MOLECULAR AND CELLULAR BIOLOGY, June 1992, p. 2534-25440270-7306/92/062534-11$02.00/0Copyright ©) 1992, American Society for Microbiology

GTPase-Activating Protein and Phosphatidylinositol 3-Kinase Bindto Distinct Regions of the Platelet-Derived Growth Factor

Receptor 1B SubunitANDRIUS KAZLAUSKAS,l* ADAM KASHISHIAN,2 JONATHAN A. COOPER,2 AND MINDAUGAS VALIUS1t

National Jewish Center for Immunology and Respiratory Medicine, 1400 Jackson Street, Denver, Colorado 80206,and Fred Hutchinson Cancer Research Center, Seattle, Washington 981042

Received 19 August 1991/Accepted 26 February 1992

In response to binding of platelet-derived growth factor (PDGF), the PDGF receptor (PDGFR) I1 subunit isphosphorylated on tyrosine residues and associates with numerous signal transduction enzymes, including theGTPase-activating protein ofras (GAP) and phosphatidylinositol 3-kinase (P13K). Previous studies have shownthat association of P13K requires phosphorylation of tyrosine 751 (Y751) in the kinase insert and that thisregion of receptor forms at least a portion of the binding site for P13K. In this study, the in vitro binding ofGAP to the PDGFR was investigated. Like PI3K, GAP associates only with receptors that have been permittedto autophosphorylate, and GAP itself does not require tyrosine phosphate in order to stably associate with thephosphorylated PDGFR. To define which tyrosine residues are required for GAP binding, a panel of PDGFRphosphorylation site mutants was tested. Mutation of Y771 reduced the amount of GAP that associates to anundetectable level. In contrast, the F771 (phenylalanine at 771) mutant bound wild-type levels of PI3K,whereas the F740 and F751 mutants bound 3 and 23%, respectively, of the wild-type levels of PI3K butwild-type levels of GAP. The F740/F751 double mutant associated with wild-type levels of GAP, but nodetectable P13K activity, while the F740/F751/F771 triple mutant could not bind either GAP or P13K. The invitro and in vivo associations of GAP and PI3K activity to these PDGFR mutants were indistinguishable. Thedistinct tyrosine residue requirements suggest that GAP and P13K bind different regions of the PDGFR. Thispossibility was also supported by the observation that the antibody to the PDGFR kinase insert Y751 regionthat blocks association of P13K had only a minor effect on the in vitro binding of GAP. In addition, highlypurified PI3K and GAP associated in the absence of other cellular proteins and neither cooperated norcompeted with each other's binding to the PDGFR. Taken together, these studies indicate that GAP and P13Kbind directly to the PDGFR and have discrete binding sites that include portions of the kinase insert domain.

Platelet-derived growth factor (PDGF) is a protein whichinitiates cell growth and chemotaxis in tissue culture cellsthat express PDGF receptors (PDGFRs). There are a num-ber of sources of PDGF, which suggest physiological rolesfor this mitogen. Platelets contain PDGF, and upon aggre-gation and degranulation at the site of injury, PDGF isreleased and contributes to the process of wound healing byrecruiting macrophages and initiating cellular proliferation(18, 53). A number of different cell types (endothelial,smooth muscle, and macrophage) also secrete PDGF, andthe transforming protein of the v-sis oncogene encodes the Bchain of PDGF ligand (19, 53), indicating that PDGF regu-lates growth via a paracrine or autocrine interaction of thegrowth factor and its target cells. In addition, PDGF hasbeen found in a number of different types of embryos (53)and recently has been localized to the developing brain (55),indicating a role in development.There are three forms of PDGF, AA, AB, and BB, all of

which bind to cell surface receptors and trigger proliferation(19, 53). The receptor for PDGF can be either a homo- orheterodimer of the a and PDGFR subunits (19, 47). Thesereceptor subunits differ in their ability to bind the differentchains of PDGF, so that PDGF-AA binds to aa PDGFRs,

* Corresponding author.t Permanent address: Institute of Biochemistry, Lithuanian

Academy of Sciences, Vilnius, Lithuania.

PDGF-AB binds to axa and ota PDGFRs, and PDGF-BBbinds all possible PDGFRs (16, 19, 47). The studies de-scribed herein focus on PDGF-BB and the 13-type PDGFR.There has been considerable effort focused on defining the

intracellular sequence of events by which PGDF mediates a

mitogenic signal. Both PDGFR subunits are protein tyrosinekinases which are greatly activated following binding ofPDGF and ultimately trigger DNA synthesis (35). This leadsto tyrosine phosphorylation of numerous proteins, includingthe receptor itself. The two major PDGF-induced tyrosinephosphorylation sites of the PDGFR 1B subunit are tyrosine751 (Y751) and Y857 (25). Other minor sites have beenidentified and include Y740 and Y771 (22). Phosphorylationof Y857 but not Y751 appears to be necessary for thePDGF-dependent increase in receptor kinase activity (10,27).

Binding of PDGF also leads to the stable association of anumber of signal transduction enzymes with the tyrosine-phosphorylated PDGFR, including phospholipase C-y (32,51), the GTPase-activating protein of ras (GAP) (20, 28),phosphatidylinositol (PI) 3-kinase (PI3K) (5, 21, 25, 52),c-Raf (39), the src family members p60csrc, p62c-Yes, andp59fyf (33), as well as a 120- and a 64-kDa protein (25, 26) ofunknown function. The identified receptor-associated pro-teins are signal transduction enzymes, which are affected byPDGF binding. Activation of the PDGFR results in thetyrosine phosphorylation and activation of phospholipase C-y(14, 30, 32, 36, 40, 41, 49, 51), increasing the levels of two

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BINDING OF GAP AND PI 3-KINASE TO THE PDGFR 1 SUBUNIT 2535

second-messenger molecules diacylglycerol and inositol-trisphosphate. The levels of activated (GTP-bound) rasincrease two- to threefold in PDGF-stimulated cells (13, 46),which may in part be due to the tyrosine phosphorylation orassociation of GAP with the PDGFR. Similarly, the concen-trations of PI 3-phosphates increase following exposure ofcells to PDGF (2), which reflects the increased activity ofPI3K. The activities of c-Raf (39), a serine/threonine kinase,as well as the protein tyrosine kinase of p60csrc (4, 33) alsoincrease in PDGF-stimulated cells. Thus, the activities of thereceptor-associated proteins are affected by PDGF stimula-tion, and it seems likely that these proteins are the intracel-lular mediators of PDGF mitogenic signal. This possibility issupported by the observation that PDGFR mutants that failto associate with the full complement of the receptor-associated proteins mediate only a partial mitogenic re-sponse (4, 27, 53).Given the putative importance of the receptor-associated

proteins for PDGF-mediated signal relay, the events leadingto the formation of the complex of activated receptor andsignal relay molecules are actively being investigated. Stud-ies with receptor mutants indicate that receptor kinaseactivity is an absolute requirement (25, 53). Furthermore,receptor variants that are kinase active but lack phosphory-lation sites have a reduced ability to bind some of theproteins (4, 9, 20, 25, 53). Reconstitution of the associationof the signaling enzymes with the PDGFR in vitro hasdemonstrated that receptor phosphorylation triggers bindingand that phosphate of Y751 in the kinase insert is requiredfor P13K binding (26). Furthermore, two different phosphor-ylated peptides inhibit in vitro binding of PI3K to the murinePDGFR (7). The sequences of these peptides resemble thosefound in the human receptor at Y70 and Y751, suggestingthat Y740 also is a phosphorylation site and, like Y751,mediates binding of PI3K.An important contribution to our understanding of how

tyrosine-phosphorylated proteins associate with cellular pro-teins has been provided by several laboratories demonstrat-ing that src homology (SH) domains, in particular SH2domains, bind certain phosphotyrosine-containing proteins(1, 7, 11, 31, 34, 36). All of the known PDGFR-associatedproteins except c-Raf have at least a single SH2 domain,which enables stable association. Given that the various SH2domains exhibit distinct affinities for tyrosine-phosphory-lated proteins (1, 31) and that the PDGFR has multipletyrosine phosphorylation sites (25, 26), it is possible that thevarious receptor-associated proteins have distinct bindingsites on the PDGFR.The data presented indicate that two PDGFR kinase insert

tyrosine residues, Y740 and Y771, differentially regulate thebinding of GAP and PI3K in vitro. From the comparison ofbinding of GAP and P13K with that of a panel of mutants, itappears that both Y751 and Y740 are required for binding ofP13K, whereas Y771 is required for binding of GAP. Inaddition, these two proteins bind the PDGFR in the absenceof other cell proteins and are unable to compete for oneanother's binding sites. These studies indicate that the invitro bindings of GAP and P13K are independent events andthat these two proteins have distinct binding sites within thePDGFR 1-subunit kinase insert region.

MATERUILS AND METHODS

Materials. Human GAP, expressed in baculovirus (BV)-infected Spodoptera frugiperda Sf9 cells, was purified tonear homogeneity and was a generous gift of Frank McCor-

mick (Cetus Corp.). Highly purified bovine PI3K activitycontaining both the 110- and 85-kDa subunits, but no otherprotein detectable by silver staining, was graciously pro-vided by Chris Carpenter and Lewis Cantley (Tufts Univer-sity). The 1G2 mouse monoclonal antiphosphotyrosine anti-body (a gift of Raymond A. Frackelton [Brown University])was purified on a protein A-Sepharose column as describedpreviously (15); the PY20 phosphotyrosine monoclonal an-tibody was purchased from ICN.

Cells. TRMP cells (canine kidney epithelial) expressing thevarious PDGFR mutants were maintained in Dulbecco'smodified Eagle's medium supplemented with 0.5 mg of G418per ml and 10% fetal bovine serum. The F740 and F771mutants were made by subjecting the wild-type PDGFR tooligonucleotide-directed mutagenesis as described previ-ously (22, 25). The F740/F751/F771 triple mutant was madeas follows. The AatII-BamHI fragment of F771 in RR4 (25)was subcloned into F740 in RR4 to generate F740/F771.Y751 of this construct was converted to phenylalanine bysite-directed mutagenesis using the oligonucleotide and ap-proach described previously (25), resulting in the F740/F751/F771 triple mutant. The F740/F751 double mutant wasobtained by subcloning the AatII-BamHI fragment of thewild-type PDGFR in RR4 into the F740/F751/F771 triplemutant described above. The RR4 insert containing thesemutations was subcloned into the full-length PDGFR, and allof the receptor constructs were subcloned in the pLXSNretroviral vector (37); the T2 and PA317 packaging cell lineswere used to obtain amphotropic virus, and the TRMP cellswere infected and selected in G418 (1 mg/ml) as describedpreviously (25). Results of quantitative Western immunoblotanalysis indicate these cells express 6 x 106 receptors percell (22).

Immunoprecipitation. TRMP cells expressing the wild-type or mutant receptors were grown to confluence in 10%fetal bovine serum, the medium was switched to Dulbecco'smodified Eagle's medium supplemented with 0.1% calf se-rum overnight, after which time the cells were lysed in EB(24) and the PDGFR was immunoprecipitated either with apolyclonal rabbit serum (2678) raised against the carboxy-terminal tail of the PDGFR 1 subunit (26) or with a mousemonoclonal antibody (PR7212) recognizing an extracellularepitope of the human PDGFR ,B subunit (17). Immunecomplexes were collected on a suspension of formalin-fixedStaphylococcus aureus, spun through a 600-,ul cushion ofEB-10% sucrose, washed twice with 1.0 ml of RIPA (23),twice with 1.0 ml of PAN plus 0.5% Nonidet P-40 (NP-40)(24), and twice with 1.0 ml of PAN, resuspended in PAN,and stored at -70°C.

In vitro binding assay. Receptor immunoprecipitates rep-resenting 106 cells (6 x 1012 receptors) were autophosphor-ylated by incubation with 50 ,uM ATP-10 mM MnCl2-20 mMpiperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES; pH7.0)-20 ,ug of aprotinin per ml for 10 min at 30°C, after whichthe ATP was removed by washing the immunoprecipitatesonce in 250 ,ul of PAN. The basal activity of the receptor inthese immunoprecipitates isolated from unstimulated cellswas high, and adding PDGF to the in vitro kinase assay didnot markedly affect the receptor's kinase activity, nor did itaffect the sites or stoichiometry of phosphorylation (25, 27);therefore, PDGF was not added to the in vitro PDGFRphosphorylation reaction mixtures. To prepare cell lysates,serum-starved TRMP cells expressing empty vector werelysed in VIB (150 mM NaCl, 10 mM PIPES [pH 7.0], 0.1%NP-40, 20 ,ug of aprotinin per ml, 3 mM dithiothreitol, 10mMEDTA, 1 mg of bovine serum albumin per ml, 1 mg of

VOL. 12, 1992

2536 KAZLAUSKAS ET AL.

C:

cD 1)( ?D

0 0.6 1.8 6.3 (a co

ATP: + + + + - + + +

_ - __GAP

1 2 3 4 5 6 7 8 9 10

FIG. 1.- Binding of GAP to phosphorylated wild-type PDGFRsfrom cell lysates. PDGFR immunoprecipitates representing 6 x 1012receptors prepared from resting TRMP cells expressing the wild-type human PDGFR ,B subunit were incubated with ATP (+) or inbuffer (-), washed to remove ATP, and then incubated withphosphatases (P'ase) or phosphatases blocked with phosphataseinhibitors (P'ase + Inhib.). The receptor immunoprecipitates were

incubated in 0.9 ml of VIB (cell lysis buffer) containing no cell lysate(lane 1) or increasing amounts of lysate representing 0, 0.6 x 106, 1.8x 106, or 6.3 x 106 TRMP cells. Each of the samples in lanes 5 to 8received lysate representing 3.5 x 106 TRMP cells. Following a 2-hincubation, unbound proteins were washed away, and the sampleswere resolved on a 7.5% acrylamide-0.193% bisacrylamide SDS-polyacrylamide gel. Proteins were transferred to Immobilon, and thereceptor-associated GAP was detected by Western blot analysis.Lanes 9 and 10 are total cell lysate representing 3.5 x 104 and 1.4 x

105 TRMP cells, respectively, expressing an empty expressionvector. Samples in lanes 1 to 4 were prepared with monoclonalantibody PR7212, and samples in lanes 5 to 8 were prepared withpolyclonal antiserum 2678; all lanes contained similar levels ofPDGFR (not shown).

ovalbumin per ml, 1 mM phenylmethylsulfonyl fluoride, 2mM Na3VO4), the insoluble debris was cleared by centrifu-gation at 15,000 x g for 30 min at 4°C, and lysate represent-ing 3.5 x 106 cells was combined with the buffer- orATP-treated PDGFR immunoprecipitate in a final volume of300 pl of VIB. This quantity of cell lysate was sufficient tosaturate all of the binding sites for GAP (Fig. 1) and PI3K(26) and was routinely used unless otherwise indicated. Celllysates and receptor immunoprecipitates were incubated at4°C for 1.5 h with mixing; then receptors were washed twicein 1.0 ml of RIPA, twice in 1.0 ml of PAN plus 0.5% NP-40,and twice in 1.0 ml of PAN and analyzed for associated P13Kor GAP as described below. The phosphorylated PDGFRwas not detectably dephosphorylated during the incubationwith cell lysate in the in vitro binding assay.To bind purified GAP or P13K, the pure proteins were

diluted in VIB and preincubated with formalin-fixed S.aureus, the S. aureus was removed by centrifugation, andthe precleared GAP or PI3K was used in place of cell lysatesexactly as described above.To prebind the receptor with the aP751 antiserum (raised

against the P751 peptide [DESVDY751VPMLDMK] [26]),the receptor was phosphorylated with ATP and washed asdescribed above, 2 RI of aP751 or 2 RI of aP751 blocked with1 mM (final concentration) of the P751 peptide was added tothe PDGFR in a total volume of 25 ,u of PAN, the mixturewas incubated in ice for 30 min, the cell lysate was added,and the in vitro binding assay was performed as describedabove.Phosphorylated PDGFR immunoprecipitates were phos-

phatase treated for 15 min at 30°C in 10 pI of phosphatasebuffer (PAN with 20 ,ug of aprotinin per ml, 20 ,uM leupeptin,and 1 mM MgCl2) containing 0.08 U of bacterial alkalinephosphatase (Sigma) and 0.1 U of potato acid phosphatase

(Sigma). In certain samples, the phosphatase activity wasblocked by adding 2 mM Na3VO4, 5 mM NaF, 5 mM1-glycerolphosphate, and 5 mM p-nitrophenyl phosphate(PNPP). A cocktail of these inhibitors was added at the startof the incubation at 30°C, again after 5 min, and then after 10min at 30°C, so that the final concentration was 6 mMNa3VO4, 15 mM NaF, 15 mM ,B-glycerolphosphate, and 15mM PNPP. Following the incubation with phosphatases orphosphatases and inhibitors, the receptor immunoprecipi-tates were washed two times in PAN supplemented with 2mM Na3VO4 and 1 mM PNPP and then used in the in vitrobinding assay without any additional modifications.

In vivo binding. The association of GAP and P13K activityin an intact cell was assayed exactly as previously described(22, 25, 26).Western blot analysis. The PDGFR and associated proteins

were resolved on a 7.5% acrylamide-0.193% bisacrylamidesodium dodecyl sulfate (SDS)-polyacrylamide gel, trans-ferred to Immobilon (Millipore), incubated for 1 h at roomtemperature in BLOTTO (1% nonfat dry milk-0.05% Tween20-0.005% NaN3 in Western rinse [155 mM NaCl, 10 mMTris, pH 7.5]), and then probed with the primary antibodyfor 3 to 4 h at room temperature or overnight at 4°C. Todetect GAP, the primary antibody was anti-GAP B (28)diluted 1/200 in BLOTTO; to detect the PDGFR, a 1,000xdilution of an antibody raised against the carboxy-terminaltail of the human PDGFR 1B subunit (2897) was used.Following incubation with the primary antibody, the blotswere rinsed with Western rinse and probe for 3 to 4 h at roomtemperature with a goat anti-rabbit antibody conjugated toalkaline phosphatase, the blot was washed again with West-ern rinse and then with APB buffer (100 mM Tris [pH 9.5],100 mM MgCl2, 50 mM NaCl), and developed in APB buffercontaining 0.33 mg of p-nitroblue tetrazolium per ml and0.165 mg of 5-bromo-4-chloro-3-indolylphosphate toluidiumsalt per ml.P13K assay. The receptor-associated P13K activity was

assayed exactly as previously described (25) except the PIsubstrate was resuspended in dimethyl sulfoxide and incu-bated with the immunoprecipitates at room temperature asdescribed previously (12) instead of by using sonicated PI inan aqueous buffer.DNA synthesis. The ability of PDGF to initiate DNA

synthesis in the TRMP cells expressing the various PDGFRmutants was assayed as previously described (27).

RESULTS

GAP binds to the phosphorylated PDGFR in vitro. We havepreviously reported that in intact cells, GAP associates withthe wild-type PDGFR and that receptor mutants that arekinase inactive or lack phosphorylation sites are less able toassociate with GAP (28). These studies suggested that re-ceptor phosphorylation is required for GAP binding. To testthis possibility, the contribution of receptor tyrosine phos-phorylation to GAP binding was determined by using the invitro binding assay employed to characterize association ofP13K with the PDGFR (26).PDGFRs that have no phosphotyrosine (isolated from

unstimulated cells) bind very little GAP, whereas preincu-bating the PDGFR with ATP dramatically increased theamount of GAP that associates (Fig. 1; compare lanes 5 and6). If the ATP-treated (i.e., autophosphorylated) PDGFRsare exposed to phosphatases, but not phosphatases in whichthe activity has been blocked by phosphatase inhibitors,prior to the addition of cell lysates, then the amount of GAP

MOL. CELL. BIOL.

BINDING OF GAP AND PI 3-KINASE TO THE PDGFR C SUBUNIT 2537

that associates is reduced to the basal level (lanes 7 and 8).Given that the PDGFR autophosphorylates exclusively ontyrosine residues, these data indicate that the in vitro bindingof GAP requires receptor tyrosine phosphorylation.

Binding of GAP in vivo occurs simultaneously with itstyrosine phosphorylation, so it is possible that tyrosinephosphorylation of GAP is a prerequisite for its associationwith the PDGFR. GAP bound to the PDGFR from lysates ofquiescent cells (Fig. 1), which contain no detectable ty-rosine-phosphorylated GAP (6, 27, 38). Furthermore, the invitro binding reaction was performed in the presence of 10mM EDTA, which inhibits the enzymatic activity of proteinkinases, so it is very unlikely that GAP became tyrosinephosphorylated during the binding assay. Thus, consistentwith the work of Anderson et al. (1), it appears that GAPdoes not need to be tyrosine phosphorylated in order tostably associate with the PDGFR.To calculate the approximate affinity for the binding of

GAP to the phosphorylated PDGFR, the maximal amount ofGAP that bound to a fixed quantity of PDGFR was deter-mined. GAP binding was saturable and reached a maximumat less than or equal to the value for lysate representing 1.8x 106 cells (Fig. 1, lanes 1 to 4 and 6). Comparison of thesignal intensity from a known amount of purified GAP (seebelow) and a known amount of cell lysate (Fig. 1, lanes 9 and10) indicates that the amount of GAP present in TRMP cellsis approximately 3.5 x 10-18 mol per cell, so that 6.3 x 10-12mol of GAP binds at saturation. The binding reactions wereperformed in 0.9 ml so that the estimated Kd is less than 3.5x 10-9 M. Note that these calculations involve severalassumptions, so that the values obtained are only approxi-mations.Our estimate ofKd for the in vitro binding of PI3K activity

is 2 x 10-11 (26), which is nearly 100 times greater than theupper limit for the Kd of GAP. To test whether P13K bindsmore tightly than GAP, the levels of binding of GAP andPI3K activity were compared in the presence of a number ofdifferent agents. GAP and P13K binding was not reduced by0.6 M urea or 2 mM phosphotyrosine (not shown). Incontrast, GAP binding was reduced to nearly the basal levelwhen the binding was done at a final NaCl final concentra-tion of 1.8 M, whereas the recovery of PI3K activity was notaffected (Fig. 2). Similar quantities of receptor were presentin all samples (not shown). Thus, it appears that P13Kactivity has a higher affinity for the PDGFR than does GAP.The observation that the phosphorylated PDGFR binds

GAP from cell lysates raises the question of whether thisinteraction is direct or involves an intermediary protein. Todistinguish between these two possibilities, highly purifiedGAP expressed in Sf9 cells (BV GAP) was tested for itsability to bind to PDGFRs in vitro. The in vitro bindingprotocol described above was used except that BV GAPdiluted in VIB (the lysis buffer) was used instead of celllysates. Mock immunoprecipitates prepared from TRMPcells expressing an empty expression vector contained noPDGFRs and bound only very low levels of GAP (Fig. 3A,lanes 1 and 2). The autophosphorylated PDGFRs boundvastly greater amounts of GAP than did receptors that haveno phosphotyrosine (lanes 3 and 4). Estimates of the Kd ofbinding of BV GAP were also in the nanomolar range. Just aswith cell lysates, the BV GAP did not bind when thephosphorylated PDGFR was preexposed to phosphatasesbut not to phosphatases that had been blocked with inhibi-tors (lanes 5 to 7). Thus, it appears that like the GAP in celllysates, highly purified BV GAP requires receptor phosphor-ylation for stable association. Furthermore, although the

A

B

l IZ~-- GAP

ATP: + + + +

NaCI 0 0.2 06 1 8

1..^.i _ PIP

......_p

ATP: - + + + 1

NaCI: 0 0.2 0.6 1 8

FIG. 2. Binding of GAP and PI3K in the presence of increasingconcentrations of salt. NaCl was added to the indicated finalconcentration (molar) to lysates of TRMP cells harboring an emptyvector. The cell lysates were then combined with PDGFR immuno-precipitates that had been preexposed to buffer (-) or ATP (+);after 1.5 h, the immunoprecipitates were washed and split into twosamples. Receptor-associated GAP was detected by Western blotanalysis (A); PDGFR-associated PI3K activity was measured in aP13K assay as described in Materials and Methods B). PI marks theposition of PI4-phosphate.

contribution of minor contaminating proteins present ineither the receptor immunoprecipitate or the preparation ofBV GAP cannot be completely ruled out, these experimentsindicate that GAP and the phosphorylated PDGFR interactdirectly, without an intermediary protein.The data presented above demonstrate that GAP associ-

ates only with receptors that have phosphotyrosine. If this istrue, then one would predict that masking the phosphoty-rosine epitopes with a phosphotyrosine antibody wouldprevent the association of GAP. Indeed, phosphorylatedPDGFRs that were preincubated with an antiphosphoty-rosine antibody prior to their exposure to BV GAP boundbarely detectable levels of GAP (Fig. 3B; compare lanes 2and 3). Phosphotyrosine antibody that had been blockedwith phosphotyrosine, a preimmune serum, or PR7212(which recognizes an extracellular epitope of the receptor)had little effect on subsequent GAP binding (lanes 4 and 5and data not shown). These observations support the notionthat receptor tyrosine phosphorylation is required for the invitro binding of GAP.

Binding of GAP to PDGFR mutants. The experimentsdescribed above indicated that receptor phosphorylation isrequired for GAP binding to the PDGFR. To identify whichtyrosine residues need to be phosphorylated in order forGAP to bind, we tested a panel of PDGFR phosphorylationsite mutants. Mutating Y740, Y751, or both of these residueshad little effect on GAP binding, whereas the F771 andF740/F751/F771 mutants failed to associate with detectableamounts of GAP (Fig. 4A; Table 1). Y716, Y763, Y775, andY778 are also in the kinase insert, but the correspondingphenylalanine substitution mutants bind wild-type levels ofGAP (data not shown). The quantities of PDGFR in thesevarious immunoprecipitates were similar (Fig. 4A), so thatthe observed differences in binding of GAP were not due todifferences in amount of receptor. GAP also failed to bindthe kinase inactive PDGFR and was consistently less able tobind the F857 mutant (data not shown). Since the F857receptor is an intrinsically poorer kinase (10, 27), the reduc-tion in GAP binding to F857 receptors may reflect a reducedability to phosphorylate Y771. Alternatively, phosphoryla-tion of Y857 may contribute to the GAP binding site.

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2538 KAZLAUSKAS ET AL.

A

0

ATP:

B

4-

ca

ir r

....~~~~~~~~~~~~~~~~~~~~~~~.l~~~~~~1 2 3 4 5 6 -7

a[ 2Y1 21 PI

_ PDGFR

-"GAP

ATP: - + + + +

_0-0- GAP

1 2 3 45

FIG. 3. Binding of highly purified BV GAP to the PDGFR. (A)Resting TRMP cells expressing an empty expression vector (0;lanes 1 and 2) or the wild-type human PDGFR subunit (WT; lanes3 to 7) were immunoprecipitated with an anti-PDGFR antibody(2678); the immunoprecipitates were incubated with buffer (-) or

buffer containing ATP (+), washed, and incubated with phosphatasebuffer (lanes 5 to 7) supplemented with nothing (lane 5), bacterialalkaline and potato acid phosphate (P'ase; lane 6), or the phos-phatases blocked with phosphatase inhibitors (P'ase + Inhib.; lane9). The immunoprecipitates were washed again and exposed to 0.2p.g of GAP purified from BV-infected Sf9 cells, the samples were

resolved on 7.5% acrylamide-0.193% bisacrylamide SDS-polyacryl-amide gel and transferred to Immobilon, and the upper and lowerhalves of the blot were probed with anti-PDGFR and anti-GAPantibodies, respectively. (B) The wild-type PDGFR was immuno-precipitated and phosphorylated as described above and then incu-bated in PAN containing no antibody (lanes 1 and 2), 0.1 ,ug of eachPY20 and 1G2 monoclonal phosphotyrosine antibody (aPY; lane 3),2 ,ug of PR7212 (lane 4), or 2 ,ul of crude preimmune serum (PI; lane5). The immunoprecipitates were then subjected to the standard invitro binding assay using 0.2 ,ug of BV GAP, and the amount of GAPthat bound was detected with an anti-GAP antibody as described forpanel A.

Analysis of these mutants indicate that the in vitro binding ofGAP absolutely requires that the receptor have kinaseactivity and tyrosine at position 771 in the kinase insert.The data presented in Fig. 4 are for in vitro binding. We

have found that GAP associates with these PDGFR mutantsin an intact cell identically as they do in vitro (Fig. 5; Table2). Note that the experimental variation in the amount ofreceptor-associated GAP and P13K (see below and reference22) was at times up to twofold and is within the estimatederror inherent to these experiments.GAP and P13K bind to distinct regions of the PDGFR. To

determine whether GAP and P13K have independent bindingsites, the requirements for association of GAP and P13Kwith the PDGFR were contrasted. Whereas the F771PDGFR mutant is greatly impaired in its ability to bind GAP(Fig. 4A and 5A, Tables 1 and 2), analysis of P13K binding to

the F771 mutant demonstrated wild-type levels of associa-tion with P13K (Fig. 4B and 5B, Tables 1 and 2). The F740mutant has the reciprocal phenotype: wild-type levels ofGAP bind, but only 3% of P13K activity associates (Fig. 4

and 5, Tables 1 and 2). In addition, the F740/F751 mutantbinds no detectable P13K activity but binds wild-type levelsof GAP (Fig. 4 and 5). The four other insert tyrosines (Y716,Y763, Y775, and Y778) were not required for binding ofPI3K (data not shown). Importantly, the levels of binding ofPI3K to the PDGFR mutants were identical in vitro and invivo (Fig. 4B and SB). Note that a small amount of P13Kassociated with the wild-type, F740, and F751 receptors inunstimulated intact cells (Fig. 5; Table 2). This probablyrepresents low levels of basal phosphorylation at Y740 andY751, since the F740/F751 and F740/F751/F771 mutantsbound no detectable P13K activity in unstimulated cells (Fig.5; Table 2). These studies indicate that associations of GAPand P13K are both dependent on receptor phosphorylationbut appear to have distinct binding sites within the kinaseinsert.The possibility that GAP and P13K bind different regions

of the PDGFR was further investigated by testing the abilityof the aP751 antibody to block binding of GAP. aP751 is anantibody raised against the 13 amino acids immediatelysurrounding Y751 (DESVDYVPMLDMK) and sharply re-duces the in vitro binding of P13K (26). PDGFR immunopre-cipitates were autophosphorylated, incubated with the aP751antibody or aP751 that had been blocked with the P751peptide, and exposed to cell lysates, and associated GAPand P13K activity were determined as described in Materialsand Methods. GAP binding was unaffected by the aP751antibody, whereas the amount of P13K activity was reducedto approximately 23% of the wild-type level (Fig. 6). Notethat the F751 receptor mutant binds 23% of the wild-typelevel of P13K (Fig. 4B; Table 1). Thus, binding of GAP andbinding of P13K activity are differentially affected by theaP751 antibody, consistent with the possibility that thesetwo proteins have distinct binding sites.GAP and PI3K bind independently to the PDGFR. The in

vitro binding of GAP and P13K permits the investigation ofthe interaction of these proteins with each other and with thereceptor. If GAP and P13K bind to the PDGFR indepen-dently, then preloading the receptor with saturating doses ofone of the proteins should not affect the subsequent bindingof the other protein. To avoid possible competition withother cell proteins, highly purified BV GAP and bovine brainP13K were used instead of cell lysates as the source of GAPand PI3K. PDGFR immunoprecipitates were incubated withincreasing concentrations of either pure GAP or P13K todetermine the saturating dose of each of these proteins.Receptors that had bound a saturating amount of GAP (Fig.7A, lanes 1 to 8) could still bind nearly as much P13K as didreceptors that had no GAP on them (Fig. 7B, lanes 7 to 10).Similarly, PDGFRs that were preloaded with PI3K couldassociate with as much GAP as did receptors that had noPI3K bound to them (Fig. 7A, lanes 7, 8, 11, and 12). Theamount of prebound P13K or GAP was not dramaticallydisplaced by subsequent exposure to the reciprocal bindingprotein (Fig. 7A, lanes 7 to 10; Fig. 7B, lanes 7, 8, 11, and12). Note that P13K appears to associate with the PDGFRmore tightly than does GAP (Fig. 2), so P13K should havebeen able to displace GAP if the two proteins had the samebinding sites. These experiments indicate that (i) purifiedGAP and PI3K bind to the PDGFR directly, without theintervention of other cell proteins, and (ii) GAP and P13Kassociate with the receptor independently, suggesting dis-tinct binding sites.

Ability of PDGFR mutants to trigger DNA synthesis. Todetermine the biological consequences of the failure of GAPor P13K to associate with the PDGFR, we measured the

MOL. CELL. BIOL.

BINDING OF GAP AND PI 3-KINASE TO THE PDGFR P SUBUNIT 2539

A

_ PDGFR

" GAP

ATP: - + - + - + - + + +

N WT F740 F751 F771 40/51 40/51/71

B

_ PIP

ATP: + - + + + + + +

N WT F740 F751 F771 40/51 40/51/71

FIG. 4. In vitro binding of GAP and PI3K to a panel of PDGFR mutants. Resting TRMP cells expressing similar levels of the variousPDGFR constructs were immunoprecipitated with a PDGFR antibody; the receptors were phosphorylated in vitro (+), exposed to lysates ofTRMP cells expressing an empty expression vector (N), washed, and subjected to Western blot analysis using PDGFR antibodies or GAPantibodies (top and bottom portions of panel A) or to a P13K assay (B). The arrowheads labeled PDGFR and GAP denote the positions ofthe immunoreactive PDGFR and GAP proteins, respectively. (B) Autoradiogram from the PI3K assay. The arrowhead labeled PIP marks theposition of PI 4-phosphate. WT, wild-type human PDGFR ,1 subunit; F740, F751, F771, 40/51 (F740/F751), and 40/51/71 (F740/F751/F771),mutants in which phenylalanine has been substituted for the corresponding tyrosine.

ability of the various PDGFR mutants to initiate DNAsynthesis in response to PDGF stimulation. Subconfluent,quiescent TRMP cells expressing similar numbers ofPDGFRs were stimulated with the indicated concentrationof PDGF, and the extent of [3H]thymidine incorporation wasdetermined as previously described (27). The ability of F740and F771 to stimulate DNA synthesis was identical to that ofthe wild-type receptor (Fig. 8A). Consistent with our previ-ous observations, the F751 mutant as well as the multiplemutants that include the F751 mutation show a small butconsistent reduction in mitogenic response. The cells ex-pressing an empty expression vector (N in Fig. 8A) failed torespond to even the highest doses of PDGF, as expected,since the TRMP cells bear no endogenous PDGFRs (25).Figure 8B is a PDGFR Western blot of total cell lysatesdemonstrating that the various cell lines express similarnumbers of PDGFR.

TABLE 1. Quantitation of the amount of GAP and PI3K thatbinds to PDGFR mutants in vitroa

Fraction of wild-type levelConstruct

PDGFR GAP PI3K activity

WiT 1.0 1.0 1.0F740 1.3 1.8 0.03F751 0.7 1.0 0.23F771 0.7 0.0 1.7F740/F751 1.0 2.3 0.0F740/F751/F771 1.4 0.0 0.0

a The data in Fig. 4 were quantitated either densitometrically (using an

Apple 1 scanner and Image 1.35 software [Fig. 4A]) or by counting theradioactivity in the PI 4-phosphate spot in a scintillation counter (Fig. 4B).Data are for the ATP-treated samples. determination. The values for GAP andP13K have been corrected for the amount of receptor present.

DISCUSSION

The in vitro association of GAP with the PDGFR requiresthat the receptor be tyrosine phosphorylated. In contrast,receptor-catalyzed tyrosine phosphorylation of GAP is notrequired for binding, since GAP present in extracts ofunstimulated cells can bind to the PDGFR, and bindingoccurs under conditions that do not allow phosphorylation.Comparison of the binding of GAP and PI3K with that of a

panel of receptor mutants, as well as the ability of anantibody to the PDGFR kinase insert to inhibit binding,indicates that GAP and P13K bind to distinct regions of thePDGFR subunit kinase insert. In addition, both GAP andP13K bind independently of one another, without the inter-vention of other cellular proteins.

Binding of the receptor-associated proteins requires thatthe PDGFR be tyrosine phosphorylated. Our previous stud-ies have indicated that Y751 is important for binding of PI3K(25, 26). We have subsequently found that Y740, which isalso a tyrosine phosphorylation site in the PDGFR kinaseinsert (22), is also important for the stable association ofPI3K activity (Fig. 4 and 5) (22). When both Y740 and Y751are mutated, we can detect no associated PI3K activity,even on long exposures of the autoradiograms. Thus, itappears that no other tyrosines can act in place of Y740 andY751 as substrates of the PDGFR and as binding sites forPI3K. In contrast, GAP binding requires that Y771 bepresent, while Y740 and Y751 are dispensable. Given thatY771 is also a PDGFR phosphorylation site (22) and that thePDGFR must be phosphorylated in order for GAP to stablyassociate, it appears that phosphorylation of Y771 triggersGAP binding.

Several lines of evidence argue that at least a portion ofthe binding sites for GAP and P13K lie within the kinaseinsert immediately surrounding the tyrosine residues thatmust be phosphorylated in order for them to associate.

I 9'.9

VOL. 12, 1992

2540 KAZLAUSKAS ET AL.

A

_ PDGFR

GAP

PDGF - + - + - + - + - + - + - +

N WT F740 F751 F771 40/51 40/51/71

B

_PIP

PDGF + + + + + - + - +N WT F740 F751 F771 40/51 40/51/71

FIG. 5. Binding of GAP and P13K to PDGFR mutants in intact cells. Confluent, quiescent cultures of TRMP cells expressing similarnumber of PDGFR mutants were left resting (-) or stimulated for 5 min at 37°C with 40 ng of PDGF-BB per ml, lysed, and immunoprecipitatedwith a PDGFR antibody. Receptor immunoprecipitates representing 1.5 x 106 cells were subjected to PDGFR (top portion of panel A) or GAP(bottom portion of panel A) Western blot analysis. (B) Receptor immunoprecipitates representing 5 x 105 cells were subjected to a PI3Kassay; the autoradiogram is presented. Arrowheads mark the positions of the PDGFR, GAP, and PI 4-phosphate (PIP).

Antibodies to the kinase insert region that includes Y751 can

block the binding of P13K (Fig. 7) (26). This possibility isfurther supported by the work of Escobedo et al. (7) dem-onstrating that a peptide with phosphate at position Y740 orY751 (Y708 or Y719, respectively, in the mouse PDGFRsequence) effectively competes the in vitro binding of PI3Kactivity. In addition, phosphorylated Y771 peptides, but notthe phosphorylated Y740 or Y751 peptide, can block the invitro binding of GAP (54). Finally, P13K activity can bepurified on a phosphorylated peptide corresponding to theregion surrounding Y751 (42). Together, these data stronglysuggest that GAP and P13K bind to the kinase insert regionof the PDGFR immediately surrounding Y771 and Y740/Y751, respectively.Recent reports indicate that one of the mechanisms by

which cellular proteins bind to phosphotyrosine-containingproteins is via SH2-containing domains (1, 7, 11, 31, 34, 36).The known SH2 domains do not have identical amino acidsequences (31), and the various SH2 domains have differentaffinities for phosphotyrosine-containing proteins (1). The

two reported 85-kDa subunits of PI3K each have two non-

identical SH2 domains (8, 42, 48). The F740 mutant routinelybinds much less P13K than does the F751 mutant (Fig. 4Band SB), consistent with the possibility that these tworegions of the kinase insert have different affinities for P13K(22). The difference in ability of P13K binding may be furtherintensified if the two SH2 domains of the 85-kDa subunit ofP13K also exit distinct affinities for the PDGFR. Like thePDGFR ,1 subunit, the kinase insert of the PDGFR a subunithas tyrosine residues homologous to both Y740 and Y751,and Yu et al. (56) recently reported that substitution of eitherof these tyrosine residues with phenylalanine dramatically

A B

_ PIP

PDGFR

GAP --_

TABLE 2. Quantitation of the amount of GAP and P13K thatassociates with PDGFR mutant in vivo

Fraction of wild-type level

Construct P13K activityPDGFR GAP

Control +PDGF

Wild type 1.0 1.0 0.02 1.00F740 1.1 1.0 0.02 0.04F751 0.9 0.7 0.03 0.12F771 1.0 0.0 0.04 0.58F740/F751 1.0 2.3 0.00 0.00F740/F751/F771 1.1 0.0 0.00 0.00

a The data presented in Fig. 5 were quantitated as described in the footnoteto Table 1. No GAP was detected in samples from unstimulated cells. Both theGAP and P13K measurements were corrected for the amount of receptorpresent.

ATP. + + +

e N)L0CO>

4 *ATP

......

r- r-

CL CLm {M

FIG. 6. Evidence that an antibody to the 751 region of the kinaseinsert does not block binding of GAP. PDGFR immunoprecipitatesprepared from resting TRMP cells expressing the wild-type PDGFRwere autophosphorylated in vitro (+), preincubated with an anti-peptide antiserum to a 13-amino-acid region surrounding Y751(aP751) or to the same antiserum that had been blocked with peptide(aP751/B), exposed to lysates of TRMP cells, and washed, and thesamples were subjected to Western blot analysis (A) and to a P13Kassay (B). PIP marks the position of PI 4-phosphate. The amount ofradioactive PI 4-phosphate was quantitated by scintillation count-ing. The aP751 antiserum reduced binding to 23% of that in lane 2;the P751/B sample (lane 4 of panel B) contained 73% of the activityin lane 2.

a!f lI'P0@RLst

L":: ;:.

. ,*a , *

MOL. CELL. BIOL.

BINDING OF GAP AND PI 3-KINASE TO THE PDGFR , SUBUNIT

1 2 3 4 5 6 7 8 9 10 11 12

m;e 4 _ --GAP

7+ I I .-

ugsGAP: 0 0.2 0.5 1.5 1.5 1.5GAP P13Kthen P13K then GAP

2 3 4 5 6 7 8 9 10 11 12 13 14--11 r----l------- --------1 --w----.. _|:.. 71

.:::: :.

.. ... '.

'X't_ - --"|--i-------jj--- - .. .. , 'f - + -

ng P13K: 0 2 6 18 18 18 18GAP P13Kthen P13K then GAP

FIG. 7. Evidence that P13K and GAP do not compete for the same binding sites on the PDGFR. Wild-type PDGFR immunoprecipitatesrepresenting 1.2 x 106 receptors were permitted to autophosphorylate (+), incubated with increasing amounts of purified GAP (panel A, lanes1 to 8) or purified PI3K (panel B, lanes 1 to 8), and subjected to a GAP Western or P13K assay. In panels A and B, lanes 9 and 10, the receptorswere first incubated with the indicated saturating dose of GAP, washed, exposed to the indicated saturating dose of P13K, and then subjectedto both GAP Western and P13K analyses. In panels A and B, lanes 11 and 12, the reciprocal experiment was performed in which receptorswere exposed first to PI3K and then to GAP. Lanes 13 and 14 of panel B are samples in which the highest dose of PI3K was incubated withmock immunoprecipitates (prepared with antibody from TRMP cells expressing an empty vector [20]) and indicate nonspecific binding of pureP13K. In panel B, lane 15, 2 ng of the pure P13K was assayed as a positive control for the P13K assay. Comparison of the intensity of thepure P13K signal and what bound to the PDGFR at saturation indicates that 1 to 2% of the input PI3K activity associated with the PDGFR.PIP, PI 4-phosphate.

reduced PI3K binding. Thus, both the PDGFR a and lsubunits have two kinase insert tyrosines that are requiredfor maximal binding of PI3K. The emerging theory is thatPI3K tightly binds to PDGFRs with phosphate at both Y751and Y740 but only weakly to receptors with only one of thesesites phosphorylated (22).GAP binding has an absolute requirement for tyrosine at

residue 771 in the kinase insert. Both c-fns and c-kit do notbind GAP (44, 45), and they do not have a Y771 homolog.The position of Y771 within the kinase insert appears not tobe critical for GAP binding, since kinase insert deletionmutants that move the Y771 region in the N- or C-terminusdirection do not affect the ability of GAP to associate (22).Thus, it seems likely that as with the putative PI3K bindingsite, the amino acids immediately surrounding Y771 are

important for GAP binding.We have previously reported that only 20% of the wild-

type level of GAP binds to the F751 PDGFR mutant (28),whereas in more recent in vitro and in vivo studies (Fig. 4and 5) (22), the same mutant associates with nearly wild-typelevels of GAP. These differences may arise from using cellswith different numbers of expressed PDGFR. The recentstudies have been with cells expressing approximately 16-fold more receptors per cell, such that the GAP/PDGFRratio is lower in these new cells. When sufficiently highconcentrations (1 to 2 ,ug/ml) of GAP are used in vitro, GAPdoes associate with the F771 mutant (unpublished observa-tions). Thus, the specificity of the association of GAP andthe PDGFR can be diminished when the binding reaction isforced with high concentrations of GAP.The observation that numerous proteins associate with the

activated PDGFR has raised the possibility that receptor-associated proteins interact with each other as well as the

receptor during the course of forming a stable complex. Ifinteractions between GAP and PI3K occur, they do not alterthe ability of these proteins to associate with the PDGFR(Fig. 7). Note that both the 85- and 110-kDa proteins of P13Kbound to the PDGFR in vitro (unpublished observations).These studies indicate that both PI3K activity and GAP binddirectly to the PDGFR without the assistance of any othercellular proteins.The distinct tyrosine requirements for binding of PI3K and

GAP and the fact that GAP and PI3K do not compete eachother's association with the PDGFR indicate that thesemolecules bind different regions of the PDGFR. An alternateexplanation of why GAP and PI3K do not compete eachother's binding is that they bind distinct PDGFR molecules.This would happen if discrete PDGFR molecules containedphosphotyrosine at Y740 or Y751 and Y771. In vivo activa-tion of the receptor leads to receptor subunit dimerizationand transphosphorylation (29), so that receptor dimers maycontain nonidentically phosphorylated receptor subunits andtherefore unique sets of associated proteins. We estimatethat the stoichiometry of phosphorylation of Y751, Y740,and Y771 is 33, 2, and 10% of the total tyrosine-phosphory-lated receptor population in PDGF-treated TRMP cells (22).Phosphopeptide maps of the PDGFR isolated with PDGFRor GAP antibodies are indistinguishable (unpublished obser-vations), strongly suggesting that PDGFRs associated withGAP also have Y857 and Y751 phosphorylated. Directevidence that GAP and P13K bind to the same PDGFRmolecule includes the observation that when PDGF-stimu-lated cells are immunoprecipitated with an anti-GAP anti-body, the complete complex of the PDGFR and all theassociated proteins can be recovered (28). It is possible thatGAP and PI3K coimmunoprecipitate as a result of their

A

B 1 5

PIP

VOL. 12, 1992 2541

_ - +

2542 KAZLAUSKAS ET AL.

A

0

c00(:Lrr0z

z

C.

PDG3:' ,g

Lnlr-

BP:D- -,0 -:

-o LO r- L

vM 1!W4 PPDGFR

FIG. 8. Ability of PDGFR mutants to stimulate DNA synthesis.TRMP cells expressing the various PDGFR constructs were platedin serum-free Dulbecco's modified Eagle's medium in triplicate at 4x 104 cells per well in a 24-well dish; after 2 days, the cells werestimulated with the indicated concentration of PDGF. All wells thatreceived PDGF also were supplemented with 2% horse serum,which was needed for the optimal response to PDGF. The responseto 2% horse serum was typically less than 20% of the maximalresponse in all of the cell lines and was subtracted in calculating theresponse to PDGF. Data are expressed as the percentage of maximalresponse, i.e., response to 10% fetal bovine serum. For a givensample, the standard error of the mean was routinely less than 10%.Similar results were obtained in a total of three independent exper-iments.

association with different PDGFR subunits within a receptorcomplex; however, it is possible that some PDGFR subunitscontain phosphate at Y740, Y751, and Y771 and therebyhave both PI3K and GAP associated.

In cells expressing the F771 mutant, PDGF fails to triggerboth the stable association of GAP with the PDGFR (Fig. 5)and the detectable tyrosine phosphorylation of GAP (22) butdoes stimulate wild-type levels of DNA synthesis (Fig. 8).This finding indicates that these events are not necessary forthe receptor to trigger a biological signal. Similarly, the F740receptor binds only 1% (Fig. 5; Table 2) of the wild-typelevel of P13K but triggers a normal DNA synthesis response.Like the ,13 ubunit, mutants of the PDGFR a subunit that donot bind PI3K activity are still able to relay the mitogenicsignal of PDGF (56). Even the F740/F751/F771 mutant,which does not bind GAP or P13K, is able to trigger a nearlymaximal DNA synthesis response. The surprisingly smalleffect on the ability of the PDGFR to relay a biological signalwhen it does not stably associate with GAP and P13K may bea quirk of the TRMP cells. However, when one takes intoaccount the fact that the estimate for in vivo stoichiometry ofphosphorylation of Y740 and Y751 together is 35%, while forY771 it is only 10%, and one assumes that only PDGFRmolecules phosphorylated at these tyrosine resides can bindPI3K or GAP, then the majority of the activated PDGFRs donot associate with GAP or PI3K. Indeed, we estimate that in

vivo only 4 to 8% of the PDGFRs have GAP bound, which isconsistent with the estimate that 10% of the PDGFRs arephosphorylated at Y771 (22). Given that the PDGFR isknown to initiate multiple signaling pathways, the reason thePDGFR mutants can still relay biological signals is probablythat the GAP and P13K-independent mitogenic cascadesremain fully operable.

ACKNOWLEDGMENTS

We thank Dan Bowen-Pope for the gift of PDGF and PR7212 andTony Pawson for generously providing the anti-GAP B antisera. Wealso thank Frank McCormick for purified BV GAP and ChrisCarpenter and Lewis Cantley for purified P13K. Finally, we thankGary Johnson and Dwight Klemm for critically reading the manu-script.

This work was supported in part by grants CA 28151 and BRSS07RR05842 and by the Cancer League of Colorado.

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