Coupling to the Adaptor Protein GRB21

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 345, No. 1, September 1, pp. 103–110, 1997 Article No. BB970245

A New Function for Phospholipase C-g1: Coupling to the Adaptor Protein GRB21

Zhendong Pei,* Judith A. Maloney,* Lijun Yang,† and John R. Williamson*,2 *Department of Biochemistry and Biophysics, School of Medicine, University of Pennsylvania, Philadelphia, PA 19104; and †Department of Pathology, Albert Einstein College of Medicine, Bronx, New York 10461

Received April 24, 1997, and in revised form June 4, 1997

Key Words: phospholipase C-g1; GRB2; epidermal Epidermal growth factor (EGF)-induced autophos- growth factor; angiotensin II; tyrosine phosphoryla- phorylation of the EGF receptor results in high-afﬁn- tion; crosstalk; WB cells. ity binding of the adaptor protein GRB2, which serves as a convergence point for multiple signaling path- ways. Present studies demonstrate that EGF induces the co-immunoprecipitation of phospholipase C (PLC)- The Ras protein plays a critical role in receptor tyro- g1 with the adaptor protein GRB2 and the guanine sine kinase-mediated mitogen-activated protein kinase nucleotide exchange factor Sos, but not with the adap- activation. Extensive evidence shows that the coupling tor protein SHC, in WB cells. Inhibition of PLC-g1 tyro- of receptor-tyrosine kinases to Ras is mediated by the sine phosphorylation by phenylarsine oxide reduces recruitment of the guanine nucleotide exchange factor the co-immunoprecipitation of PLC-g1 with GRB2. Sos to the plasma membrane via the adaptor molecule Furthermore, angiotensin II, a G protein-coupled re- GRB2 (1–5). The adaptor protein GRB2 is composed ceptor agonist, also induces the tyrosine phosphoryla- almost entirely of one SH2 domain and two SH3 do- tion of PLC-g1 and its co-immunoprecipitation with mains. Upon cell stimulation, GRB2 interacts via its GRB2 in WB cells. Interestingly, angiotensin II stimu- SH2 domain with the autophosphorylated carboxyl-ter- lation also causes tyrosine phosphorylation of the EGF minal tail of activated tyrosine kinase receptors such receptor, suggesting that angiotensin II-induced PLC- as the epidermal growth factor (EGF)3 receptor and g1 tyrosine phosphorylation in WB cells may be via recruits the nucleotide exchange factor Sos to the EGF receptor tyrosine kinase activation. In addition, plasma membrane via its SH3 domains (6), which in there is some level of association between PLC-g1 and turn leads to the activation of the Ras protein (7). More GRB2 that is independent of the tyrosine phosphoryla- recent studies indicate that another adaptor protein, tion of PLC-g1 in both in vivo and in vitro studies. In vitro studies further demonstrate that the Tyr771 and SHC, also binds to tyrosine autophosphorylation sites Tyr783 region of PLC-g1 and the SH2 domain of GRB2 of the activated receptors via its SH2 domain and be- are potentially involved in the tyrosine phosphoryla- comes tyrosine phosphorylated in stimulated cells. tion-dependent association between PLC-g1 and Upon phosphorylation, SHC interacts with the SH2 do- GRB2. The association of PLC-g1 with GRB2 and Sos main of GRB2 and functions as an alternative docking suggests that PLC-g1 may be directly involved in the site for GRB2–Sos complexes (8). The existence of two Ras signaling pathway and that GRB2 may be involved adaptor proteins has invoked the suggestion that GRB2 in the translocation of PLC-g1 from cytosol to the may have other roles besides Ras activation (9). Indeed, plasma membrane as a necessary step for its effect on in addition to Sos, GRB2 has also been shown to bind inositol lipid hydrolysis. ᭧ 1997 Academic Press to Vav, c-Abl, dynamin, Gab1, phosphotyrosine phos-

1 This work was supported in part by National Institute of Health 3 Abbreviations used: Ang II, angiotensin II; BSA, bovine serum Grants DK-15120 and DK-48493 to J.R.W. Z.P. was supported by albumin; EGF, epidermal growth factor; EGTA, ethylene glycol bis(b- National Institute of Health Institutional Training Grant DK-07314. aminoethyl ether)-N,N؅-tetraacetic acid; GPCR, G protein-coupled 2 To whom correspondence should be addressed at Department receptor; GST, glutathione S-transferase; PAO, phenylarsine oxide; of Biochemistry and Biophysics, School of Medicine, University of PBS, phosphate-buffered saline; PBST, phosphate-buffered saline Pennsylvania, 37th & Hamilton Walk, Philadelphia, PA 19104. Fax: with 0.1% Tween 20; PLC, phospholipase C; SDS–PAGE, sodium (215) 898-9918. dodecyl sulfate–polyacrylamide gel electrophoresis.

0003-9861/97 $25.00 103 Copyright ᭧ 1997 by Academic Press All rights of reproduction in any form reserved.

AID ABB 0245 / 6b3e$$$421 07-31-97 06:25:58 arca 104 PEI ET AL. phatase SH-PTP2, and p85 subunit of phosphatidylino- of PLC-g1 further increases this association. Further- sitol 3-kinase (10–17). more, angiotensin (Ang) II, a G protein-coupled recep- The activation of phospholipase C (PLC) is one of tor agonist, also induces the tyrosine phosphorylation several early cellular responses to various growth fac- of the EGF receptor and PLC-g1, and the association tors and peptide mitogens (18). PLC catalyzes the gen- of PLC-g1 with GRB2. eration of two second messengers from the hydrolysis of phosphatidylinositol 4,5-bisphosphate. One, diacyl- glycerol, induces activation of members of the protein MATERIALS AND METHODS kinase C family of serine/threonine-specific kinases, Materials. The monoclonal antibody against PLC-g1 was a gift while the second, inositol 1,4,5-trisphosphate, causes from Dr. S. G. Rhee (National Institute of Health). Polyclonal anti- Ca2/ release from specialized intracellular organelles bodies against the EGF receptor, PLC-g1 and Sos, the monoclonal antibody against GST, mouse and rabbit IgG, and protein A/G plus (19). PLC-g1, one of the PLC isoforms that contains agarose were purchased from Santa Cruz Biotechnology, Inc. Mono- the Src homology regions in its structure with two SH2 clonal antibodies against GRB2 and phosphotyrosine were purchased domains and one SH3 domain, has been shown to be a from Transduction Laboratories. EGF and polyclonal antibody against SHC were purchased from Upstate Biotechnology, Inc. An- physiological substrate for tyrosine phosphorylation by 32 tyrosine kinase receptors such as the EGF receptor, giotensin II and phenylarsine oxide (PAO) were from Sigma. [g- P]- ATP was obtained from Amersham. platelet-derived growth factor receptor, and nerve Cell culture. WB cells, a rat liver epithelial cell line, and CCL- growth factor receptor (reviewed in Ref. 18). PLC-g1 39 cells that are derived from Chinese hamster lung fibroblasts were forms stable complexes with these tyrosine kinase re- maintained at 37ЊC in a humidified 5% CO2 atmosphere in Richter’s ceptors and has been implicated as a signal transducer improved essential medium containing insulin (Irvine Scientific, for these receptors (20). A number of studies have indi- Santa Ana, CA) and McCoy’s 5A medium (Life Technologies, Inc) cated that PLC-g1 is involved in cell mitogenesis. Mi- supplemented with 10% fetal bovine serum, respectively. Prior to the start of experiments, cells were grown to 80% confluence and croinjection of purified PLC-g1 has been shown to in- starved for 20 h in the maintaining medium without serum. duce DNA synthesis (21). It was also suggested that Fusion proteins. The plasmids expressing GST-GRB2, GST- PLC-g1 is upstream of Ras in the nerve growth factor GRB2-SH2, GST-p85-C-SH2, GST-22Y3 (derived from bovine PLC- receptor signal transduction pathway (22). Further- g1 cDNA residues 545–850 containing the two SH2 domains and more, PLC-g1 has been found to be involved in the one SH3 domain together with the two potential tyrosine phosphory- 771 783 platelet-derived growth factor receptor downstream lation sites Tyr and Tyr between the C-terminal SH2 domain and the SH3 domain of PLC-g1), GST-N/C (residues 545–759 con- mitogenic signaling (23–26). In addition, studies have taining the N-SH2 and C-SH2 domains of PLC-g1), and GST-SH3 shown that PLC-g1 can induce cell mitogenesis inde- (residues 798–850 containing the SH3 domain of PLC-g1) were pendently of its catalytic activity (27–29). kindly provided by Drs. Gerald Gish and Tony Pawson (Samuel Lu- Tyrosine phosphorylation of PLC-g1 has been sug- nenfeld Research Institute, Mount Sinai Hospital, Toronto, Canada). gested to be necessary for its activation in intact cells, Fusion proteins were purified by binding to glutathione–agarose beads and eluted with reduced glutathione (34). but the detailed mechanisms involved are still not clear. EGF stimulation elicited a redistribution of PLC- Immunoprecipitation and Western blotting. Cells in 100-mm tis- sue culture plates were stimulated with 200 ng/ml of EGF or 1 mM g1 from the cytosol to the plasma membrane in A-431 Ang II for 2 min. The stimulation was terminated by washing with epidermoid carcinoma cells (30). However, it appears 10 ml phosphate-buffered saline (PBS), and the cells were lysed in that binding to the activated receptor is insufficient for PLC lysis buffer containing 50 mM Hepes (pH 7.4), 1% Triton X-100, PLC-g1 activation, and that tyrosine phosphorylation 10% glycerol, 150 mM NaCl, 100 mM NaF, 1 mM EGTA, 50 mM pyrophosphate, 1.5 mM MgCl2 ,10mMdithiothreitol with 10 mg/ml is required for PLC-g1 activation in intact cells (31– leupeptin and aprotinin, 1 mM phenylmethylsulfonyl fluoride, and

33), although not for in vitro assays (31). This differ- 1mM Na3VO4 for 30 min on ice. The supernatants were collected by ence may be accounted for by substrate availability centrifugation at 14,000 rpm for 10 min at 4ЊC. The amount of protein since the substrate of PLC-g1, phosphatidylinositol in the supernatants was measured by the method of Bradford using 4,5-bisphosphate, is located on the inner leaﬂet of the bovine serum albumin (BSA) as a standard. For immunoprecipitation, 2 mg of monoclonal antibodies was incubated with 1 mg of cell plasma membrane. Therefore, a possible function for lysates for 2 h at 4ЊC with shaking and then incubated with 40 ml phosphotyrosine residues of PLC-g1 in its activation is of protein A/G plus agarose beads for 1 h at 4ЊC with shaking. After to bind to the SH2 domain of a coupling factor which incubation, the agarose beads were washed three times with PLC is responsible for bringing PLC-g1 to the plasma mem- lysis buffer and twice with PBS. The beads were then resuspended brane. in SDS–PAGE sample buffer and heated at 95ЊC for 5 min. For Western blotting, the immunocomplexes were resolved by In an effort to understand the mechanisms for PLC- SDS–PAGE and transferred to nitrocellulose membranes which g1 enzymatic activation and its mitogenic effect in WB were blocked with 3% BSA or 5% nonfat dry milk in PBS containing cells, we studied the tyrosine phosphorylation of PLC- 0.1% Tween 20 (PBST) for 1 h. The blots were then incubated with g1 and its interaction with signaling proteins upstream primary antibodies for1hatroom temperature. Bound primary antibodies were visualized by the ECL detection system, using horse- of the Ras protein. In this report, we demonstrate that radish peroxidase-conjugated secondary antibodies at a 1:3000 dilu- PLC-g1 is associated with the adaptor protein GRB2 tion. When needed, the membranes were stripped in ImmunoPure constitutively, and that the tyrosine phosphorylation Elution Buffer (from Pierce) for 30 min at 55ЊC. In some cases, pro-

AID ABB 0245 / 6b3e$$$421 07-31-97 06:25:58 arca PHOSPHOLIPASE-g1 BINDS DIRECTLY TO GRB2 105 tein bands were quantitated following densitometric scanning using Sigma Gel. In vitro binding assays. For the direct binding assay, PLC-g1 immunocomplexes were separated by SDS–PAGE and transferred to nitrocellulose membrane. After blocking with PBST containing 5% nonfat dry milk for 1 h at room temperature, the blot was incubated with 0.05 mg of GST or GST-GRB2 per microliter in PBST containing 3% BSA for1hatroom temperature. The blot was then incubated with primary anti-GST monoclonal antibody for 1 h at room temperature, following the Western blotting procedures as described above. In order to eliminate the nonspecific binding from the glutathione– agarose beads, the beads conjugated with fusion proteins were prepared by mixing an equal amount of excess of purified fusion proteins with an equal volume of 50% slurry of glutathione–agarose beads FIG. 1. EGF induces the association of PLC-g1 with GRB2 and Sos (5 mg of fusion protein/10 ml of beads) overnight at 4ЊC and then in WB cells. Serum-starved WB cells were stimulated with 200 ng/ washed twice with PBS. For PLC-g1 and GRB2 fusion proteins pre- ml EGF for 2 min, and cell lysates were immunoprecipitated (IP) cipitating assays, 20 ml of glutathione–agarose beads conjugated with the indicated antibodies as described under Materials and with fusion proteins were mixed with 2 mg of cell lysates from EGF- Methods. The immunoprecipitates were separated by 10% SDS– stimulated or unstimulated cells. After incubation for4hat4ЊC with PAGE and blotted with the indicated antibodies. Similar results shaking, the beads were washed three times with PLC lysis buffer were obtained in two other experiments. plus 2% Triton X-100 and two times with PBS and then analyzed by SDS–PAGE and Western blotting as described above. For the GRB2 fusion protein binding 22Y3 assay, purified GST- 22Y3 protein was first digested with thrombin to delete GST. The tyrosine phosphorylation of PLC-g1 correlates with its GST-free 22Y3 protein was then tyrosine phosphorylated in vitro co-immunoprecipitation with GRB2, we employed a using purified c-Src (obtained from Upstate Biotechnology, Inc.) by pharmacological approach. Previous studies indicated following the manufacturer’s protocols. The 32P-labeled 22Y3 protein was then shaken with 10 ml of glutathione–agarose beads conjugated that PAO at 1 mM for 30 s selectively inhibited PLC- with fusion proteins for 2 h. After incubation, the mixtures were g isoform tyrosine phosphorylation in some cell lines washed three times with PLC lysis buffer and one time with PBS. whilst not inhibiting tyrosine phosphorylation of up- The mixtures were then added to sample buffer, heated for 5 min at stream tyrosine kinases such as receptor tyrosine ki- 95ЊC, and separated by SDS–PAGE. The binding was detected by autoradiography. nases (36). We first determined whether we could re- produce that inhibition in WB cells. WB cells were RESULTS stimulated with EGF for 2 min after pretreatment with GRB2 and Sos co-immunoprecipitation with PLC-g1 or without 1 mM PAO for 30 s. The cell lysates were in WB cells. To identify whether PLC-g1 could co- immunoprecipitated with anti-phosphotyrosine anti- immunoprecipitate with GRB2 in intact WB cells upon body. Figure 2A shows that PAO had very little effect EGF stimulation, EGF-stimulated or unstimulated on the whole anti-phosphotyrosine blot. Furthermore, cells were lysed and immunoprecipitated with 2 mgof we compared the effect of PAO on tyrosine phosphory- monoclonal anti-PLC-g1 or anti-GRB2 antibodies, re- lation of the EGF receptor, SHC, and PLC-g1. As spectively. The immunocomplexes were resolved by shown in Fig. 2A, PAO had very little effect on tyrosine SDS–PAGE and transferred to nitrocellulose mem- phosphorylation of the EGF receptor and SHC. In con- branes. Appropriate membrane fractions were cut and trast, tyrosine phosphorylation of PLC-g1 was almost immunoblotted with either anti-PLC-g1 or anti-GRB2 totally inhibited. Therefore, PAO is a relatively selec- antibodies. Figure 1A shows that EGF stimulation re- tive inhibitor of PLC-g1 tyrosine phosphorylation when sulted in a large tyrosine phosphorylation of PLC-g1 used at 1 mM for 30 s. We then studied whether the and an increased co-immunoprecipitation with GRB2. inhibition of PLC-g1 tyrosine phosphorylation would Also, PLC-g1 was found to be increased approximately affect the association between PLC-g1 and GRB2. As fourfold in anti-GRB2 immunocomplexes from EGF- shown in Fig. 2B, the association between PLC-g1 and treated WB cells (Fig. 1B). Furthermore, PLC-g1 im- GRB2 was indeed correspondingly reduced. The results munocomplexes also included Sos (Fig. 1C), which con- support the suggestion that tyrosine phosphorylation stitutively binds to GRB2 (35). The basal level of co- of PLC-g1 is responsible for the increased co-immuno- immunoprecipitation of PLC-g1 with GRB2 and Sos precipitation of PLC-g1 with GRB2 after EGF stimula- suggests that non-tyrosine phosphorylated PLC-g1 tion. may constitutively bind to GRB2 without growth factor Ang II induces tyrosine phosphorylation of PLC-g1 stimulation. However, these results also demonstrate and its co-immunoprecipitation with GRB2 in WB cells. that EGF stimulation further increases the association Recent studies indicated that some G protein-coupled of PLC-g1 with the GRB2-Sos complex in WB cells. receptor (GPCR) agonists, such as Ang II and throm- Phenylarsine oxide reduces the co-immunoprecipita- bin, were able to induce tyrosine phosphorylation of tion of GRB2 with PLC-g1. To examine whether the PLC-g1 in several cell types (37, 38). We wondered

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FIG. 3. Ang II induces PLC-g1 tyrosine phosphorylation and co- immunoprecipitation with GRB2. Serum-starved WB cells were stim- FIG. 2. Effects of phenylarsine oxide (PAO) on PLC-g1 tyrosine ulated with 1 mM Ang II for the indicated times, and cell lysates were phosphorylation and association with GRB2. Serum-starved WB immunoprecipitated (IP) with monoclonal anti-PLC-g1 antibody. cells were stimulated alone or following pretreatment with 1 mM The immunocomplexes were resolved by SDS–PAGE and blotted PAO or vehicle for 30 s. The anti-phosphotyrosine (A) or anti-PLC- with the indicated antibodies. g1 (B) immunocomplexes were resolved by SDS–PAGE and transferred to nitrocellulose membrane as described under Materials and Methods. The membranes were ﬁrst blotted with anti-phosphotyrosine and then the corresponding fragments were cut, stripped, and Ang II induces tyrosine phosphorylation of EGF re- blotted with the indicated antibodies. The results are representative ceptor in WB cells. GRB2 is an adaptor protein that of three independent experiments. links the EGF receptor to the Ras signaling pathway (6). It has been shown that EGF stimulation induces tyrosine auto-phosphorylation of the EGF receptor, whether these GPCR agonists could also induce tyrosine phosphorylation of PLC-g1 and affect its co-immunoprecipitation with GRB2 in WB cells. As shown in Fig. 3, Ang II (1 mM for 2 min) was able to induce tyrosine phosphorylation of PLC-g1, albeit much less than after EGF stimulation. Thrombin, however, was not able to induce the tyrosine phosphorylation of PLC- g1 in WB cells. Since Ang II increased the tyrosine phosphorylation of PLC-g1 in WB cells, we examined whether it was also able to induce the co-immunoprecipitation of PLC-g1 with GRB2. As shown in Fig. 3, Ang II indeed did cause an increased co-immunoprecipitation of PLC-g1 with GRB2 in WB cells. EGF induces co-immunoprecipitation of PLC-g1 with GRB2 in CCL-39 cells. After establishing that tyrosine phosphorylation of PLC-g1 correlates with its co- immunoprecipitation with GRB2, we studied whether this phenomenon could also occur in another cell type. CCL-39 cells, derived from Chinese hamster lung ﬁ- broblasts, were treated with EGF and Ang II respectively, and then lysed and immunoprecipitated with 2 FIG. 4. EGF induces association of PLC-g1 with GRB2 in the CCL- mg of polyclonal anti-PLC-g1 antibody. As shown in 39 cells. The CCL-39 cells were starved overnight and stimulated Fig. 4, EGF was able to induce tyrosine phosphoryla- with 1 mM Ang II or 200 ng/ml EGF for 2 min. The cell lysates were immunoprecipitated (IP) with polyclonal anti-PLC-g1 antibody and tion of PLC-g1 and its co-immunoprecipitation with analyzed by SDS–PAGE and Western blotting with the indicated GRB2, but Ang II was not. antibodies.

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tion with PLC-g1. However, the EGF receptor was present in the EGF-stimulated anti-PLC-g1 immunoprecipitates (Fig. 5C). Therefore, PLC-g1 may still bind to activated EGF receptors but to a much lower degree than GRB2 and SHC, which is consistent with previous observations that 10 times more SHC than PLC-g1 could be detected in association with activated EGF receptors (39, 40). Figure 5C also shows that SHC isoforms could not be detected in the anti-PLC-g1 immunoprecipitates. Similarly, PLC-g1 could not be detected in the anti-SHC immunoprecipitates (data not shown). PLC-g1 binds to GRB2 directly in vitro. To deter- mine whether PLC-g1 could bind to GRB2 directly, in vitro assays were employed. Anti-PLC-g1 immunoprecipitates from the EGF-treated and untreated cell lysates were prepared and probed with purified GST or GST-GRB2 proteins respectively. The membranes were then blotted with a monoclonal anti-GST antibody. As controls, the membrane was also blotted with antibodies against anti-phosphotyrosine and anti-PLC-g1 antibodies, respectively. Figure 6 shows that GST-GRB2 FIG. 5. Ang II induces the tyrosine phosphorylation of the EGF protein but not GST was able to bind directly to PLC- receptor in WB cells. Serum-starved WB cells were stimulated with g1, and that EGF stimulation increased this binding. 1 mM Ang II or 200 ng/ml EGF and immunoprecipitated with 2 mg of polyclonal anti-EGF receptor (EGFR) antibody (A, B) or 2 mgof These results support the immunoprecipitation data in polyclonal anti-PLC-g1 antibody (C). The immunocomplexes were showing that there was a weak constitutive binding separated by SDS–PAGE and transferred to nitrocellulose mem- between PLC-g1 and GRB2, and that tyrosine phos- brane as described under Materials and Methods. The nitrocellulose phorylation of PLC-g1 elevated this interaction. membrane was first blotted with anti-phosphotyrosine antibody and 771 783 then stripped and segmented according to the molecular weight The Tyr and Tyr region of PLC-g1 binds directly markers, followed by blotting with the indicated antibodies. It needs to the SH2 domain of GRB2. To find out which region to be noted that, in lane 2 of B, the EGFR band blotted with the of PLC-g1 or GRB2 was responsible for their interac- rabbit polyclonal anti-EGFR antibody was weak due to a possible tion, we used a fusion protein binding approach. We incomplete stripping of the mouse monoclonal anti-phosphotyrosine first examined whether GST-GRB2-SH2 fusion protein antibody. This possibility is supported by the fact that the EGFR band appeared when blotted with the mouse monoclonal anti-PLC- could precipitate tyrosine phosphorylated-PLC-g1from g1 antibody. the Ang II or EGF-stimulated cell lysates. As shown in Fig. 7A, the GST-Grb2-SH2 fusion protein bound to tyrosine phosphorylated PLC-g1, which was increased thereby providing docking sites for a number of signaling molecules, including SHC, GRB2, and PLC-g1 (20). Therefore, we wondered how the EGF receptor could be involved in both EGF- and Ang II-induced co-immunoprecipitation of PLC-g1 with GRB2. Polyclonal anti- EGF receptor antibody (2 mg) was used for immunoprecipitation of cell lysates treated or untreated with EGF and Ang II for 2 min, respectively. Interestingly, it was found that besides EGF, Ang II also induced tyrosine phosphorylation of the EGF receptor (Fig. 5A). These results suggest that Ang II and EGF receptor signaling cascades in WB cells converge at some point downstream of the EGF receptor. We then examined whether GRB2 and PLC-g1 were co-immunoprecipi- FIG. 6. Direct binding of PLC-g1 to GRB2. The PLC-g1 immuno- tated with the EGF receptor after EGF and Ang II precipitates were separated by 10% SDS–PAGE and transferred to stimulation. As shown in Fig. 5B, both EGF and, to a nitrocellulose membrane. The membrane was successively probed with anti-phosphotyrosine antibody, GST and anti-GST antibody, lesser extent, Ang II stimulation caused a co-immuno- GST-GRB2 and anti-GST antibody, and anti-PLC-g1 antibody, re- precipitation of the EGF receptor with GRB2 and SHC, spectively. The results are representative of three independent ex- but only a weak and nonspecific co-immunoprecipita- periments.

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FIG. 7. Association of PLC-g1 with GRB2 in vitro. In A and B, GST fusion proteins were isolated and then incubated with Ang II or EGF- stimulated or unstimulated cell lysates as described under Materials and Methods. The precipitates of GST fusion proteins were then blotted with indicated antibodies, respectively. In C, GST-free 22Y3 protein was first tyrosine phosphorylated by c-Src in vitro and the 32P- labeled 22Y3 was then incubated with the indicated fusion proteins at 4ЊC for 2 h. The bound 22Y3 was detected by autoradiography. Aliquots of the indicated fusion proteins were analyzed by Western blotting to check the protein loading: lane 1, GST; lane 2, GST-p85-C- SH2; lane 3, GST-GRB2-SH2. in the Ang II-stimulated cell lysates and even further used to tyrosine-phosphorylate GST-free 22Y3 protein increased in the EGF-stimulated cell lysates. We then in vitro. As shown in Fig. 7C, tyrosine-phosphorylated tested three different GST-truncated PLC-g1 fusion 22Y3 protein was able to bind directly to the purified proteins for binding to GRB2 from WB cell lysates. The GST fusion protein GST-GRB2 SH2, but not to the GST fusion protein GST-22Y3 includes the whole Src-homol- control or to the GST-C-SH2 domain of the p85 subunit ogy region (the two SH2 domains and one SH3 domain) of phosphatidylinositol 3-kinase, suggesting that the and the two tyrosine phosphorylation sites Tyr771 and binding between the tyrosine phosphorylated 22Y3 pro- Tyr783 of PLC-g1. The fusion proteins, GST-N/C which tein and GRB2-SH2 fusion protein was specific. contains the two SH2 domains, and GST-SH3 which contains the SH3 domain, have no known tyrosine DISCUSSION phosphorylation sites in PLC-g1. Figure 7B shows that, in unstimulated WB cells, no GRB2 proteins were The present studies provide several lines of evidence bound by the fusion proteins. However, in the EGF- to show that PLC-g1 associates with the adaptor pro- stimulated WB cells, the GST-22Y3 fusion protein was tein GRB2 in both a constitutive and tyrosine phos- able to bind appreciably to GRB2, while very little phorylation-dependent fashion. The constitutive asso- GRB2 protein was bound to GST-N/C and GST-SH3 ciation between PLC-g1 and GRB2 was first observed fusion proteins. The fusion protein loading for each on reciprocal immunoprecipitation experiments using sample was quite similar (data not shown). Therefore, anti-PLC-g1 and anti-GRB2 antibodies (Fig. 1). This the precipitation of GRB2 by GST-22Y3 in the EGF- tyrosine phosphorylation-independent binding was fur- stimulated cell lysate may not be due to the binding of ther revealed by the direct binding assay (Fig. 6). How- SH2 domains of 22Y3 to the activated EGF receptor ever, this constitutive binding appears not to involve which associates with GRB2 since GST-N/C also con- the region of the two SH2 domains and one SH3 domain tains SH2 domains. The differences between GST-22Y3 of PLC-g1, since the three fusion proteins, GST-22Y3, and GST-N/C and GST-SH3 relate to the presence GST-N/C, and GST-SH3, precipitated absolutely no of two potential phosphotyrosine residues Tyr771 and GRB2 from the unstimulated WB cell lysates (Fig. 7B). Tyr783 in GST-22Y3. To examine whether the GST- Based on other studies (16, 17), the constitutive bind- 22Y3 fusion protein was tyrosine phosphorylated by ing between PLC-g1 and GRB2 may involve a proline- the EGF-stimulated cell lysate, the membrane was rich motif of PLC-g1 and one of the two SH3 domains blotted with anti-phosphotyrosine antibody. As shown of GRB2. More interestingly, both EGF and Ang II in Fig. 7B, GST-22Y3 was indeed tyrosine phosphory- stimulation further increased the association between lated by the cell lysate, suggesting that the Tyr771 and PLC-g1 and GRB2 (Figs. 1 and 3), and the stimulation- Tyr783 region may be responsible for the binding of dependent association appeared to correlate with tyro- GRB2 to the GST-22Y3 fusion protein. sine phosphorylation of PLC-g1 (Fig. 2B). In addition, In order to confirm whether tyrosine phosphorylated in vitro studies (Fig. 7) indicated that the Tyr771 and 22Y3 could bind directly to GRB2, purified c-Src was Tyr783 region of PLC-g1 and the SH2 domain of GRB2

AID ABB 0245 / 6b3e$$$423 07-31-97 06:25:58 arca PHOSPHOLIPASE-g1 BINDS DIRECTLY TO GRB2 109 were potentially involved in the tyrosine phosphoryla- lated associations between PLC-g1 and GRB2 are tion-dependent interaction between PLC-g1 and mediated by the activated EGF receptor. However, GRB2. since only a small amount of PLC-g1 was detected in The physiological significance of PLC-g1-GRB2 asso- anti-EGF receptor immunoprecipitates (Fig. 5), the ciation is currently speculative. One possibility is that co-immunoprecipitation of PLC-g1 with GRB2 may GRB2 may be involved in the translocation of PLC-g1 not be mainly due to the binding of PLC-g1 to acti- from the cytosol to the plasma membrane. Since the vated EGF receptors which associate with GRB2. substrate for PLC-g1 is a membrane lipid, phosphati- Furthermore, our data shows that the adaptor pro- dylinositol 4,5-bisphosphate, the translocation of PLC- tein SHC was not detected in the anti-PLC-g1 immu- g1 to the plasma membrane would be critical for its noprecipitates although both SHC and GRB2 had a enzymatic effect. Currently, an established function for large amount of binding to the EGF receptor. There- GRB2 in tyrosine kinase receptor-mediated signaling fore, a direct interaction between PLC-g1 and GRB2 is to recruit Sos via its two SH3 domains to the plasma in vivo is possible. Thus, a possible mechanism is membrane where it can activate Ras (6). Other studies that Ang II and EGF cause the autophosphorylation indicate that phosphotyrosine phosphatase SH-PTP2 and activation of EGF receptors, which induces the and phosphatidylinositol 3-kinase can also bind to binding of SH2-containing molecules, including PLC- GRB2 in a growth-factor-dependent and independent g1 and GRB2, to the phosphotyrosine residues of fashion (14–17). Taking these findings altogether with EGF receptors, which, in turn, promote the associa- our results, it is possible that GRB2 may function as a tion between tyrosine phosphorylated-PLC-g1 and general translocation factor that facilitates association GRB2. Although our data cannot exclude that other of other signaling molecules with the plasma mem- factors may be involved in the interaction of PLC- brane, thereby initiating multiple signaling cascades. g1 and GRB2 in vivo, our in vitro data suggest that It is worthwhile to note that, in T cells, ligation of T- tyrosine-phosphorylated PLC-g1 is able to bind di- cell antigen receptor caused PLC-g1 redistribution rectly to GRB2 (Figs. 6 and 7). from cytosol to a particulate compartment that paral- It has been suggested that Gbg subunit mediates leled GRB2 redistribution (41, 42). Furthermore, Tyr783 pertussis toxin-sensitive GPCR-mediated mitogenesis, phosphorylation is required for growth-factor-induced but the mechanism by which Gbg activates the tyro- PLC-g1 enzymatic activity in intact cells but appears sine phosphorylation-dependent signaling cascade to not to be very important for in vitro enzymatic activity Ras and mitogen-activated protein kinases is still not (31). This observation is in accordance with our finding clear (47). The pleckstrin homology domain of PLC-g1 that the Tyr771 and Tyr783 region is involved in the di- has been shown to have a high affinity for the Gbg rect binding of PLC-g1 to GRB2 in vitro. subunit and to inhibit the Gbg-mediated activation of In addition, the finding that PLC-g1 co-immunopre- mitogen-activated protein kinase (48, 49). Further- cipitated with GRB2 and Sos also suggest that PLC- more, PLC-g1 was found to bind directly to the Src g1 may be involved in cell mitogenesis by directly inter- tyrosine kinase both in vivo and in vitro (50), and inhi- acting with the Ras signaling pathway. Inhibition of bition of Src family tyrosine kinases has been shown PLC-g1 tyrosine phosphorylation by PAO at 1 mM for to inhibit Ang II-stimulated PLC-g1 tyrosine phosphor- 30 s caused about a 30% decrease in Ras activation ylation (51) and Ras activation (52) in rat aortic smooth (data not shown). Furthermore, a recent report indi- muscle cells. Our present studies demonstrate the asso- cated that PLC-g1 appeared to be essential in em- ciation of tyrosine phosphorylated PLC-g1 with GRB2. bryonal development (43), further supporting the Therefore, PLC-g1 may be one of the linkers that medi- involvement of PLC-g1 in cell mitogenesis. ate the GPCR agonist signals to the Ras signaling path- Another finding of significance in the present study way for their mitogenic effects. is that Ang II also induced the tyrosine phosphorylation of the EGF receptor in WB cells. This finding is ACKNOWLEDGMENTS in agreement with other studies showing crosstalk We thank Dr. Sue Goo Rhee for the monoclonal anti-PLC-g1 anti- between the platelet-derived growth factor or the body and Drs. Gerald Gish and Tony Pawson for the GST-fused GRB2 EGF receptor and GPCR-mediated signaling path- and PLC-g1 plasmids. ways (44–46). 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