Ceil, Vol. 65, 75-82, April5, 1991,Copyright© 1991 by Cell Press

cDNA Cloning of a Novel 85 kd Protein That Has SH2 Domains and Regulates Binding of PI3-Kinase to the PDGF 13-Receptor Jaime A. Escobedo,*t Sutip Navankasattusss,t W. Michael Kavanaugh,t Dale Milfay,* Victor A. Fried,$ and Lewis T. Williams*t *Howard Hughes Medical Institute tCardiovascular Research Institute University of California San Francisco San Francisco, California 94143 SDepartment of Anatomy and Cell Biology New York Medical College Val Halla, New York 10595

Summary Using immobilized PDGF receptor as an affinity reagent, we purified an 85 kd protein (p85) from cell lysates and we cloned its cDNA. The protein contains an SH3 domain and two SH2 domains that are homologous to domains found in several receptor-associated enzymes. Recombinant p85 overexpressed in maremalian cells inhibited the binding of endogenous p85 and a 110 kd protein to the receptor and also blocked the association of PI3-kinase activity with the receptor. Experiments with receptor mutants and with short peptides derived from the kinese insert region of the PDGF receptor showed that the recombinant p85 binds to a well-defined phosphotyrosine-containing sequence of the receptor, p85 appears to be the subunit of PI3kinase that links the enzyme to the ligand-activated receptor. Introduction When platelet-derived growth factor (PDGF) binds to its receptor, the receptor phosphorylates itself on tyrosine residues and physically associates with several cytoplasmic molecules. These signaling molecules, which are likely to mediate the mitogenic effects of the PDGF, include tyrosine and serine kinases (Kypta et al., 1990; Morrison et al., 1989), phospholipase C-7 (Kumjian et al., 1989; Meisenhelder et al., 1989; Morrison et al., 1990; Wahl et al., 1989), GTPase activating protein (GAP) (Kaplan et al., 1990; Kazlauskas et al., 1990; Molloy et al., 1989), and phosphatidlyinositol 3-kinase (PI3-kinase) (Coughlin et al., 1989). An 85 kd phosphoprotein and a 110 kd phosphoprotein were also found to bind to the PDGF I~-receptor, but the identities of these proteins are not known (Coughlin et al., 1989; Kaplan et al., 1990; Kazlauskas and Cooper, 1989; Morrison et al., 1990; Escobedo et al., 1991). PI3-kinase phosphorylates PI at the 3 position of the inositol ring (Auger et al., 1989; Whitman et al., 1987, 1988). This enzyme was first found associated with middle-T antigen in polyoma virus-transformed cells (Whitman et al., 1985; Kaplan et al., 1986, 1987) and with v-src protein in v-src-transformed cells (Courtneidge and Heber, 1987; Fukui and Hanafusa, 1989). PI3-kinase activity was also found in anti-phosphotyrosine immunoprecipitates of cells stimulated by growth factors (Kaplan et al.,

1987; Vartikovski et al., 1989), suggesting that it plays a role in the growth of normal cells and that tyrosine phosphorylation might regulate its activity. We have shown previously that ligand stimulation of PDGF receptors induces association of the receptor with PI3-kinase (Coughlin et al., 1989). In anti-phosphotyrosine, anti-PDGF receptor, and anti-middle-T immunoprecipitates, the presence of PI3-kinase activity has been coincident with the presence of a phosphorylated p85, leading to speculation that the PI3-kinase and p85 phosphoprotein are identical (Courtneidge and Heber, 1987; Coughlin et al., 1989; Kaplan et al., 1987; Morrison et al., 1990; Escobedo et al., 1991). Recently, PI3-kinase activity was found to be associated with an 85 kd protein, supporting the notion that the p85 is an integral part of this enzyme (Carpenter et al., 1990; Morgan et al., 1990). Using an in vitro system to study the binding of PI3kinase to the receptor, we found that the PI3-kinase from BALB/c 3T3 cell lysates could bind to tyrosine-phosphorylated PDGF I~-receptors but not to dephosphorylated receptors (Coughlin et al., 1989; Escobedo et al., 1991). As in the in vivo studies, the binding of PI3-kinase enzyme activity to the receptor correlated with the presence of p85. A 110 kd protein (p110) was also found to associate with the receptor both in vivo and in vitro (Escobedo et al., 1991). Recent experiments have shown that p85, p110, and PI3-kinase bind to the same region of the PDGF receptor, suggesting that the p85 and p110 proteins are subunits of the PI3-kinase (Escobedo et al., 1991). Conventional chromatographic purification of PI3-kinase activity from bovine brain or rat liver yielded 85 kd and 110 kd proteins, adding further evidence that these proteins are subunits of the enzyme, p85 appears to be the subunit that binds directly to the PDGF receptor (Escobedo et al., 1991). In this study we have used tyrosine-phosphorylated PDGF 13-receptoras an affinity reagent to purity p85 from 3T3 cell lysates. Partial amino acid sequence was used to clone a cDNA sequence that encodes a p85 that binds directly to tyrosine-phosphorylated PDGF I~-receptors. The cDNA sequence of p85 includes sequences for one SH3 domain and two SH2 domains (Sadowski et al., 1986; Pawson, 1988). Similar domains are also found in some of the other cytoplasmic molecules that bind to the ligand-activated PDGF receptor. The recombinant p85 bound to the receptor at the same site of the endogenous p85. Overexpression of the recombinant p85 blocked the association of PI3-kinase activity and the 110 kd subunit with the receptor. These studies show that the PI3-kinase associates with the PDGF receptor by way of its 85 kd subunit, and is composed of at least one additional subunit.

Results Association of a p85 Phosphoprotein, p110 Phosphoprotein, and PI3-Kinase with PDGF Receptors Previous studies have shown that in PDGF-stimulated 3T3

Cell 76

kDa

1

2

--200

--116

PDGF:

~

kDa ~200

~

-

-

116

--

96

96

--

68

68

Table 1. Amino Acid Sequence of the 85 kd Tryptic Fragments Peptide

Sequence

K30 K194 K32 K114a K46

XXXTADGTFLVRDAXT XXFSDPLTFNXVVELIN MNDIKPDLIQL XNSlQPDLIQLR XXEYDRLYEEY

Letter X represents an unidentified amino acid residue.

--

Figure 1. PDGF-DependentAssociationofp85,p110,and PI3-Kinase Activitywith the PDGFReceptor (A) Lysatesfrom PDGF-stimulated(+) or unstimulatedcells(-) were mixed with immobilizedPDGF e-receptors. The complexeswere washed extensivelyand the receptor-associatedproteinswere detected by Coomassiestain. (B)The receptor-associatedproteinswerephosphorylatedin vitrousing [~P-13]ATP(Morrisonet al,, 1989).A Coomassiestain(lane1) and autoradiographof the samegel (lane2) are shownfor comparison. Arrowheadsindicatethe positionsof p85 and p110.

cells, unidentified 85 kd and 110 kd phosphoproteins, as well as PI3-kinase activity, coimmunoprecipitate with the PDGF receptor (Kaplan et al., 1987; Coughlin et al., 1989; Kazlauskas and Cooper, 1989; Morrison et al., 1990; Escobedo et al., 1991). These proteins were not found in PDGF receptor immunoprecipitates from unstimulated cells. Using an in vitro system we previously showed that p85 phosphoprotein, p110 phosphoprotein, and PI3kinase activity derived from lysates of unstimulated 3T3 cells bound to activated PDGF receptors immobilized on Sepharose beads (see Escobedo et al., 1991, and Figures 1A and 1B). However, when 3T3 cells were stimulated by PDGF, p85, p110, and PI3-kinase in the lysates were no longer capable of binding to the receptor in vitro, possibly because they had previously been phosphorylated by receptors prior to lysis of the cells (Escobedo et al., 1991; W. M. K., J. A. E., and L. T. W., unpublished data). This finding (Figure 1A) showed that the in vitro association is a specific PDGF-dependent process. To purify the receptor-associated proteins we incubated large quantities of cell lysates with large amounts of recombinant receptor (Figure-'lA). Under these conditions p85 and p110 that associated with the PDGF receptor were visualized by Coomassie stain (Figures 1A and 1B, lane 1) and comigrated with the radioactively labeled p85 (Figure 1B, lane 2). These findings showed that a large scale preparative procedure could be used to purify p85, which was reported previously to associate with ligand-activated PDGF receptors (Kaplan et al., 1987; Coughlin et al., 1989; Morrison et al., 1990; Escobedo et al., 1991).

Purification of the Receptor-Associated p85 and Determination of Its Amino Acid Sequences p85 was purified by affinity chromatography. Tyrosinephosphorylated PDGF receptors were immobilized on protein A-Sepharose using a PDGF receptor polyclonal antiserum (AB 77) (Keating et al., 1988). The receptorantibody complex was incubated with BALB/c 3T3 lysates.

Proteins that associated with the receptor were identified in polyacrylamide gels as shown in Figure 1A. p85 was electroeluted from the polyacrylamide gel and 20 p~g of p85 was digested with trypsin. Peptide fragments were separated by reverse-phase high pressure liquid chromatography, and the amino acid sequences of these peptides were determined by gas-phase amino acid sequencing. Several peptide sequences were obtained (see Table 1). The peptide K30, TADGTFLVRDAXT (each letter is a single amino acid representation and "X" indicates an undetermined amino acid), was used in the preparation of degenerate oligonucleotide probes for the screening of a mouse BALB/c 3T3 cDNA library (see Experimental Procedures).

Cloning of a cDNA Encoding the Receptor-Associated p85 Protein Computer-assisted comparison of the K30 peptide to the Genbank data base showed that the peptide sequence is similar to part of an SH2 sequence motif (Sadowski et al., 1986). We prepared probes from four pools of partially degenerate oligonucleotides based on the K30 sequence. Screening of 400,000 recombinant phages from a mouse BALB/c 3T3 cDNA library yielded three positive clones. The sequence of a 3.0 kb cDNA, clone P85-1, included a large 5' untranslated region (556 nucleotides) followed by an ATG codon that meets the translation initiator criteria (Kozak, 1989) and a long open reading frame (724 codons) that would encode a protein of 83,700 kd (Figure 2A). In clone P85-1 the coding sequence is followed by a short 3' untranslated region (270 nucleotides), but there is no polyadenylation signal. Five of the eight peptide sequences derived from the tryptic digest of the purified p85 were found in the coding sequences of the P85-1 cDNA (Table 1). The other peptide sequences were of low yield and their significance is not known. The P85-1 cDNA encodes a sequence homologous to an SH3 domain (amino acids 10 to 62) and two SH2 domains, one located in the middle of the coding sequence (amino acids 332 to 370) and the other at the carboxyl terminus (amino acids 623 to 659) (see underlined sequences, Figure 2A). A comparison of the amino acid sequence of the SH2 domains present in p85 with other SH2 domains contained in other proteins revealed a 200-30% sequence identity (Figure 2B). The probe that we used to isolate our cDNA clone encoded the sequence TADGTFLVRDAXT. Approximately half of this sequence is highly conserved in the group of SH2 domain-containing proteins shown in Figure 2B and the other half is relatively

p85, a Subunit of the PI3-KinaseThat Binds PDGF Receptor 77

| 7t

141 211 281 351 42! 491 56! 63| 70!

MSAEGYQYRA GTYVEYIGRK

LYDYKKEREE RISPPTPKPR

DIDLHLGDIL PPRPLPVAPG

TVNKGSLVAL SSKTEADT/Q

GFSDGPEARP QALPLPDLAE

EDIGWLNGYN QFAPPDVAPP

[TTGERGDFP LLIKLLEA[E

KKGLECSTLY RTQSSSNPAE LRQLLDCDAA SVDLEMIOVH VLADAFKRYL ADLPNPVIPV AVYNEIV~ISLA QELQSPEDCl QLLKKLIRLP NIPHQCWLTL QYLLKHFFKL SQASSKNLLN ARVLSEIFSP VLFRFPAASS DNTEHLIKAI EILISTEWNE RQPAPALPPK PPKPITVANN SMNNNMSLQD AEWYWGD[SR EEVNEKLRDT ADGTFLVRDA ~ ILRKGGNNKL IKIFHRDGKY GFSDPLTFNS VVELINHYRN ESLAQYNPKL DVKLLYPVSK ~ V V K E D NIEAVGKKLH EYNTQFQEKS REYDRLYEEY IRISQEIQMK RIAIEAFN[I [KIFEEQCQT QERYSKEYIG KFKREGNEKE [QR]MHNHDK LKSRISEIID SRRRLEEDIK KQAAEYREID KRMNSIKPDL IQLRKTRDOY LMWLTQKGVR QKKLNEWLGN ENTEDQYSLV EDDEDLPHHD [KTWNVGSSN RNKAENLLRG ~ SSK~GCYAC~ VVVDGEVKHCVINKTATGYG FAEPY~IYSS [KELVIIIYQII TSLVQHNDSL NVTLAYPVYA QQRR

B v-SRC n-85K C-85K n-GAP c-GAP n-PLC c-PLC v-CRK

1147] [333] [624] [178] [348] [550] [668] [263]

F

~N

~

- -~R m - - S ~ [ -

VI~S N " -I~N K H~(~D - -II~T FH S - -KQ ~HH~(~AG~DGHI ~ H A S L T - -~IA Q ~IA~R L S " -~GD

N P E N ....

[]R

G - K AmC~

* * AIJ~N~R G K R ...... O ~ S -K -I AI~ER~RQAGKS ..... t~GJSYJL,'I ~ ] O RR -EAYN MTVGQAC ..... S DN A ~ T E Y Cl ET G A ~ D ~ S S ~ V - G - AIEIH~M ...... R V~D[6[AIF~K RN -e - -AVS~QGQRH ......

unique in the SH2 domain that is in the amino-terminal half of our p85 (see amino acids 350-362 in the second line of Figure 2B). p85 also contained an amino-terminal sequence that is homologous to SH3 domains (Sadowski et al., 1986). It is noteworthy that the predicted sequence of the p85 contained no consensus sequences for an ATPbinding site or other conserved sequence motifs common to known serine/threonine or tyrosine kinases (Hanks et al., 1988).

~_~o '~Da ~00

p85:

4-

4-

kDa D0

96

96

68

68

--

Figure 3. Specificityof Binding of Recombinantp85 Proteinto PDGF Receptors In Vitro (A) Associationof recombinantp85 with wild-typeand mutatedPDGF receptors.Immunoblotanalysisof receptor-associatedcomplexesusing anti-phosphotyrosineantibodies. Immunoprecipitatesof baculovirus-expressedwild-typereceptor(bcR) and kinase insert mutant of the receptor(bcAki) were incubatedwith p85 expressedin the insect cell system.Followingassociation,the complexeswerewashedextensively and subjected to in vitro kinase reaction. The associatedp85 and PDGF receptor were detected by anti-phosphotyrosineimmunoblot. The last lane represents immunoprecipitatedreceptor incubatedwith uninfectedSf9 lysate.The arrowheadindicatesthe position of p85. (B) Specific inhibition of binding of the recombinantp85 to the PDGF receptorby a phosphorylatedpeptidefrom the kinase insert region of the PDGFreceptor.Associationof the baculovirus-expressedp85 with baculovirus-expressedwild-typePDGFreceptorwas performedas describedin (A), lane 3, exceptthat the incubationof immobilizedreceptor and the insect cell lysate was done in the presenceof unphosphorylated peptide Y719 from the kinase insert region (lane 1), tyrosinephosphorylated peptide (Y719P) (lane 2), or no peptide (lane 3). Receptor-boundp85 was detectedby the in vitro kinasereaction.The arrowhead indicatesthe position of p85.

Q - O C~A~ P GSFV~ P -GD S Y D ~ T ~ P " N S ~ A I ~1 P -GDFV~

Figure 2. Predicted Amino Acid Sequenceof p85 That Associateswith the PDGF Receptor (A) The cDNA clone P85-1 contained a 724 aminoacid open readingframe.The predicted amino acid sequence of this open reading frame is shown. The positionsof the two SH2 regions are underlined. (B) Computerassistedcomparisonof the p85 predictedamino acidsequencewith other SH2 domain-containingproteins.Gaps introduced for optimal alignment are indicated by hyphens. The letters n, c, and v referto the N-terminus, C-terminus,and viral, respectively.The numbers in brackets refer to the amino acid position in the respective proteins. Identical amino acid residues are stippled,

Functional Properties of Baculovirus-Expressed Recombinant p85 p85 produced by an insect cell expression system (Summers and Smith, 1987) bound to the wild-type PDG F receptor in vitro (Figure 3A). In this experiment the recombinant p85 protein was incubated with phosphorylated receptor, the complex was washed extensively, and an in vitro kinase reaction was performed on the complex to detect the associated p85. The estimated concentration of p85 that occupied half of the binding sites on the receptor was less than 1 nM (data not shown). Like its mammalian counterpart, the baculovirus-expressed p85 failed to bind to the kinase insert deletion mutant receptor (Figure 3A). Previous studies showed that binding of the endogenous mammalian p85, p110, and PI3-kinase activity to PDGF receptors could be blocked by a 20 amino acid peptide consisting of sequences in the kinase insert region including phosphotyrosine at position 719 (Escobedo et al., 1991). The peptide did not block binding of phospholipase C-y or GAP (Escobedo et al., 1991) to the receptor. This finding suggested that p85, p110, and PI3-kinase bind as a complex to the same 20 amino acid portion of the receptor (Escobedo et al., 1991). We tested whether the recombinant p85 bound to the same portion of the receptor. A 20 amino acid peptide that included a phosphorylated tyrosine at position 719 blocked the binding of recombinant p85 to the PDGF receptor (Figure 3). The unphosphorylated form of this peptide did not block binding of the recombinant p85 (Figure 3B). The recombinant p85 was metabolically labeled by incubation of Sf9 cells infected with recombinant p85 baculovirus with [3sS]methionine. A tryptic map of the labeled recombinant p85 that binds to the receptor in vitro was identical to a tryptic map of [35S]methionine-labeledendogenous p85 from 3T3 cells (data not shown). Using a modification of the ligand-blotting technique ("receptor blot," Escobedo et al., 1991), we showed that baculovirus-expressed p85 binds directly to the phosphorylated receptor (Figure 4). In this experiment baculovirusexpressed and immunoprecipitated PDGF receptor was labeled by autophosphorylation in vitro in the presence of [y-32P]ATP and Mn 2+. The radiolabeled receptor probe was

Cell 78

1 2 3 Lysates:

m

~

4

kDa

¢~

--200

m I

--

96

68

m68

Figure 4. PhosphorylatedPDGF ReceptorInteracts Directlywith p85 The ability of the radiolabeled PDGF receptor to bind directly with recombinantp85 was analyzedby receptorblot (Cohenet al., 1990a). Immunoprecipitatedbaculovirus-expressedreceptorwas radiolabeled in vitro by incubationwith [~P.3,]ATPand Mg2.. This radiolabeledreceptor was extracted and used to probe filters containing proteins fractionatedby SDS-PAGE. Lane 1, Sf9 cell lysate; lane 2, lysatesof Sf9 cells expressingthe recombinantp85; lane 3, lysatesof quiescent 3T3 cells. The arrow indicatesthe position of p85.

bac 8 5 k

.

1/4

--

i then incubated with electrophoretically separated cell lysates that had been transferred to nitrocellulose paper. When the incubation with the probe was performed under very stringent conditions (0.4% SDS), the receptor probe recognized p85 as its major binding protein (Escobedo et al., 1991). This result shows that the binding of p85 to the receptor is a direct interaction and does not require another intermediate protein(s). Taken together, these findings showed that the recombinant p85 has the expected properties of the endogenous p85: it binds directly to the receptor, it can be tyrosinephosphorylated by the receptor in vitro, it does not bind to a mutant receptor with deleted kinase insert sequences, it appears to bind to a short sequence in the kinase insert region, and its interaction with receptor requires phosphorylation of tyrosine 719. As predicted from the absence of an ATP-binding motif in the cDNA coding sequence, there was no PI3-kinase activity in the preparation of recombinant p85 expressed using the baculovirus system (Figure 5, lane 5). Since the recombinant p85 expressed in insect cells bound to the same small region of the receptor that is known to bind PI3-kinase activity, we tested whether p85 could block the binding of PI3-kinase to the receptor (Figure 5). As the amount of recombinant p85 added to the receptor in vitro was increased, the binding of 3T3 lysate-derived PI3kinase activity to the receptor and the binding of 3T3 cellderived p110 decreased (Figure 5).

Activity of Recombinant p85 Expressed in Mammalian Cells The recombinant p85 was expressed in COS cells by cDNA transfection. Untransfected COS cells have endogenous p85 (as well as a protein with slightly slower mobility on SDS-PAGE), p110 (Figure 6, last lane), and PI3-kinase activity that bound to the PDGF receptor when the cell lysates from the COS transfectants were mixed with immo-

~

~

•

PiP

-- Origin

Figure 5. Baculovirus-Expressedp85 Inhibits Binding of Cell Lysate PI3-Kinase to PDGF Receptors Five hundred microlitersof crude 3T3 cell lysateswas incubatedwith immobilizedbaculo-expressedPDGF receptor in the presenceof lysates of baculovirus-infectedcells expressing recombinantp85. The baculovirus-expressed85 kd proteinwas addedas 500 p.Iof crudeSf9 baculovirus-infectedcell lysates (bac 85k) (see ExperimentalProcedures) or dilutions (1/8, 1/4, and 1/2) of this lysate (lanes2-4) as indicated. These amountsof baculovirus-expressedp85 are estimatedto be 20-fold, 40-fold, and 80-fold in excess over the endogenous3T3 p85. In the first lane only 3T3 cell lysate was added (no recombinant p85) and in lane 5 only recombinant p85, but no 3T3 lysates, was included. In these experimentsthe amount of receptor availablefor association was limiting. The receptor-associatedproteins were detected by in vitro kinaseassayand the receptor-associatedPI3-kinase activitywas measuredby TLC as describedin Figure 1 and the Experimental Procedures.The positionsof p85 and p110 are indicated by the arrow and arrowhead,respectively.The origin of the chromatogramand the positionof the PI3 phosphateare indicatedby Origin and PIP, respectively.All lanesof the in vitro kinaseexperimentswere from the same gel and were exposedfor the same amount of time.

bilized receptors in vitro. Lysates of COS cells transfected with the p85 cDNA had more p85 that could bind to PDGF receptors than did lysates of untransfected cells (Figure 6, middle lane). However, the transfected cell lysates had less PI3-kinase and p110 that could bind to the PDGF receptor (Figure 6, bottom panel, and middle lane). It is likely that the excess recombinant p85 competed for the site on the receptor that normally binds p85, p110, and PI3-kinase activity as a complex. The endogenous COS cell pl 10 apparently was unable to form a complex with the recombinant p85. Another possible explanation of these results is that the recombinant p85 inhibits PI3-kinase activity through a direct interaction with a catalytic subunit of PI3-kinase and serves as a repressing subunit. However, addition of p85 to preformed complexes of receptor and PI3-kinase from cell lysates failed to inhibit PI3-kinase activity, making this possibility less likely (data not shown).

p85, a Subunit of the PI3-Kinase That Binds PDGF Receptor 79

o~

~- o O

o O

B

200

m

96

--

68

PIP

Origin

Figure 6. Overexpressionof p85 in COS-7 Cells Inhibits the Binding of COS-7 PI3-Kinaseto PDGF Receptors Lysates of 3T3 cells, COS cells transfected with the pBJ expression vector containingthe p85 cDNA insert,and COS cellstransfectedwith the controlpBJ vectorwereincubatedwith immobilizedPDGFreceptor as described in Figure 1. The receptor-associatedproteins were detected by an in vitro kinase assay (top) and PI3-kinase activity was measured (bottom) as described in Figure 1. The arrow indicatesthe positionof p85. Originand PIP referto the origin and the phosphoinositol phosphate, respectively.

Discussion

In this study we purified an 85 kd protein that is known to be one of the major receptor-associated proteins in PDGF-stimulated cells. This protein appears to bind tightly to the PDGF I~-receptor after ligand activation of intact cells (Coughlin et al., 1989; Kaplan et al., 1987; Morrison et al., 1990; Escobedo et al., 1991). Recently, this protein was shown to bind to a short phosphotyrosine-containing sequence in the receptor (Escobedo et al., 1991). The remarkable ability of the p85 to bind the receptor in receptor-blotting experiments (Escobedo et al., 1991 and Figure 4) suggests that the p85 binds with unusually high affinity to the receptor under these conditions as compared with other proteins known to bind to PDGF receptors. Using the in vitro system described here we have estimated the apparent dissociation constant of p85 and the receptor to be below 1 nM (data not shown). In this study we cloned the cDNA for p85 and characterized some of the functions of the recombinant version of the p85. We found that the recombinant p85 interacts with the receptor in the same way as the endogenous 3T3 cell p85. The recombinant protein associated tightly with the activated wild-type

PDGF receptor, but did not associate with a PDGF receptor mutant lacking the kinase insert region (Escobedo et al., 1991 and Figure 3A). The direct binding of recombinant p85 to the receptor was shown by receptor-blotting experiments (Figure 4). Recombinant p85 bound to the same receptor sequence of 20 amino acids as did endogenous 3T3 cell p85 (Figure 3B). The interaction of the recombinant p85 and the receptor peptide required phosphorylation of tyrosine 719 in the receptor sequence (Figure 3B). Other investigators have proposed that p85 that associates with the PDGF receptor is identical to the 81-85 kd protein that was found associated with middle-T antigen and the c-src protein in polyoma virus-transformed cells (Courtneidge and Heber, 1987; Kaplan et al., 1987; Cohen et al., 1990a). These investigators also proposed that this p85 is PI3-kinase (Courtneidge and Herber, 1987; Kaplan et al., 1987; Cohen et al., 1990a). An 85 kd protein copurities with PI3-kinase activity from bovine brain (Morgan et al., 1990) and rat liver (Carpenter et al., 1990). The p85 encoded by our cDNA clone had no measurable PI3kinase activity when bound to tyrosine-phosphorylated PDGF receptors and did not have a consensus sequence for an ATP-binding site. However, this p85 may be a subunit of PI3-kinase. The finding that the recombinant p85 from the baculovirus expression system competes with PI3-kinase activity from 3T3 lysates for binding to the receptor, and that in COS cell transfectants the recombinant protein competes with the endogenous COS cell PI3kinase for binding to the receptor, establishes that there is a relationship between the p85 and the PI3-kinase enzyme. It is possible, for example, that the PI3-kinase activity requires a multisubunit structure and that in the presence of an excess of one subunit (p85), the multisubunit structure is prevented from interacting with the receptor. The function of the PI3-kinase bound to the receptor is not understood. Recently, it was reported that PI3-kinase localizes to the plasma membrane after growth factor stimulation of cells (Cohen et al., 1990b). This is not surprising since the substrate for the enzyme is a membrane-bound molecule. Thus, receptor localization may bring the PI3kinase into proximity with its substrate or with other regulatory molecules that are also complexed with the PDGF receptor. The most striking feature of the sequence of the p85 is that it contained two SH2 domains. This sequence motif found in src protein, the src homology 2 domain (SH2), was first recognized in noncatalytic regions of cytoplasmic tyrosine kinases (Sadowski et al., 1986). Experiments performed in mutants of these kinases suggested that the SH2 domains regulated the kinases through intramolecular interactions (Koch et al., 1989). Recent studies of the protein encoded by the c-crk proto-oncogene have shown that its SH2 domain binds to tyrosine-phosphorylated proteins (Mayer and Hanafusa, 1990). In addition to p85, there are several cytoplasmic molecules that have SH2 domains and bind to tyrosine-phosphorylated PDGF receptors. These molecules include phospholipase C-7, GAP, pp60 src, pp59 fyn, and pp62 c-yes. Experiments on the binding of some SH2-containing proteins (PLC~, GAP, and p85) to mutants of the PDGF

Cell 80

receptor have suggested that each of these proteins binds to a distinct region of the receptor and that the proteins do not c o m p e t e with each other for their respective binding sites (Morrison et al., 1989; E s c o b e d o et al., 1991). The most plausible m o d e l to explain these findings is that each of these SH2-containing signaling molecules binds to the receptor through the SH2 domains, and that each SH2 domain interacts with a distinct phosphotyrosine-containing s e q u e n c e in the receptor. Recent studies with mutants of the GAP protein have suggested that the SH2 domains of GAP mediate the interaction of GAP with the PDGF receptor (W. Fantl, G. Martin, F. McCormick, and L. T. W., unpublished data; Anderson etal., 1990). Thus the role of SH2 d o m a i n s in the interaction of signaling molecules m a y be to recognize phosphotyrosine residues in a specific s e q u e n c e context. Since there are multiple tyrosines in the receptor that are phosphorylated, there are several potential sites for interaction with SH2 domains. Studies currently underway will determine w h e t h e r the binding of p85 to the receptor is through the SH2 domains of p85. The biochemical role of PI3-kinase in regulating cell proliferation is not understood. The studies of this e n z y m e in the context of mitogenesis have been largely correlative. In the case of p o l y o m a v i r u s - t r a n s f o r m e d cells, the ability of the middle-TIc-src c o m p l e x to transform cells correlates with the presence of PI3-kinase activity, suggesting that the PI3-kinase is necessary, but perhaps not sufficient, for cell transformation (Courtneidge and Heber, 1987). Similar correlative studies have been performed with mutants of the PDGF receptor and indicate the potential role of the PI3-kinase in growth factor-stimulated cell proliferation (Coughlin et al., 1989; Escobedo and Williams, 1988). The finding that in PDGF-stimulated cells there is a large transient increase in the a m o u n t of PI-3,4-biphosphate and PI-3,4,5-triphosphate suggests that the PI3-kinase and its products are mediators of PDGF mitogenic responses (Auger et al., 1989). The precise roles of these phospholipids and their corresponding inositol phosphates in the signaling process remains to be determined. The availability of molecules that regulate the interaction of PI3-kinase activity with growth factor receptors will help elucidate the importance of this e n z y m e in the growth of normal and transformed cells. Experimental Procedures Cells and Viruses

PDGF receptor-associated proteins were obtained from BALB/c 3T3 cell lysates (C. Scher, Philadelphia Women Hospital, PA). Transient expression was done in COS-7 cells. Insect cells Spodoptera frugiperda (Sf9) and baculovirus vector pVL1393 were obtained from M. Summers (Texas A & M, Texas). Baculovirus vector pAcC4 was obtained from Dr. R. Clark from Cetus Corporation (Emeryville, California). Recombinant PDGF receptor baculovirus was prepared by cotransfection of Sf9 cells with a baculovirus vector containing the PDGF receptor cDNA and wild-type baculovirus DNA as described by Summers and Smith (1987). Preparation of BALB/c 3T3 Cell Lysates

BALB/c 3T3 cells cultured in Dulbecco's modified Eagle's medium supplemented with 100/0bovine serum, 50 p.g/ml penicillin, and 50 I~g/ ml streptomycin were washed with cold phosphate-buffered saline. Cell lysates were prepared by incubating the cells with lysis buffer containing 20 mM Tris (pH 8.0), 137 mM NaCI, 2 mM EDTA, 1 mM

phenylmethylsulfonyl fluoride, aprotinin (O.15 U/ml), 20 p.M leupeptin, 1 mM sodium vanadate, and 1°h Triton X-100 as previously described (Morrison et al., 1989). Lysates were cleared by centrifugation and added to immune complexes containing phosphorylated PDGF receptors. PDGF-stimulated BALB/c 3T3 cell lysates were prepared by lysis of BALB/c 3T3 cells previously incubated with 2 nM PDGF for 10 min at 370C. Isolation of PDGF Receptor-Associated Proteins

Sf9 cells (107)were infected with PDGF receptor baculovirus for 4860 hr. Tyrosine-phosphorylated PDGF receptors from Sf9 cell lysates were precipitated by incubation with a receptor antibody directed to a sequence in the extracellulardomain. Immu ne complexes were precipitated using protein A-Sepharose. Precipitates were sequentially washed with lysis buffer, with 0.5 M LiCI, 0.1 M Tris (pH 7.4), twice with 10 mM Tris (pH 7.4), and once with distilled water. The immunoprecipitated receptors were further phosphorylated by incubation of the immune complexes with kinase buffer (30 mM Tris [pH 7.4], 10 mM MnCI2, 50 mM ATP) for 15 min at 25°C. Association between the immobilized receptor (from 106cells) and 3T3 cell lysate proteins (from 107 cells) was carried out as previously described (Morrison et al., 1989). Following association, immune complexes were washed sequentiallyas indicated above. The presence of the receptor-associated proteins in the complex was determined by in vitro labeling of the receptor-associated proteins (Morrison et al., 1989). Radiolabeled proteins were resolved on a 7% SDS-polyacrylamide gel and detected by radioautography. Determination of PI3-Klnase Activity in Receptor Immune Complexes

Cell lysates from PDGF-treated and control cells were prepared as previously described (Morrison et al., 1989). Lysates were incubated with PDGF receptor antibodies. The receptor immune complex was precipitated with protein A-Sepharose (Pharmacia). After a series of sequential washes (see above), the presence of PI3-kinase activity on the precipitates was determined by incubating the precipitates with PI3-kinase buffer containing 30 mM HEPES (pH 7.4), 30 mM MgCI2, 0.2 mM adenosine, 40 I~M ATP, 0.2 mg/ml sonicated phosphatidylinositel, and 10 p.Ciof [~P-y]ATP (3000 Ci/mmol) for 10 m in at room temperature. The samples were extracted with chloroform, and aliquots of the radiolabeled mixture were spotted onto a thin layer chromatography (TLC) plate as previously described (Kaplan etal., 1987). After onedimensional chromatography the plates were exposed to X-ray film. Purification and Sequencing of p85

Immobilized baculovirus-expressed PDGF receptor (50-100 I~g)was incubated with 3T3 cell lysates (from 1500 15 cm culture dishes) for 3 hr. After several washes the immune complex containing the receptor-associated proteins was run on preparative SDS-polyacrylamide gels. Discrete Coomassie-stained bands containing the desired proteins were cut from the gel and electroeluted as previously described (Hunkapiller et al., 1983). We obtained 50 p.gof p85 and similar amounts of pl 10. Twenty micrograms of gel-eluted p85 were subjected to trypsin digestion as previously described (Yarden et al., 1986). Individual peptides were separated by high pressur e liquid chromatography and sequenced in a gas-phase sequenator using Edman degradation procedures. Cloning of the cDNA for the Receptor-Associated p85

The amino acid sequence of peptide K30 (TADGTFLVRDAXT) obtained from p85 was used to synthesize a set of four pools of degenerate (256 degeneracies) oligonucleotides (48 nucleotides long). These oligomers were ~P-labeled at the 5'end using T4 polynucleotidekinase (Boehringer and Mannheim) and [~P-7]ATP (5000 Ci/mmol). Plaques (5 x 10s) from a ;Lgtl 1 mouse BALB/c 3T3 cDNA library (kindly provided by Dr. Dan Nathans, John Hopkins University, MD) were screened using the following hybridization conditions: 20% formamide, 5x SSC at 45°C, and wash at 55°C with 2x SSC. Under these conditions three cDNA clones were isolated with one of the pool probes (5'AC-(CIA)-GIC(CIT)-GAT-GG(CIG)..AC(CIA)-TI-T-CT(GIC)GT(G/C)-CG(NG)-GAC-GC(C/T)-GAA-AC-3'). Two of the clones contained inserts of 3.0 kb. Restriction analysis of these two clones showed that they were identical. The remaining clone contained a 2.8 kb cDNA

p85, a Subunit of the PI3-Kinase That Binds PDGF Receptor 81

insert. Restriction endonuclease analysis of this clone indicated that it corresponded to a truncated version of the P85-1 cDNA clone. The DNA insert of one of the 3.0 kb clones was sequenced by the dideoxyterminator technique using T7 DNA polymerase from UBI.

Transient Expression of the Recombinant p85 in COS-7 Cells The cDNA that encodes p85 was cloned into a mammalian expression vector, pBJ-1. In this vector the expression of the p85 is under the transcriptional control of a SV40/HIV-I hybrid promoter (Takebe et al., 1988). The plasmid was transfected into COS-7 cells using Lipofectin (Bethesda Research Laboratories, Bethesda, MD) following the manufacturer's protocol. Sixty hours after transfection the cells were lysed and assayed for expression of the p85 by a receptor association assay (see above). Expresslon of p85 In Sf9 Cells Using the Baculovirus System The full-length cDNA was cloned into a baculovirus expression plasmid pVL1393. Using the calcium precipitation technique the vector was cotransfected with wild-type baculovirus DNA into Sf9 cells. The resuiting recombinant virus was used to infect fresh Sf9 cells. Four days later, positive clones were identified and plaque purified as previously described (Summers and Smith, 1987). Larger amounts of p85 were obtained by infecting 106Sf9 cells with recombinant baculovirus encoding the p85 at a multiplicity of infection of 10. Forty-eight hours later the cells were lysed as described above and lysates were used in the receptor association assay. Probing of p85 by Receptor Blot The presence of p85 in cell lysates of 3T3 and Sf9 cells infected with p85 recombinant baculovirus was detected by probing nitrocellulose filters containing the SDS-PAGE fractionated proteins with in vitro radiolabeled PDGF receptors under previously described conditions (Cohen et al., 1990a; Escobedo et al., 1991). The presence of p85 was detected by radioautography of the nitrocellulose filter. Acknowledgments We thank Dr. David Kaplan for his valuable suggestions and Dr. Harold Varmus, Angus MacNicol, and Michael Pazin for their critical reading of the manuscript. We are grateful to Mercedita del Rosario for her technical help and Marie Doherty for the preparation of oligonucleotides. W. M. K. was supported by National Institutes of Health grant K11-HI02410. This work was supported by NIH grant R01 HL-32898 (L. T. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC Section 1734 solely to indicate this fact. Received December 4, 1990; revised January 23, 1991.

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The accession number for the sequence reported in this paper is M60651.