Sequence Specificity in Recognition of the Epidermal Growth Factor ...

THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1993 by The American Society for Biochemintry and Molecular Biology, Inc

Vol. 268,No. 31,Issue of November 5 , pp. 23634-23639,1993 Printed in U.S.A.

Sequence Specificity in Recognition of the Epidermal Growth Factor Receptor by Protein Tyrosine PhosphataselB* (Received for publication, April 30, 1993, and in revised form, July 16, 1993)

Kim L.Milamkit, Guochang ZhuSQ,Cathryn G. Pearl$, Dennis J. McNamarall, Ellen M. Dobrusinll, Derek MacLeanll, Andrea Thieme-Seflerll, Zhong-Yin Zhangll, Tomi Sawyer(, Stuart J. Decker$**, Jack E.Dixonll , and Alan R. Saltiel$Q$S From the Departmentsof $Signal Transduction and TChemistry, Park-Davis Pharmaceutical Research Division, WarnerLambert Company,Ann Arbor, Michigan48105 and the Departments of §Physiology, **Microbiology, and IlBiological Chemistry, University of Michigan School of Medicine, Ann Arbor, Michigan 48109

Proteintyrosine phosphatases all contain a conserved cysteine that forms an intermediate thiophosphate esterbond during tyrosinephosphate hydrolysis. A bacterial glutathione S-transferase fusion protein containing rat brain phosphatase P T P l b was constructed inwhich this conserved cysteine was mutated to serine. The resulting catalytically inactive enzyme was labeled in vivo to high specific activity with 36S, and the binding of this labeled fusion protein to the immunoprecipitated epidermal growth factor (EGF) receptor was evaluated. The binding was ligand-dependent, and saturation analysis revealeda nonlinear Scatchard plot, with a K , for high affinity bindingof approximately 100 nM. A number of glutathione S transferase fusion proteins containing arc homology 2 (SH2)domains attenuated phosphatase binding in a concentration-dependentmanner. Phospholipase C (PLC)y and the GTPase-activating protein of raa were the most potent inhibitors. Tyrosine-phosphorylated EGF receptor peptide fragments were evaluated for specific inhibition of P T P l b and PLCy SH2 binding to the activated receptor. One such peptide, modeled on EGF receptor tyrosine992, blocked the bindingof both fusion proteins. Another phosphopeptide, modeled on tyrosine 1148, inhibited the bindingof P T P l b but not the PLCy fusion protein. This sitespecificity was confirmed by analysis of equilibrium bindingof the fusion proteins to EGF receptors mutated in each of these phosphorylation sites. The results revealed clear sequence specificity in the binding of proteins involved in theregulation of intracellular signalingby receptor tyrosine kinases.

that contain src homology 2 (SH2) domains, regions of approximately 100 amino acids that direct binding to tyrosinephosphorylated proteins (4). SH2-containing proteins constitute a functionally diverse family, including several proteins with enzyme activity, as well as others with regulatory functions (5-13). The one feature common to all of these proteins is their presumed role in signal transduction for cellular growth and differentiation. The catalytic activity of many receptor tyrosine kinases and theirinteractions with SH2 proteins are tightlygoverned by protein tyrosine phosphatases (PTPases). Like tyrosine kinases, PTPases constitute a large family of enzymes that can be divided into two classes (14). One is characterized by extracellular and transmembrane regions, with two cytoplasmic catalytic domains (15, 16); the second cytosolic class has a single catalytic domain. The cytosolic enzymes can be further classified by sequences outside the catalytic domain. These can contain SH2 domains (17-21), cytoskeletal elements (22, 23), or hydrophobic carboxyl-terminal segments (24-26). Although the precise roles of the different PTPases have yet to be determined, analysis of their tissue or subcellulardistribution suggests some degree of selectivity with respect to substrates. Forexample, within the transmembrane family of PTPases, numerous tissue-specific enzymes have been identified. CD45 is expressed in lymphocytes and appears to be involved in modulating T cell receptor signaling via dephosphorylation of regulatory domains in the nonreceptor tyrosinekinases (27,28). Expression of DLAR, a receptorlike Drosophila PTPase, is restricted to neuronal cells, indicating a role in neural development (29, 30). Tissue-specific expression of the PTPases containing SH2 domains has also been described. SHPTP-1 is found primarily in hematopoietic cells (21), butthe related SHPTP-2 appears to bemore Protein tyrosine phosphorylation plays a critical role in the ubiquitous (17). Despite these examples of tissue-specific expression, there control of cellular proliferation anddifferentiation (1, 2). Virtually all growth and differentiation factors bind to cell has been little biochemical characterization of the mechasurface receptors that either possess or indirectly activate nisms underlying substrate specificity for PTPases. Progress protein tyrosine kinase activity, leading to tyrosine phos- has been hampered by the difficulty in discriminatingbetween phorylation of the receptors themselves, as well as several substrate recognition and catalytic activity. To investigate intracellular substrates (3).Although the precise mechanisms the binding of a PTPase to a tyrosine-phosphorylated subinvolved in signal initiation from these receptors remain strate, we have exploited previous observations that all undefined, many receptors interact with signaling proteins PTPases contain aconserved cysteine residue that is essential for activity (16, 31, 32). Mutation of this conserved catalytic * The costs of publication of this article were defrayed in part by cysteine (Cys215)of rat brain P T P l b (33) rendersthe protein the payment of page charges. This article must therefore be hereby enzymatically inactive (32) but still allows substrate binding. marked “advertisement” in accordance with 18 U.S.C. Section 1734 We expressed the mutated PTPlbC215S as a bacterial glutasolely to indicate this fact. $$ To whom correspondence should be addressed Dept. of Signal thione S-transferase fusion protein (34) and labeled it tohigh specific activity in vivo using 35S.The radiolabeled fusion Transduction, Parke-Davis Research Division, 2800 Plymouth Rd., protein has been used to characterize the binding of P T P l b Ann Arbor, MI 48105. Fax: 313-996-5668.

23634

PTPl b Binding to the Activated EGF Receptor

23635

to the epidermal growth factor (EGF)' receptor and to compare the kinetics of this interaction with those involving proteins containing SH2 domains.

quantitatively evaluatethe binding of the mutantphosphatase PTPlbC215S atopotential tyrosine-phosphorylated substrate, we utilized a rapidand simple binding assay described recently for the binding of SH2 domains to the EGFreceptor (36,37). EXPERIMENTALPROCEDURES PTP1bc2lSs was expressed inbacteria as aglutathione SMaterials-Mouse EGF was from Collaborative Research and anti- transferase fusion protein and purified by affinity chromatogEGF receptor antibody (Ab-1) was from Oncogene Science. Anti- raphy on glutathione-Sepharosebeads. To obtain radiolabeled phosphotyrosine antisera have been described previously (35). fusion protein, Tran%-label was added to the bacterial culTran%-label was from ICN Radiochemicals. Glutathione-Sepharose tures during induction. The purified protein was analyzed by was purchased from Pharmacia LKB Biotechnology Inc. All other SDS-polyacrylamide gel electrophoresis, followedbyCooreagents were from Sigma and were the highest quality available. Cloning of the glutathione S-transferase-PTPlbCzlssfusion protein massie Blue staining and autoradiography. The resulting fu(34) and other glutathione S-transferase-SH2fusion proteins (36,37) sion protein was over 90% pure, with a specific activity of approximately 20,000 cpm/pg (Fig. lA). have been described. Expression and Purification of Glutathione S-transferase Fusion To evaluate the binding properties of the [3sS]PTPlbC215S Proteins-Bacteria harboring a plasmid encoding the glutathione S- fusion protein, we utilized an immobilized preparation of t r a n s f e r a ~ e - P T P l b fusion ~ * ~ ~ protein ~ (34) were grown at 37 "C until activated EGF receptor. NIH 3T3cells expressing the human A- was approximately 0.5. Fusion protein expression was induced by addition of 0.2 mM isopropyl-1-thio-P-D-galactopyranoside EGF receptor (3T3/hEGFR) (35) were treated with or without (IPTG), and the induced cultures were incubated a t 37 "C for an EGF toinduce tyrosine phosphorylation. The phosphorylated additional 3 h. The cells were harvested by centrifugation, washed receptor was immunoprecipitated and immobilized on protein with phosphate-buffered saline, and lysed by sonication. The lysate A-agarose beads. The beads were washed several times with was clarified by centrifugation, and the supernatant was incubated binding buffer and incubated with radiolabeled PTPlbC215S in with glutathione-agarose beads a t 4 "C for 1 h. The beads were the presence or absence of excess unlabeled fusion protein. washed, and the fusion protein was eluted with 5 mM glutathione. Bound ligand was separated from free by filtration, and the The resulting purified protein was devoid of tyrosine phosphatase was quantitated by scintillation countactivity. 35S-labeledfusion protein was prepared by addition of 2 mCi bound [3sS]PTPlbc21ss ing (Table I). Nonspecific binding was determined by precipTran3'S-label during the induction period. Site-directed Mutagenesis of the Human EGF Receptor-The hu- itating withpre-immune serum in place of the anti-EGF man EGF receptor cDNA wassubject to site-directed mutagenesis by receptor antibody. The [35S]PTPlbc21ss fusion protein specifsubstitution of phenylalanine for tyrosine a t positions 992,1068, ically bound to the EGF receptor, and thisbinding was almost 1086, 1148,and 1178, using the Bio-Rad mutagenesis kit. Immunoprecipitation of Phosphorylated EGF Receptor"3T3 cells completely displaced by excess unlabeled PTPlbC215s. Binding of the fusion protein to the EGF receptor was stably transfected with the human EGF receptor (35) or receptor mutants were incubated with 150 ng/ml EGF for 10 min a t 37 "C. greatly enhanced by treatment of cells with EGF, although The cells were washed with phosphate-buffered saline and lysed in some binding was observed in the absence of EGF treatment. HY buffer containing protease inhibitors asdescribed previously (38). To determine whetherthis growth factor-independent binding Lysates were clarified by centrifugation and incubated with anti-EGF was due to residual tyrosine phosphorylation,we analyzed the receptor antibody for 45 min a t 4 "C, with rocking. Immune complexes were harvested by addition of protein A-agarose beads. The beads phosphotyrosine content of the receptor before and after EGF were washed three times with Tris binding buffer (50 mM Tris-HC1, treatment by immunoblotting with anti-phosphotyrosine pH 7.4, 10 mMMgC12, 100 mM NaC1, 0.1% Triton X-100) and used antisera. 3T3/hEGFR cells were treated with or without EGF, for binding assays. EGF-dependent tyrosine phosphorylation of the and the EGF receptor was immunoprecipitated with antireceptor was confirmed by SDS-polyacrylamide gel electrophoresis followed by blotting with anti-phosphotyrosine antisera as described previously (35). Receptor Binding Assays-Binding of the '%-labeled PTPlbC21'S fusion protein to the immunoprecipitated EGF receptor was assayed as described previously for the binding of labeled glutathione Stransferase-SH2 fusion proteins (36, 37). Briefly, binding assays fusion contained approximately 1 pg receptor, 30 nM [3sS]PTPlbC215S protein (20,000cpmlpg), and theindicated concentration of unlabeled fusion protein. Binding was carried out a t 4"C for 30 min, and unbound ligand was separated from the beads by filtration on Millipore 96-well filtration plates containing 0.65 p DVPP filters. The beads were washed three times with binding buffer, and the bound ligand was quantitated by scintillation counting. For competition binding assays, the activated receptor was preincubated with the unlabeled competitor for 10 min prior tothe addition of [3sS] PTPlbC21hS. Synthesis of Phosphorylated Peptides-Phosphotyrosine-containing peptides were synthesized, purified, and characterized as described previously (39). RESULTS

All mammalian PTPases containa conserved cysteine residue in the catalytic domain that is absolutely required for enzymatic activity (16, 31, 32). Substitution of Cys2lSof rat brain P T P l b with serine produces an inactive enzyme (32) that nevertheless retains itsability to recognize substrate. T o The abbreviations used are: EGF, epidermal growth factor; GAP, GTPase-activating protein;PLCr, phospholipase C r l ; PTPase, protein tyrosine phosphatase; SH2, src homology domain 2; NGF, nerve growth factor.

A

B

1 2 1 200 9760 -

43 -

2

3 200 -

- EGFR 9768-

29

43

-

29 -

FIG. 1. Purification of the glutathione S-transferasePTPlbC215S fusion protein and immunoblot analysis of tyrosine-phosphorylated EGF receptor. A, the [JsS]PTP1b"YISs"gl~tathione S-transferase fusion protein was purified by affinity chromatography on glutathione-Sepharose as described under "Experimental Procedures." The resulting protein was analyzed by SDSpolyacrylamide gel electrophoresis followed by Coomassie Blue staining and autoradiography. Lane 1, molecular weight standards. Lane 2, Coomassie Blue staining. Lane 3, autoradiograph. The molecular mass of the fusion protein is approximately 69 kDa. B, 3T3/hEGFR cells were treated with or without 150 ng/ml EGF for 10 min at 37 "C. The EGF receptor was immunoprecipitated from cell lysates as described under "Experimental Procedures." The extent of tyrosine phosphorylation was analyzed by immunoblotting with anti-phosphotyrosine antibodies followed by 12sI-labeledprotein A. Lane 1, untreated cell lysates. Lane 2, EGF-treated cell lysates. The arrow indicates the tyrosine-phosphorylated EGF receptor.

PTPl b Binding to

23636

the Activated EGF Receptor

TABLE I

IP CXEGFR

Binding of thef5S]PTPlbC215S fusion protein to the EGF receptor 3T3/hEGFR cells were treated with or without 150 ng/ml EGF for 10 min at 37 'C. The cell lysates were incubated with preimmune serum or anti-EGFreceptor antibodies followed byprotein A-agarose. The immune complexes were incubated for 30 min with 30 nM ["SI PTPlbCZ15S in the absence or presence of 100-fold excess unlabeled fusion protein.Bound was separated fromfree by filtrationand quantitated by scintillation counting. The results are expressed as the means of triplicate determinations f S.D. Control

Immune precipitate

Anti-EGFR Preimmune

'

EGF

+

PTPlbC2'5S

-

+

200 + EGFR

97 -

EGF Label alone

Excess unlabeled

1854 f 89 290 f 81 1 5 2 f13167 f 25

Label alone

68-

Excess unlabeled

756 f 17 250 f 49 1 1 9 f12018 f 3 4

receptor antibody as described above. The immune complexes were fractionated by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and probed with a rabbit polyclonal anti-phosphotyrosine antiserum followedby l2'1-labeled protein A. Autoradiography of the immunoblot (Fig. 1B) revealed a modest tyrosine phosphorylation of the receptor prior to EGF treatment, although this phosphorylation was increased severalfold by exposure of cells to EGF. These results explain the residual binding of PTPlbczl'stothe receptor innontreated cells and suggest that the mutant phosphatase binds specifically to the EGFreceptor in a phosphorylation-dependent manner. To confirm the specificity of this interaction, we immobilized the PTPlbc21's fusion protein on glutathione-Sepharose beads and incubated the beads with lysates from cells treated with or without EGF. The beads were washed several times, and the bound proteins eluted with sample buffer and analyzed by SDS-polyacrylamide gel electrophoresis and immunoblotting with anti-phosphotyrosine antibodies (Fig. 2). As a control, the EGF receptor was immunoprecipitated with anti-EGF receptor antibodies and probed by anti-phosphotyrosine immunoblotting. Treatment of cells with EGF stimulated the binding of the phosphorylated EGF receptor to the PTPlbC215S beads. In addition, the PTPlbC215Sprotein was apparently heavily phosphorylated on tyrosine when incubated withthe lysates, although this phosphorylation was not growth factor-dependent. These results suggest theEGFdependent association of PTP1bc2'" with the tyrosine-phosphorylated EGF receptor, although it is possible that the increase in bound receptor reflects only increased content of phosphotyrosine with unchanged receptor levels. Using the immobilized, activated EGF receptor we evaluated some of the characteristics of [35S]PTPlbC215S binding. As was the case for PLC-y SH2 domains (36), the pHdependence of binding of PTPlbC2'5s to the EGFreceptor was not remarkable. Varying the pHfrom 5.5 to 9.0 had no significant effect on binding. Divalent cations such as calcium, magnesium, manganese, and zinc had no appreciable effect on binding at concentrations up to5 mM. In contrast, thisinteraction was quite sensitive to sodium orthovanadate, a known phosphatase inhibitor. [35S]PTPlbc21ss binding was inhibited by vanadate over a concentration range of 0.1 to 1.0 mM. These concentrations of vanadate had no effect on the binding of the PLC-y or abl SH2 domains to theactivated EGF receptor (data notshown). To determine the affinity of PTPlbC215S for the EGFreceptor, competition assays were performed under equilibrium binding conditions. [35S]PTP1bcz'sswas incubated with tyrosine-phosphorylated EGF receptor in the presence of the indicated concentrations of unlabeled fusion protein,and bound ligand was quantitated (Fig. 3). [3sS]PTPlbcz1's spe-

43 -

29 -

FIG. 2. Immunoblot analysis of proteins bound to PTPlbC2'5"beads. 3T3/hEGFR cells were treated with or without 150 ndml EGF for 10 min at 37 "C. The cells were lysed with HY buffer and incubated with either PTP1bCz1" immobilized on glutathione-Sepharose beads or anti-EGFreceptor (EGFR)antibodies and protein A-agarose. The beads were washed several times with HY buffer and boiled in SDS sample buffer. The eluted proteins were analyzed by SDS-polyacrylamide gel electrophoresis and immunoblotting with anti-phosphotyrosineantibodies. Molecular weight standards are indicated on the left. The arrow indicates the position of the tyrosine-phosphorylated EGF receptor.

0

100

200

300

400

500

bound (nM) FIG.3. Scatchard analysis of PTPlbC215Sfusion protein binding to theEGF receptor. 3T3/hEGFR cells were treated with 150 ng/ml EGF for 10 min a t 37 "C. EGF receptors were immunoprecipitated, and equilibrium binding of the PTPlbcz'ss fusion protein to the immune complexes was performed. The immunoprecipitated fusion protein receptors were incubated with 30 nM [nsS]PTPlbC215S in the presence of the indicated concentrations of unlabeled fusion protein. The incubation was carried out a t room temperature for 30 min, and bound was separated from free by filtration and quantitated by scintillation counting. The results were transformed to a Scatchard plot.

cifically bound to theactivated receptor and was displaced by unlabeled fusion protein in a concentration-dependent manner. Scatchard analysis of the results revealed a curvilinear plot with two apparent binding sites. The high affinity site exhibiteda Kd of approximately 100 nM with a BmaXof approximately 12 pmol/pg of receptor. The affinity of the

PTPl b Binding to the Activated EGF Receptor second site was much lower. Analysis of high affinity binding alone to the receptor indicates a stoichiometry of approximately 2:l. The affinity of PTPlbC215S for the EGF receptor is similar to that demonstrated previously for binding of a glutathione S-transferase fusion protein containing both SH2 domainsof PLCr (36). To determine whether SH2 proteins and PTPlb bind to similar sites on the receptor, we examined the ability of several fusion proteins containing SH2 domains to displace [3sS]PTPlbC215S binding (Fig. 4A). Equilibrium binding of PTPlbC2'5Sto the EGFreceptor was assayed in the presence of glutathione S-transferase fusion proteins containing the combined amino-and carboxyl-terminal SH2domains of PLCy (PLCr NCSH2), the amino-terminal SH2 domain of ras GAP (GAPNSHS), the ab1 SH2 domain (ablSH2), andthe amino-terminal SH2 domain of the p85 subunit of phosphatidylinositol 3-kinase (p85NSH2) (36, 37). Each of the SH2 domain-containing fusion proteins displaced PTPlbC215S

"tPTPase

0

0.001

0.01

1

0.1

100

10

Fusion Protein @M) I

I

o

0.001

I

I

\

I

d-

0.01

0.1

1

10

100

Fusion Protein @M) FIG. 4. Equilibrium binding of glutathione S-transferase fusion proteins to tyrosine-phosphorylated EGF receptor. A , theEGF receptor was immunoprecipitated from 3T3/hEGFR cells treated with 150 ng/ml EGF for 10 min at 37"C. The immune complexes were incubated with 30 nM [36S]PTPlCZ15S in the presence of the indicated concentrations of the following unlabeled fusion proteins: PLCr NCSH2, GAP NSH2, p85 NSH2, ab1 SH2, or PTPlc21ss. The incubation was performed in Tris binding buffer at room temperature for 30 min. was separatedfromfree by filtration and quantitated by scintillation counting. B, immunoprecipitated EGF receptor was incubated with 25 nM [35S]PLCrNCSH2 inthe presence of the indicated concentrations of unlabeled pTplbCZ16S. Thebinding assay was performed and quantitated as described above.

23637

binding in a concentration-dependent manner, although the potencies varied greatly (Fig. 4A).The PLCy and GAP SH2 domains were most effective in the displacement of PTPlbC215S, with IC50values of 0.18 and 0.10 pM, respectively. The ab1 and p85 SH2 domains were much less efficient at displacing PTPlbC215S,with IC50 values of20 and 10 p M , respectively. Interestingly, the displacement of [35S]PTPlbC215S by unlabeled PTPlbC215Soccurred with an IC,,of approximately 0.17 p ~ similar , to that observed with the PLCy and GAP SH2 fusion proteins. Because of these quantitative similarities, we examined whether PTPlbC215S could displace PLCy binding to the receptor. A fusion protein containing the two SH2 domains of PLC-y (PLCy NCSH2) was labeled with 35S as described for PTPlbC215S, and [35S]PLCy the was incubated with activated EGF receptor in the presence of unlabeled PTPlbC215S. Fig. 4B shows that PTPlbC215Sdisplaced PLCy binding in a concentration-dependent manner, with an ICs0 of approximately 0.13 p ~ These . results demonstratethat the affinities of PTPlbC215S and the PLCy SH2 domains for the phosphorylated EGF receptor are similar. Furthermore, the reciprocal displacement of both PTPlbC215S and PLCy SH2 domains suggests that these proteinsmay bind to similar sites on the receptor. To directly address this issue, we compared the displacement of both PTPlbC215S and PLCy binding to the EGF receptor by phosphorylated peptides. Peptides modeled around four of the major autophosphorylation sites of the EGF receptor (tyrosines 992,1068,1148, and 1173) were tested for their ability to displace the binding of [35S]PLCy or [35S]PTPlbC215Sto the phosphorylated EGF receptor in equilibrium binding assays (Fig. 5). As described previously, all four peptides displaced the binding of the PLCy SH2 domains (39); however, the peptides modeled around Tyrgg2 and Tyr"73 were clearly most effective (Fig. 5A). Similarly, PTPlbC215S bindingwas displaced by all four peptides, but peptides modeledonTyr"48 andTyrgg2 were much more efficient than those including TyrlW and T y P 3 (Fig. 5B). The unphosphorylated peptides had no effect on binding of either fusion protein to thereceptor (data not shown). These results indicate that both PLCy SH2 domains and PTPlb have preferred binding sites on the EGF receptor, suggesting that these interactions occur in a sequence-specific manner. To further explore the site specificity of EGF receptor binding by the PTPlbC215S and PLCySH2 fusion proteins, we constructeda series of single pointmutations of the phosphorylation sites in the carboxyl-terminal domain of the EGF receptor. These mutant receptors were transfected into 3T3 cells and were isolated after treatment with EGF, as described above. Equilibrium binding of both [35S]PTPlbC215S and [35S]PLCy to these receptors was evaluated (Table 11). Mutation of all five phosphorylation sites reduced the binding of both fusion proteins to background levels (data notshown). Substitution of Tyr114'with Dhenvlalanine did not aDDreciablv affect the binding PLcy. However, mutation of -f'yr'068 0; TyrlOM caused a 5-8-fold reduction in the affinity of PLCy for thephosphory~ated receptor. Mutation of ~ ~ ~ and 9 9 2 T y P 3in thereceptor caused a greater decrease in the affinity of this interaction, resulting in a 200-300-fold reduction; with a Kd of approximately 100 pM. With respect toPTPlbC215S binding, substitution of Tyr106', TyrlW, or Tyr1173with phenylalanine hadlittle effect, althoughmutations at both Tyrgg2 and Tyr114' caused 3-4-fold reductions in affinity. A direct comparison of the effect of thesemutations on the relative binding affinities Of P T P l b and P L c y is difficult, since the latter fusioncontains protein two SH2 domains, maywhich

PTPl b Binding Activated to the

23638

i

" t 1173

1068 992

1148

EGF Receptor

TABLE I1 Comparison of binding affinities ofPTPlbC215S and PLCySH2 fusion proteins to mutated EGF receptor NIH 3T3cells expressing wild-type or mutated EGFreceptors were treated with 150 ng/ml EGF for 10 min, and receptors were immunoprecipitated as described in Fig. 2. Equilibrium binding of13?3] PTPlbC215Sand[36S]PLCr SH2 fusion proteins tothe different receptors was analyzed as described in Fig. 3. Each assay contained approximately equal numbers of receptors. Equilibrium dissociation constants (I&) were calculated using the LIGAND program by weighted molecular regression curve fitting to the mass action equation.Resultsare representative of a single experiment that was repeated three times. K d

Receptor

PLC-y SH2

PTPlbC21ss M

100

so 60 40 20

0 0.1

0.01

1 0 0 0 11 0 0

10

Phosphopeptide @M)

I

FIG. 5. Inhibition of fusion protein-EGF receptor binding by tyrosine-phosphorylated peptides. A, immunoprecipitated EGF receptor was incubated with 25nM [36S]PLCyNCSH2 in the presence of the indicated concentrations of tyrosine-phosphorylated peptides modeled after the following EGF receptor autophosphorylation sites: Tyrse2,Tyr'"@, T ~ r l ' ~and ~ , Tyr"". The incubation was performed in Tris binding buffer for 30 min at room temperature. Bound was separated from free by filtration and quantitated by scintillation counting. B , immunoprecipitated EGF receptor was incubated with [35S]PTPlbcz15s the in presence of the indicated concentrations of the following tyrosine-phosphorylated peptides: TyrS2, TyrlW, T Y ~ " ' ~and , Tyr'"'. Bound material was quantitated as described above.

bind to different sites on the receptor. Nevetheless, these data are generally consistent with the observed sensitivity of these proteins to syntheticphosphopeptides. As both fusion proteins exhibited specificity for Tyrsz, we used this site to assess the contribution of adjacent amino acids to theinteraction of the fusion proteins with the receptor. Phosphopeptides were synthesized with alanine substitution of GluW'and Leus3, and thesewere compared with the wild-type phosphopeptide for inhibition of both PLCy SH2 and PTPlbC216S binding (Table 111).This conservative modification of the sequence proximate to the phosphotyrosine discriminated the binding specificities between these two ligands. Substitution of Leugg3with alanine decreased the ability of the phosphopeptide to compete for binding of both fusion proteins. Substitution of GluW', however,increased by over %fold the affinity of the peptide for PTPlbcz15s, withno effect on PLCy binding. Thus, while both fusion proteins prefer the Tyrgg2 binding site, the mechanisms of phosphotyrosine recognition at this site appear to be distinctive.

3.95 X 1.65 X 5.10 X 10" 4.95 X 10-~ 2.43 X 9.52 X 10-~

Wild-type Y992F Y1068F Y 1086F Y1148F Y1173F

4.80 X 9.86 X 2.24 X 4.37 x 8.20 x 1.35 x

10-7 10-5 10-7 10-4

TABLEI11 Displacement of PTPlb"'5S and PLCy SH2 binding by alaninesubstituted EGF receptor phosphopeptides 3T3/hEGFR cells were treated with 150 ng/ml EGF for 10 min, and receptors were immunoprecipitated as described in Fig. 2. Equilibrium binding of [35S]PTPlbC216S and [36S]PLCySH2 fusion proteins was performed, as described in Fig. 5, in the presence of the EGF receptor peptides listed below, and the ICm values were determined. Peptide 1 represents the wild-type EGF receptor sequence, peptides 2 and 3 are the alaninesubstitutions, and peptide 4 is nonphosphorylated. IC60 Sequence PTPlbCZ1sS PLC-y SH2 W

1.DADqYLIPQQG 2. DADA,YLIPQQG 3. DADE,YAIPQQG 4. DADEYLIPQQG

0.1 0.04 0.30 No effect

10 10 20 No effect

DISCUSSION

The activities of receptor tyrosine kinases, as well as their interactions with endogenous signaling proteins, are under tight regulation. In the case of the pp140trkNGF receptor in PC-12 cells, ligand-dependent receptor phosphorylation and association with the SH2 domains of PLCy are transient, despite the continued presence of the growth factor (40). In contrast,tyrosine phosphorylation of the insulin receptor persists as long as insulin remains bound. The receptor subsequently undergoes dephosphorylation upon removal of the ligand (41). Although the precise molecular events that define these differences in phosphorylation and dephosphorylation arenot well understood, the accessibility of receptors to PTPases and other phosphotyrosine-binding proteins may play an important role. It has been suggested (42) that PTPases and certain SH2 proteins may exist in a dynamic equilibrium with respect to binding receptor tyrosine kinases. Such competition between these signaling molecules would require a critical level of specificity in recognition of tyrosine phosphorylation sequences on the part of both PTPases and SH2 domains. One of the problems in evaluating the substrate specificity of PTPases has arisen from the difficulty in distinguishing between catalytic activity and substratebinding. This enigma has been obviated by the generation of a site-directed mutant at Cys215 ofPTPlb, which lacks catalytic activity but retains

EGF Receptor

PTPl Activated bthe Binding to phosphotyrosine recognition (32). We have exploited this finding to develop an equilibrium binding assay to analyze the association of this mutant PTPase with the activated EGF receptor. This in vitro binding assay has allowed quantitative assessment of the interaction between this PTPase andtheEGF receptor, as well asa comparison with the receptor binding properties of various SH2 domains. These two types of interactions were surprisingly similar with regard to pH and cation dependence and kinetics of association. to the EGF receptor is Moreover, the binding of PTPlbC215S characterized by an affinity comparable with that observed for the SH2 domains of PLCy and GAP and was displaced by fusion proteins containing these two SH2 domains. Although the molecular basis for these interactions is not well defined, many PTPases share some sequence homology with a region of SH2 domains containing the sequence FLVRESES, which was found to be essential for phosphotyrosine recognition in the ab1 oncogene (8, 37). A similar sequence, FKVRESGS, is found 19 residues to the amino terminus of the essential Cys215in P T P l b (33) and might be involved in phosphotyrosine recognition. Despite the many similarities in the characteristics of SH2 and PTPasebinding to the EGF receptor, we uncovered some differences in sequence specificity around the phosphorylated residues. Analysis of binding toEGF receptors that had undergone site-directed mutation at themajor autophosphorylation sites revealed that the PLCy SH2 domains interact with the receptor mainly at Tyrgg2,whereas the GAP aminoterminalSH2 'domain appeared to bindpredominantly at Tyr114'.' These data are consistent with displacement of binding of the labeled fusion proteins with phosphopeptides modeled on these phosphorylation sites. Similar experimentswith PTPlbC215S revealed that both Tyrgg2 and Tyr114' represented high affinity binding sites on the EGFreceptor, whereas other sites were less critical for binding. Further comparison of the EGF receptor fragment modeled on Tyrgg2by alanine substitution of Glugglor Leugg3 revealed differences between PLCy SH2 and PTPase binding to the receptor, illustrating the complexity of sequence specificity regarding phosphopeptide recognition by these two proteins. The complex substrate specificity of P T P l b for the EGF receptor and the relative differences and similarities to the binding of the PLCy and GAP SH2 domains suggest that interactions such as these may play an important role in the regulation of signal transduction for growth and differentiation. As suggested by Rotin et al. (42), SH2 domains may function,in part, to regulate the accessibility of receptor tyrosine kinases to PTPases. This would account for the general increase in tyrosine phosphorylation following overexpression of SH2-containing oncogenes, such as v-crk (7). SH2-containing proteinsthat contain enzymatic activity may also modulate receptor phosphorylation in this manner. A possible example is the interaction of PLCy with the NGF receptor. Although these two proteins associate in an NGFdependent manner in PC-12 cells, NGF does not cause an appreciable stimulation of PI turnover or calcium mobilization (43), suggesting another regulatory role for this interaction. The molecular mechanisms involved in substratespecificity for PTPases may be under the control of many factors. Intracellular localization is likely to limit the access of G. Zhu and A. R. Saltiel, unpublished observations. D. MacLean, A. M. Seffler, D. McNamara, E. Dobrusin, G. Zhu, Z. Y. Zhang, J. Dixon, S. J. Decker, A. R. Saltiel, T. K. Sawyer, and J. A. Bristol, manuscript in preparation.

23639

PTPases topotential substrates.Moreover, the PTPasesshow distinctive patterns of tissue distribution (18, 21, 22, 29, 30, 44). Data presented here indicate that specific recognition motifs may also exist for different PTPases. Thus, although the precise mechanisms involved in the regulation of PTPases remain unknown, there is clearly greater substrate specificity than was originally presumed. REFERENCES 1. Hunter, T., and Cooper, J. A. (1985) Annu. Reu. Bbchem. 64,897-930 2. Yarden, Y., and Ullrich, A. (1988) Annu. Reu. Bwchem. 67,443-478 3. Ullrich, A., and Schlessinger, J. (1990) Cell 61,203-212 4. Koch, C., Anderson, D., Moran, M., Ellis, C., and Pawson,T. (1991) Science 252,668-674 5. Escohedo, J. A., Navankasattusas, S., Kavanaugh, W. M., Milfay, D., Fried, V. A,, and Williams, L. T. (1991) Cell 6 6 , 75-82 6. Lehmann, J., Riethmuller, G., and Johnson, J. (1990) Nucleic Acids Res. 18,1048 7. Mayer, B. J., Hamaguchi, M., and Hanafusa, H. (1988) Nature 3 3 2 , 272976

I.-

8. Mayer, B. J., Jackson, P. K., VanEtten, R. A., and Baltimore, D. (1992) Mol. Cell. Biol. 1 2 , 609-618 9. Otsu, M., Hiles, I., Gout, I., Fry, M. J., Ruiz-Larrea, F., Panayotou, G., Thompson, A,, Dband, R., Hsuan, J., Totty, N., Smith, A. D., Morgan, S. J., Courtneidge, S. A,, Parker, P. J., and Waterfield, M. D. (1991) Cell 65.91-104 "," ---

10. Sadowski, I., Stone, J. C., and Pawson, T. (1986) Mol. Cell. Bid. 6 , 43964408 11. Skolnik, E. Y., Margolis, B., Mohammadi, M., Lowenstein, E., Fischer, R., Drepps, A., Ullrich, A., and Schlessinger, J. (1991) Cell 66,83-90 12. Stahl, M. L., Ferenz, C. R., Kelleher, K. L., Kriz, R. W., and Knopf, J. L. (1988) Nature 3 3 2 , 269-272 13. Suh, P. G., Ryn, S. H., Moon, K. H., Suh, H. W., and Rhee, S. G. (1988) Proc. Nutl. Acud. Sci. U. S. A. 8 6 , 5419-5423 14. Fischer, E. H., Charbonneau, H., and Tonks, N. K. (1991) Science 2 6 3 , 401-406 15. Charbonneau. H.. Tonks. N. K.. Walsh. K. A,. and Fischer. E. H. (1988) . . Proc. Natl. A c d . Sci. U . S. A. '86, 7182-7186 16. Streuli, M., Krueger, N. X., Hall, L. R., Scblossman, S. F., and Saito, H. (1988) J. Exp. Med. 168,1523-1530 17. Freeman, R. M., Jr., Plutzky, J., and Neel, B. G. (1992) Proc. Nutl. Acud. Sci. U. S. A. 8 9 , 11239-11243 18. Matthews, R. J., Bowne, D. B., Flores, E., and Thomas, M. L. (1992) Mol. Cell. Bid. 1 2 , 2396-2405 19. Plutzky, J., Neel, B. G., and Rosenherg, R. (1991) Proc. Nutl. Acad. Sci. U. S. A. 8 9 , 1123-1127 20. Shen, S., Bastien, L., Posner, B. I., and Chretien, P. (1991) Nature 3 6 2 , 736-739 21. Yi, T., Cleveland, J. L., and Ihle, J. N. (1992) Mol. Cell. Biol. 12,836-846 22. Gu, M., York, J. D., Warshawsky, I., and Majerus, P. W. (1991) Proc. Nutl. Acud. Sci. U. S. A. 88,5867-5871 23. Yang, Q., and Tonks, N. K. (1991) Proc. Nutl. Acud. Sei. U. S. A. 88.59495953 24. Cool, D. E., Tonks, N. K., Charbonneau, H., Walsh, K. A,, Fischer, E. H., and Krebs, E. G. (1989) Proc. Nutl. Acud. Sci. U. S. A. 86,5257-5261 25. Chernoff, J., Schievella, A. R., Jost, C. A,, Erickson, R. L., and Neel, B. G. (1990) Proc. Nutl. Acud. Sci. U. S. A. 8 7 , 2735-2739 26. Brown-Shimer, S., Johnson, K. A,, Lawrence, J. B., Johnson, C., Bruskin, A,, Green, N. R., and Hill, D. E. (1990) Proc. Nutl. Acud. Sci. U. S. A. 87.5148-5152 27. Ostergard, H. L., Shackelford, D. A,, Hurley, T. R., Johnson, P., Hyman, R., Sefton, B. M., and Trowhridge, I. S. (1989) Proc. Nutl. Acud. Sci. U. S. A. 86,8959-8963 28. Ostergard, H. L., and Trowbridge, I. S. (1990) J.Exp. Med. 172,347-350 29. Yang, X., Seow, K. T., Bahri, S. M., Oon, S. H., and Chia, W. (1991) Cell 67,661-673 30. Tian, S.-S.,Tsoulfas, P., and Zinn, K. (1991) Cell 67,675-685 31. Streuli, M., Krueger, N. X., Thai, T., Tang,M., and Saito,H. (1990) EMBO J. 9,2399-2407 32. Guan, K., and Dixon, J. E. (1990) Science 249,553-556 33. Guan, K., Haun, R. S., Watson, S. J., Geahlen, R.L., and Dixon, J. E. (1990) Proc. Nutl. Acud. Sci. U. S. A. 87,1501-1505 34. Guan, K., and Dixon, J. E. (1991) Anal. Biochem. 1 9 2 , 262-267 35. Decker, S. J., Ellis, C., Pawson, T., andVelu, T. (1990) J. Biol. Chem. 2 6 6 , 7009-7015 36. Zhu, G., Decker, S. J., andSaltiel, A.R. (1992) Proc. Nutl. Acud. Sci. U. S. A. 89,9559-9563 37. Zhu, G., Decker, S. J., Mayer, B. J., and Saltiel, A. R. (1993) J.Bid. Chem. 2 6 8 , 1775-1779 38. Ohmichi, M., Decker, S. J., Pang, L., andSaltiel, A. R. (1991) J. Bid. Chem. 2 6 6 , 14858-14861 39. McNamara, D. J., Dobrusm, E. M., Zhu, G., Decker, S. J., and Saltiel, A. R. (1993) Int. J. Pept. Protein Res. in press 40. Ohmichi, M., Decker, S. J., Pang, L., and Saltiel, A. R. (1991) Biochem. Biophys. Res. Commun. 1 7 9 , 217-223 41. Haring, H. U., Kasuga, M., White, M.F., Crettaz, M., and Kahn, C. R. (1984) Biochemist? 23,3298-3306 42. Rotin, D., Margolis, B., Mohammadi, M., Daly, R. J., Daum, G., Li, N., Fischer, E. H., Burgess, W. H., Ullrich, A., and Schlessinger, J. (1992) EMBO J. 11,559-567 43. Chan, B. L., Chao, M. V., and Saltiel, A. R. (1989) Proc. Nutl. Acud. Sci. U. S. A. 86,1756-1760 44. Streuli, M., Krueger, N. X., Tsai, A. Y. M., and Saito, H.(1989) Proc. Nutl. Acud. Sci. U. S. A. 86,8698-8702