potential protein-protein interaction motifs, analternative possibility is that SHP2 directly recruited to the PDGFβRplays a positive role in focus formation of HDFs by acting as asignaling adaptor. In support of this notion, we showed that SHP2formed a stable complex with the 66 kDa form of Shc in responseto E5 or v-Sis expression (Fig. 5A). Unlike the SHP2-Gab1interaction, the SHP2-p66Shc interaction correlated with focusformation and recruitment of SHP2 to the PDGFβR. In addition,we also observed an interaction between p66Shc andp190BRhoGAP in response to sustained PDGFβR activation, andthis interaction also correlated with focus formation (Fig. 8). To thebest of our knowledge, this is the first report demonstrating theability of p66Shc to interact with SHP2 and p190B. Thus, wepropose that at least two different complexes containing SHP2 arerecruited to the activated PDGFβR in HDFs. One is a Grb2-Gab1-SHP2 complex, which is recruited to the receptor via Grb2 andlikely to be involved in enhanced growth. The other is a SHP2-p66Shc-p190B complex, which is recruited to the receptor via SHP2and relays a signal specifically required for focus formation andnot growth.

It is possible that the SHP2-p66Shc complex bound to theactivated PDGFβR in HDFs recruits and inactivates p190BRhoGAP,resulting in activation of a Rho family GTPase. Others have shownthat p190B is a substrate for SHP2 and that SHP2-mediatedtyrosine dephosphorylation of p190B inactivates it, leading tosubsequent activation of RhoA (Kontaridis et al., 2004; Sordella etal., 2003). We were unable to detect a change in tyrosinephosphorylation of p190B or Rho activity correlating with focusformation in our system (data not shown). Nonetheless, wedemonstrated that the activity of ROCK, a downstream effector ofRho, was required for focus formation and not for enhanced growthof HDFs, implying a specific role for Rho in focus formation. It ispossible that the fraction of active Rho involved focus formationis located at a specific subcellular site and thus would be difficultto detect by the standard pull-down assay for Rho activity. Therefore,in the E5-expressing HDFs, the SHP2-p66Shc complex may recruitp190B residing at a specialized site for focus formation. SHP2 boundto p66Shc could then dephosphorylate and inactivate p190B,resulting in Rho activation at this site. Experiments are planned todetermine more precisely the role of p190B and Rho in focusformation of HDFs.

One intriguing possibility is that RhoA plays a role in focusformation by altering cytokinesis. In a previous report, it was shownthat overexpression of a constitutively activated RhoA proteincaused improper orientation of the mitotic spindle, thereby alteringthe plane for cytokinesis such that it was parallel rather thanperpendicular to the substrate (Vasiliev et al., 2004). Furthermore,

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other studies showed that overactivation of RhoA atsites of cell-cell contact can destabilize adhernsjunctions (Sahai and Marshall, 2002), which in turn canchange the position of the centrosome and alter the planeof cytokinesis (Siegrist and Doe, 2006). Based on thesereports, the following scenario could explain the roleof Rho in E5-induced focus formation of HDFs:activation of the PDGFβR at sites of cell-cell contactcould result in recruitment of a SHP2-p66Shc-p190Bcomplex to the receptor followed by localizedinactivation of p190B and activation of RhoA at thesesites. This could result in destabilization of cell-cellcontacts followed by cleavage furrow formation at thesesites. Consequently, cell division would occur parallelto the substrate, resulting in one daughter cell sittingon top of its sister cell. Successive rounds of this processcould generate foci of piled up cells.

Our evidence suggests that the Ser36 phosphorylatedform of p66Shc plays a specific role in the focusforming phenotype. First, Ser36 phosphorylation of p66Shccorrelated with focus formation but not growth (Fig. 5B). Second,the nonphosphorylatable S36A p66Shc mutant impaired focusformation but not growth induced by E5 or v-Sis (Fig. 7). As S36Acould still bind to SHP2 and p190B (Fig. 8B), it is possible that itinhibited focus formation by competing with endogenous wild-typep66Shc for binding to SHP2 and p190B, thereby sequestering theseproteins in an inactive complex. Focus formation was not completelyinhibited by S36A, perhaps because the amount of S36A expressedwas insufficient for sequestering all of the available SHP2 andp190B. Therefore, we believe that the Ser36 phosphorylated formof p66Shc is a necessary component of an active signaling complexwith SHP2 and/or p190B. For example, the phospho-Ser36 residuemay be part of a recognition motif for another signaling proteinrequired for focus formation. Alternatively, as the Ser36phosphorylated form of p66Shc has been shown to promote thegeneration of intracellular reactive oxygen species (Migliaccio etal., 1999; Smith et al., 2005; Tiberi et al., 2006), it could act throughan oxidant-dependent mechanism to affect the expression or activityof proteins involved in a focus-forming process. Identification ofproteins that interact with p66Shc in a Ser36-specific manner inthe E5-expressing HDFs will provide further insight into the roleof p66Shc in focus formation.

It is important to note that overexpression of wild-type p66Shcdid not enable the C39S or W32S mutant to induce focus formation(data not shown). This suggests that an additional pathway isrequired for focus formation of HDFs. As the C39S and W32Smutants were also defective for promoting recruitment of PLC-γand p120RasGAP to the PDGFβR, signals promoted by one or bothof these substrates could also contribute to focus formation of HDFs.

In conclusion, we propose the following model outlining thePDGFβR signaling pathways that contribute to enhanced growthand focus formation of HDFs (Fig. 10): first, PI3K recruited to theactivated PDGFβR promotes activation of AKT, which in turn leadsto increased expression of cyclin D3 followed by enhanced growth.Grb2 bound to the receptor may also promote a signal for enhancedgrowth by recruiting Gab1, which in turn becomes tyrosinephosphorylated and recruits SHP2, leading to activation of JNK.Proliferation promoted by PI3K and Grb2 pathways is necessarybut insufficient for focus formation. An additional signal providedby SHP2 recruited directly to the PDGFβR is also required forfocus formation. Specifically, SHP2 bound to the receptor forms

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a complex with p66Shc, which in turn binds to p190BRhoGAPand enables SHP2 to inactivate p190B. This would result inactivation of Rho, which could stimulate a cytoskeletal remodelingprocess (such as altered cytokinesis, adhesion or motility) thatallows cells to grow in a third dimension. Although some aspectsof this model will require further elucidation, we have begun tocharacterize the signaling pathways downstream of the PDGFβRthat play a role in transformation of human cells. These pathwaysalso may be involved in maintenance or dissemination of humantumors in which overstimulation of the PDGFβR has beenimplicated, and thus may represent pertinent targets for cancertherapy.

Materials and MethodsCells and cell cultureYoung HSF4012 (NHDF4012) human foreskin fibroblasts (HDFs) were purchasedfrom Cambrex (formerly Clonetics) at passage 1. The Phoenix amphotropic producercell line was obtained from the American Type Culture Collection (ATCC) withpermission from G. Nolan (Stanford University) and was used to produce high titeramphotropic retrovirus after transient transfection. Cells were maintained as previouslydescribed (Petti and Ray, 2000).

Plasmid DNAsThe E5, W32S, C39S and v-sis open reading frames (ORFs) were subcloned fromplasmids obtained from Daniel DiMaio (Yale University, New Haven, CT) into thepBabepuro retroviral vector (which contains the puromycin resistance marker) bystandard methods. Plasmids containing the S36A mutant or wild-type p66Shc ORFwere obtained from Addgene [plasmids 10972 and 10973, respectively (Nemoto andFinkel, 2002)] and subcloned into the LXSN retroviral vector (which carries the G418resistance marker).

Stable expression of foreign genes in HDFsHDF strains stably expressing wild-type E5, mutant E5, v-sis, wild-type p66Shc orthe S36A (Ser36 to Ala substitution) p66Shc mutant were established using hightiter recombinant retroviruses expressing these genes to infect HSF4012 cells atpassage 7 or 8 as described previously (Petti and Ray, 2000). Briefly, transienttransfection of plasmid DNA into Phoenix amphotropic producer cells using thecalcium chloride method resulted in production of virus in the tissue culturesupernatant. Approximately 2-4�105 colony forming units of retrovirus plus 4 μg/mlpolybrene was added to ~1.5�106 HSF4012 cells. After 1 day, the infected cellswere trypsinized and split 1:5. The next day, puromycin (Sigma) or G418 (Gemini)was added to the cells at a final concentration of 0.5 μg/ml or 400 μg/ml, respectively.After 1-2 weeks of selection, stable cell strains were established. Control cells wereestablished using an empty retroviral vector (pBabepuro or LXSN). HDFs stablyco-expressing exogenous wild-type p66Shc or S36A with E5 or v-Sis wereestablished by sequential retroviral infection and selection. Specifically, cellsexpressing wild-type p66Shc, S36A or LXSN were first established and thensubjected to a second round of infection and selection for E5, v-Sis or pBabepuroexpression.

Fig. 10. Model of the PDGFβR signaling pathways involved in enhanced growth andfocus formation of HDFs. See text for explanation.

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Antibodies and inhibitorsPR4, a rabbit polyclonal antiserum recognizing the C-terminal 13 amino acids of thehuman PDGFβR, and an E5 rabbit polyclonal antiserum recognizing the C-terminal16 amino acids of the BPV E5 protein were obtained from Daniel DiMaio (YaleUniversity, New Haven, CT). A second PDGFβR rabbit polyclonal antiserum(against the C-terminal domain) was obtained from BD Pharmingen and used forimmunoprecipitation in some cases. Antibodies against p85-PI3K, phospho-Jun(pSer73), Gab1, β-tubulin and p66Shc were purchased from Upstate Biotechnology(UBI). Phospho-AKT (pSer 473), total AKT, phospho-ERK1/2 (pThr202/pTyr204),total ERK1/2, actin, anti-phosphotyrosine (P-Tyr-100) and actin antibodies werepurchased from Cell Signaling Technology (CST). SHP2 and p120RasGAP antibodieswere purchased from Santa Cruz Biotechnology. A phospho-Ser36-p66Shc-specificantibody was obtained from Calbiochem. All other antibodies were obtained fromBD Pharmingen Laboratories. For SHP2 detection, rabbit polyclonal antibody fromSanta Cruz (SC-280) was used for immunoprecipitation, while a mouse monoclonalantibody from BD Pharmingen (610621) was used for immunoblotting. LY294002,wortmannin, SP600125, Y27632 were purchased from CST, UBI, BioMol andCalbiochem, respectively.

Focus forming assayHDF monolayers in 60 mm dishes were infected with E5-, W32S-, C39S- or v-Sis-expressing retrovirus at a low multiplicity (m.o.i.) of infection. The next day, infectedcells were trypsinized, split ~1:3 and then maintained with bi-weekly media changesas described previously (Petti and Ray, 2000). Virus titer was determined within thesame experiment by seeding a small fraction of the trypsinized infected cells intotwo 100 mm dish, selecting for puromycin resistance and then counting the numberof drug-resistant colonies that formed. Three weeks after infection, foci werevisualized by staining monolayers with 1% Crystal Violet (w/v) in 70% ethanol.Stained foci were counted, averaged and normalized for virus titer. For Fig. 9C,LY294002, wortmannin, SP600125 or Y27632 was added to the cells just prior toconfluence at a final concentration of 20 μM, 233 nM, 10-20 μM or 10 μM,respectively. In this case, confluent cells were maintained with media changes every3 days in the absence or presence of fresh inhibitor.

Growth curvesHDFs seeded at an equal density were trypsinized and counted in triplicate using ahemocytometer at various intervals after plating. For Fig. 9A-C, 2 days after plating,the cells were either left untreated or treated with 20 μM LY294002, 233 nMwortmannin, 10 μM SP600125 or 10 μM Y27632 by adding the inhibitor to theexisting medium.

Transient siRNA-mediated knockdown of SHP2E5-expressing HDFs were transiently transfected with a control or SHP2 siRNA (SantaCruz products sc-37007 and sc-36488, respectively) using the protocol and transfectionreagents specified by the manufacturer. Cells were lysed 48 hours after transfectionfor examination of specific protein expression by immunoblotting.

Immunoprecipitation and immunoblottingTo prepare HDF extracts, cell monolayers were washed twice in PBS and then lysedin cold EBC buffer [50 mM Tris-HCl (pH 8.0), 120 mM NaCl, 0.5% NP40] or RIPAbuffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% NP40, 1 mM EDTA, 1%deoxycholic acid, 0.1% SDS] supplemented with inhibitors (10 mg/ml leupeptin, 10mg/ml aprotinin, 1 mM phenylmethysulfonylfluoride and 2 mM sodiumorthovanadate) as previously described (Petti and Ray, 2000). Immunoprecipitationwas performed as previously described. To prepare whole-cell extracts, cellmonolayers in 60 mm dishes were washed twice in PBS and then lysed in 200 μl ofhot 2� Laemmli sample buffer. Lysates were passed through a 24-gauge needle toshear cellular nucleic acid.

Immunoprecipitates or whole-cell lysates were boiled, subjected to SDS-PAGEand then transferred to polyvinylidene difluoride or nitrocellulose. Immunoblottingwas performed as previously described (Petti and Ray, 2000) using 5% nonfat drymilk in TBS-T [10 mM Tris-HCl (pH 7.4), 167 mM NaCl, 0.5% Tween-20] as theblocking buffer and primary antibody diluted as recommended by the manufacturerin blocking buffer. For E5 and PDGF receptor blots, E5 or PR4 antiserum was diluted1:500 and 1:250, respectively. A 1:5000-1:10,000 dilution of horseradish peroxidase(HRP)-conjugated protein A (Pierce) or HRP-conjugated goat anti-mouse IgG(Jackson ImmunoResearch Labs) was used as a secondary reagent. Proteins weredetected by enhanced chemiluminescence (ECL) using detection reagents purchasedfrom Pierce or Amersham, as described by the manufacturer. In some cases, blotswere stripped using Restore western blot stripping buffer (Pierce) and then reprobedwith a different antibody. For some blots, densitometry of band intensity wasperformed using the Image J program (NIH).

We thank Daniel DiMaio for the PDGF receptor and E5 antisera,as well as the C39S and W32S E5 mutants. This work was supportedby Public Health Service grant CA-073682 from the NationalCancer Institute, as well as by awards from the Concern Foundation

(Young Investigator Award) and Albany Medical College (BridgeGrant).

ReferencesBeckmann, M. P., Betsholtz, C., Heldin, C. H., Westermark, B., Di Marco, E., Di Fiore,

P. P., Robbins, K. C. and Aaronson, S. A. (1988). Comparison of biological propertiesand transforming potential of human PDGF-A and PDGF-B chains. Science 241, 1346-1349.

Bergman, P., Ustav, M., Sedman, J., Moreno-Lopez, J., Vennstrom, B. and Pettersson,U. (1988). The E5 gene of bovine papillomavirus type 1 is sufficient for completeoncogenic transformation of mouse fibroblasts. Oncogene 2, 453-459.

Betsholtz, C., Nister, M., Rorsman, F., Heldin, C. H. and Westermark, B. (1989).Structural and functional aspects of platelet-derived growth factor and its role in thepathogenesis of glioblastoma. Mol. Chem. Neuropathol. 10, 27-36.

Burkhardt, A., Willingham, M., Gay, C., Jeang, K. T. and Schlegel, R. (1989). The E5oncoprotein of bovine papillomavirus is oriented asymmetrically in Golgi and plasmamembranes. Virology 170, 334-339.

Clarke, M. F., Westin, E., Schmidt, D., Josephs, S. F., Ratner, L., Wong-Staal, F., Gallo,R. C. and Reitz, M. S., Jr (1984). Transformation of NIH 3T3 cells by a human c-siscDNA clone. Nature 308, 464-467.

DeMali, K. A., Whiteford, C. C., Ulug, E. T. and Kazlauskas, A. (1997). Platelet-derivedgrowth factor-dependent cellular transformation requires either phospholipase Cgammaor phosphatidylinositol 3 kinase. J. Biol. Chem. 272, 9011-9018.

DeMali, K. A., Balciunaite, E. and Kazlauskas, A. (1999). Integrins enhance platelet-derived growth factor (PDGF)-dependent responses by altering the signal relay enzymesthat are recruited to the PDGF beta receptor. J. Biol. Chem. 274, 19551-19558.

DiMaio, D., Guralski, D. and Schiller, J. T. (1986). Translation of open reading frameE5 of bovine papillomavirus is required for its transforming activity. Proc. Natl. Acad.Sci. USA 83, 1797-1801.

Doolittle, R. F., Hunkapiller, M. W., Hood, L. E., Devare, S. G., Robbins, K. C.,Aaronson, S. A. and Antoniades, H. N. (1983). Simian sarcoma virus onc gene, v-sis,is derived from the gene (or genes) encoding a platelet-derived growth factor. Science221, 275-277.

Feng, L. X., Ravindranath, N. and Dym, M. (2000). Stem cell factor/c-kit up-regulatescyclin D3 and promotes cell cycle progression via the phosphoinositide 3-kinase/p70S6 kinase pathway in spermatogonia. J. Biol. Chem. 275, 25572-25576.

Fleming, T. P., Saxena, A., Clark, W. C., Robertson, J. T., Oldfield, E. H., Aaronson,S. A. and Ali, I. U. (1992). Amplification and/or overexpression of platelet-derivedgrowth factor receptors and epidermal growth factor receptor in human glial tumors.Cancer Res. 52, 4550-4553.

Garcia-Guzman, M., Dolfi, F., Zeh, K. and Vuori, K. (1999). Met-induced JNK activationis mediated by the adapter protein Crk and correlates with the Gab1-Crk signalingcomplex formation. Oncogene 18, 7775-7786.

Garcia-Morales, P., Hernando, E., Carrasco-Garcia, E., Menendez-Gutierrez, M. P.,Saceda, M. and Martinez-Lacaci, I. (2006). Cyclin D3 is down-regulated byrapamycin in HER-2-overexpressing breast cancer cells. Mol. Cancer Ther. 5, 2172-2181.

Heldin, C. H. and Westermark, B. (1999). Mechanism of action and in vivo role of platelet-derived growth factor. Physiol. Rev. 79, 1283-1316.

Heldin, C. H., Ostman, A. and Ronnstrand, L. (1998). Signal transduction via platelet-derived growth factor receptors. Biochim. Biophys. Acta 1378, F79-F113.

Hermanson, M., Funa, K., Hartman, M., Claesson-Welsh, L., Heldin, C. H.,Westermark, B. and Nister, M. (1992). Platelet-derived growth factor and its receptorsin human glioma tissue: expression of messenger RNA and protein suggests the presenceof autocrine and paracrine loops. Cancer Res. 52, 3213-3219.

Holgado-Madruga, M. and Wong, A. J. (2003). Gab1 is an integrator of cell death versuscell survival signals in oxidative stress. Mol. Cell. Biol. 23, 4471-4484.

Holgado-Madruga, M., Emlet, D. R., Moscatello, D. K., Godwin, A. K. and Wong, A.J. (1996). A Grb2-associated docking protein in EGF- and insulin-receptor signalling.Nature 379, 560-564.

Horwitz, B. H., Burkhardt, A. L., Schlegel, R. and DiMaio, D. (1988). 44-amino-acidE5 transforming protein of bovine papillomavirus requires a hydrophobic core andspecific carboxyl-terminal amino acids. Mol. Cell. Biol. 8, 4071-4078.

Kallin, A., Demoulin, J. B., Nishida, K., Hirano, T., Ronnstrand, L. and Heldin, C.H. (2004). Gab1 contributes to cytoskeletal reorganization and chemotaxis in responseto platelet-derived growth factor. J. Biol. Chem. 279, 17897-17904.

Klein, O., Polack, G. W., Surti, T., Kegler-Ebo, D., Smith, S. O. and DiMaio, D. (1998).Role of glutamine 17 of the bovine papillomavirus E5 protein in platelet-derived growthfactor beta receptor activation and cell transformation. J. Virol. 72, 8921-8932.

Klein, O., Kegler-Ebo, D., Su, J., Smith, S. and DiMaio, D. (1999). The bovinepapillomavirus E5 protein requires a juxtamembrane negative charge for activation ofthe platelet-derived growth factor beta receptor and transformation of C127 cells. J.Virol. 73, 3264-3272.

Kontaridis, M. I., Eminaga, S., Fornaro, M., Zito, C. I., Sordella, R., Settleman, J.and Bennett, A. M. (2004). SHP-2 positively regulates myogenesis by coupling to theRho GTPase signaling pathway. Mol. Cell. Biol. 24, 5340-5352.

Lai, C. C., Henningson, C. and DiMaio, D. (1998). Bovine papillomavirus E5 proteininduces oligomerization and trans-phosphorylation of the platelet-derived growth factorbeta receptor. Proc. Natl. Acad. Sci. USA 95, 15241-15246.

Li, W., Nishimura, R., Kashishian, A., Batzer, A. G., Kim, W. J., Cooper, J. A. andSchlessinger, J. (1994). A new function for a phosphotyrosine phosphatase: linkingGRB2-Sos to a receptor tyrosine kinase. Mol. Cell. Biol. 14, 509-517.

Jour

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Migliaccio, E., Giorgio, M., Mele, S., Pelicci, G., Reboldi, P., Pandolfi, P. P.,Lanfrancone, L. and Pelicci, P. G. (1999). The p66shc adaptor protein controls oxidativestress response and life span in mammals. Nature 402, 309-313.

Nappi, V. M. and Petti, L. M. (2002). Multiple transmembrane amino acid requirementssuggest a highly specific interaction between the bovine papillomavirus E5 oncoproteinand the platelet-derived growth factor beta receptor. J. Virol. 76, 7976-7986.

Nappi, V. M., Schaefer, J. A. and Petti, L. M. (2002). Molecular examination of thetransmembrane requirements of the platelet-derived growth factor beta receptor for aproductive interaction with the bovine papillomavirus E5 oncoprotein. J. Biol. Chem.277, 47149-47159.

Nemoto, S. and Finkel, T. (2002). Redox regulation of forkhead proteins through a p66shc-dependent signaling pathway. Science 295, 2450-2452.

Nilson, L. A. and DiMaio, D. (1993). Platelet-derived growth factor receptor can mediatetumorigenic transformation by the bovine papillomavirus E5 protein. Mol. Cell. Biol.13, 4137-4145.

Nilson, L. A., Gottlieb, R. L., Polack, G. W. and DiMaio, D. (1995). Mutational analysisof the interaction between the bovine papillomavirus E5 transforming protein and theendogenous beta receptor for platelet-derived growth factor in mouse C127 cells. J.Virol. 69, 5869-5874.

Pellegrini, M., Pacini, S. and Baldari, C. T. (2005). p66SHC: the apoptotic side of Shcproteins. Apoptosis 10, 13-18.

Petti, L. and DiMaio, D. (1992). Stable association between the bovine papillomavirusE5 transforming protein and activated platelet-derived growth factor receptor intransformed mouse cells. Proc. Natl. Acad. Sci. USA 89, 6736-6740.

Petti, L. and DiMaio, D. (1994). Specific interaction between the bovine papillomavirusE5 transforming protein and the beta receptor for platelet-derived growth factor in stablytransformed and acutely transfected cells. J. Virol. 68, 3582-3592.

Petti, L. M. and Ray, F. A. (2000). Transformation of mortal human fibroblasts andactivation of a growth inhibitory pathway by the bovine papillomavirus E5 oncoprotein.Cell Growth Differ. 11, 395-408.

Petti, L., Nilson, L. A. and DiMaio, D. (1991). Activation of the platelet-derived growthfactor receptor by the bovine papillomavirus E5 transforming protein. EMBO J. 10, 845-855.

Petti, L. M., Reddy, V., Smith, S. O. and DiMaio, D. (1997). Identification of aminoacids in the transmembrane and juxtamembrane domains of the platelet-derived growthfactor receptor required for productive interaction with the bovine papillomavirus E5protein. J. Virol. 71, 7318-7327.

Poole, A. W. and Jones, M. L. (2005). A SHPing tale: perspectives on the regulation ofSHP-1 and SHP-2 tyrosine phosphatases by the C-terminal tail. Cell. Signal. 17, 1323-1332.

Riese, D. J., 2nd and DiMaio, D. (1995). An intact PDGF signaling pathway is requiredfor efficient growth transformation of mouse C127 cells by the bovine papillomavirusE5 protein. Oncogene 10, 1431-1439.

Roche, S., McGlade, J., Jones, M., Gish, G. D., Pawson, T. and Courtneidge, S. A.(1996). Requirement of phospholipase C gamma, the tyrosine phosphatase Syp andthe adaptor proteins Shc and Nck for PDGF-induced DNA synthesis: evidence forthe existence of Ras-dependent and Ras-independent pathways. EMBO J. 15, 4940-4948.

Sahai, E. and Marshall, C. J. (2002). ROCK and Dia have opposing effects on adherensjunctions downstream of Rho. Nat. Cell Biol. 4, 408-415.

Schiller, J. T., Vass, W. C., Vousden, K. H. and Lowy, D. R. (1986). E5 open readingframe of bovine papillomavirus type 1 encodes a transforming gene. J. Virol. 57, 1-6.

Schlegel, R., Wade-Glass, M., Rabson, M. S. and Yang, Y. C. (1986). The E5transforming gene of bovine papillomavirus encodes a small, hydrophobic polypeptide.Science 233, 464-467.

Siegrist, S. E. and Doe, C. Q. (2006). Extrinsic cues orient the cell division axis inDrosophila embryonic neuroblasts. Development 133, 529-536.

Smith, W. W., Norton, D. D., Gorospe, M., Jiang, H., Nemoto, S., Holbrook, N. J.,Finkel, T. and Kusiak, J. W. (2005). Phosphorylation of p66Shc and forkhead proteinsmediates Abeta toxicity. J. Cell Biol. 169, 331-339.

Sordella, R., Jiang, W., Chen, G. C., Curto, M. and Settleman, J. (2003). Modulationof Rho GTPase signaling regulates a switch between adipogenesis and myogenesis. Cell113, 147-158.

Spofford, L. S., Abel, E. V., Boisvert-Adamo, K. and Aplin, A. E. (2006). Cyclin D3expression in melanoma cells is regulated by adhesion-dependent phosphatidylinositol3-kinase signaling and contributes to G1-S progression. J. Biol. Chem. 281, 25644-25651.

Sun, Y., Yuan, J., Liu, H., Shi, Z., Baker, K., Vuori, K., Wu, J. and Feng, G. S. (2004).Role of Gab1 in UV-induced c-Jun NH2-terminal kinase activation and cell apoptosis.Mol. Cell. Biol. 24, 1531-1539.

Tiberi, L., Faisal, A., Rossi, M., Di Tella, L., Franceschi, C. and Salvioli, S. (2006).p66(Shc) gene has a pro-apoptotic role in human cell lines and it is activated by a p53-independent pathway. Biochem. Biophys. Res. Commun. 342, 503-508.

Uren, A., Yu, J. C., Li, W., Chung, I. Y., Mahadevan, D., Pierce, J. H. and Heidaran,M. A. (1996). Identification of a domain within the carboxyl-terminal region of the betaplatelet-derived growth factor (PDGF) receptor that mediates the high transformingactivity of PDGF. J. Biol. Chem. 271, 11051-11054.

Vasiliev, J. M., Omelchenko, T., Gelfand, I. M., Feder, H. H. and Bonder, E. M. (2004).Rho overexpression leads to mitosis-associated detachment of cells from epithelial sheets:a link to the mechanism of cancer dissemination. Proc. Natl. Acad. Sci. USA 101, 12526-12530.

Vincent, S. and Settleman, J. (1999). Inhibition of RhoGAP activity is sufficient for theinduction of Rho-mediated actin reorganization. Eur. J. Cell Biol. 78, 539-548.

Waterfield, M. D., Scrace, G. T., Whittle, N., Stroobant, P., Johnsson, A., Wasteson,A., Westermark, B., Heldin, C. H., Huang, J. S. and Deuel, T. F. (1983). Platelet-derived growth factor is structurally related to the putative transforming protein p28sisof simian sarcoma virus. Nature 304, 35-39.

Yu, M., Luo, J., Yang, W., Wang, Y., Mizuki, M., Kanakura, Y., Besmer, P., Neel, B.G. and Gu, H. (2006). The scaffolding adapter Gab2, via Shp-2, regulates Kit-evokedmast cell proliferation by activating the Rac/JNK pathway. J. Biol. Chem. 281, 28615-28626.

Zhang, Y., Lehman, J. M. and Petti, L. M. (2002). Apoptosis of mortal human fibroblaststransformed by the bovine papillomavirus E5 oncoprotein. Mol. Cancer Res. 1, 122-136.

Zhu, X., Kwon, C. H., Schlosshauer, P. W., Ellenson, L. H. and Baker, S. J. (2001).PTEN induces G(1) cell cycle arrest and decreases cyclin D3 levels in endometrialcarcinoma cells. Cancer Res. 61, 4569-4575.

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