©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Ligation of the T-cell Antigen Receptor (TCR) Induces Association of hSos1, ZAP-70, Phospolipase C-1, and Other Phosphoproteins with Grb2 and the -Chain of the TCR (*)

(Received for publication, March 1, 1995; and in revised form, June 1, 1995)

AndrE. Nel (1)(§) Shalini Gupta (1) Leo Lee (1) Jeffrey A. Ledbetter (2) Steven B. Kanner (2)

From the  (1)Division of Clinical Immunology and Allergy, Department of Medicine, UCLA School of Medicine, Los Angeles, California 90024 and (2)Bristol-Myers Squibb Pharmaceutical Research Institute, Seattle, Washington 98121

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Signaling by the T-cell antigen receptor (TCR) involves both phospholipase C (PLC)-1 and p21 activation. While failing to induce Shc/Grb2 association, ligation of the TCR/CD3 receptor in Jurkat T-cells induced hSos1-Grb2 complexes. In addition to hSos1, Grb2 participates in the formation of a tyrosine phosphoprotein complex that includes 145-, 95-, 70-, 54-, and 36-38-kDa proteins. p145 was identified as PLC-1 and p70 as the protein tyrosine kinase, ZAP-70. Although of the same molecular weight, p95 was not recognized by an antiserum to p95 Vav. The SH2 domains of Grb2 and PLC-1 were required for the formation of this protein complex. In anti-CD3-treated cells, Grb2 redistributed from the cytosol to a particulate cell compartment along with p36/p38, ZAP-70, and PLC-1. Part of the Grb2 complex associated with the particulate compartment could be extracted with Nonidet P-40, while the rest was Nonidet P-40 insoluble. In both the detergent-soluble and -insoluble fractions, Grb2 coimmunoprecipitated with the -chain of the TCR. Taken together, these results indicate that anti-CD3 induces Grb2-hSos1-PLC-1-p36/p38-ZAP70 complexes, which localize in the vicinity of TCR-.


INTRODUCTION

Ligation of the T cell antigen receptor (TCR) (^1)initiates signals at the cell membrane that control nuclear events such as induction of immediate early response genes and cytokine gene expression(1) . Two examples of membrane-to-nuclear signaling are the Ras/mitogen-activated protein kinase/c-Jun and Ca/calcineurin/NF-AT cascades, which both exert control over transcriptional initiation of the interleukin-2 gene(2, 3, 4, 5, 6, 7, 8, 9) . Both pathways, in turn, are dependent on a TCR-associated protein tyrosine kinase (PTK) network, which minimally include 3 PTKs: p56, p59, and ZAP-70(10, 11, 12, 13, 14) . The link between [Ca] flux and PTK activity is the activation of phospholipase C-1 (PLC-1) by TCR-induced tyrosine phosphorylation(15, 16) . PLC-1 initiates the breakdown of inositol phospholipids, thereby generating inositol trisphosphate, which acts as an intracellular Ca ionophore. PLC-1 activation is impaired in an Lck-deficient Jurkat cell line, suggesting a kinase-substrate interaction(17, 18) . Moreover, PCL-1 has been shown to coprecipitate with an unidentified 36-38-kDa tyrosine phosphoprotein (p36/p38), which is responsive to TCR signaling (19, 20, 21) .

Although the role of PTKs in the TCR-activated Ras pathway still needs to be clarified, studies on receptor tyrosine protein kinases (RTK) have shown that a guanine nucleotide exchange factor (GNEF), human son-of-sevenless (hSos), is involved(22, 23) . hSos activates p21 by the exchange of GDP for GTP(22, 23) . hSos is linked to RTKs through the interposition of the adaptor proteins, Shc and Grb2 (24, 25) . Grb2 is comprised wholly of an SH2 domain flanked by two SH3 domains(26) . The SH2 domain of Grb2 binds tightly to phosphotyrosine residues on RTKs or an interposed Shc molecule(25) . Shc, in turn, recognizes tyrosine-phosphorylated domains on RTKs via its own SH2 domain(25, 27) . Grb2 binding to Shc requires Shc to be tyrosine phosphorylated by the RTK or a receptor-associated PTK(25, 27) . By way of one of its SH3 domains, Grb2 binds and recruits hSos to the cell membrane(25, 28) . In addition to hSos, T-cells contain another putative GNEF, Vav, but these data are still controversial(29) . While we and others have previously shown that TCR ligation induces tyrosine phosphorylation of p95 in Jurkat cells without inducing Shc/Grb2 complexes, there is growing evidence for involvement of hSos in TCR signaling(2, 20, 21, 23, 30, 31) . It is possible, therefore, that Grb2 may be involved by binding to a receptor-associated tyrosine phosphoprotein other than Shc. To this end, our own preliminary data as well as published data from other groups have shown that Grb2 associates with several tyrosine phosphoproteins upon TCR ligation(20, 21, 28, 32) . Included among these is p36/p38, which is likely identical to the protein that binds to PLC-1(20, 21) . These findings suggest that Grb2 play a role in the assembly of a TCR-associated signaling complex, which serves to initiate both the inositol phospholipid as well as the Ras signaling pathways.

To understand the involvement of Grb2 in TCR-mediated signaling, we wished to identify the phosphoproteins in the Grb2 complex, including the presence of PLC-1. In addition, we were interested in determining the localization of this complex with respect to the TCR. We show here that anti-CD3 stimulation of Jurkat cells induces Grb2-hSos1-PLC-1-p36/p38-ZAP-70 complexes, which do not include Vav or Shc. Grb2, which is predominantly a cytosolic protein, could be seen to redistribute to both the detergent-soluble and detergent-insoluble cellular fractions during TCR ligation. Moreover, in both fractions Grb2 coimmunoprecipitated with the -chain of the TCR(33, 34) . These results imply that Grb2 is intimately involved in TCR signaling and participates in formation of a TCR-associated signal complex, which induces a convergence of components linked to multiple signaling pathways.


EXPERIMENTAL PROCEDURES

Antisera and Reagents

The monoclonal antibody (mAb) OKT3 (anti-CD3) was from Ortho Pharmaceuticals (Raritan, NJ). Biotinylated goat-anti-human IgM was purchased from Southern Biotechnology Associates (Birmingham, AL). The anti-phosphotyrosine mAb, 4G10, was from Upstate Biotechnology Inc. (Lake Placid, NY). Other mAbs used in this study included anti-Grb2 from Transduction Laboratories (Lexington, KY) and anti-PLC-1 from Upstate Biotechnology Inc. The polyclonal antisera to ZAP-70 (antiserum 833) was previously described(35) . The polyclonal anti--chain antibody, N-40, was kindly provided by Dr. C. Terhorst (Harvard Medical School, Boston, MA)(30) , while a monoclonal anti--antibody was kindly provided by Dr. R. Kubo (Cytel Corp., San Diego, CA)(36) . Polyclonal anti-Grb2 used for immunoprecipitation studies and anti-hSOS1 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), while anti-Shc was obtained from Upstate Biotechnology Inc. A polyclonal spectrin antiserum was purchased from Sigma. The PLC-1 SH2-Ig fusion protein was previously described(19) .

Bacterially Produced Fusion Proteins

The cDNA encoding GST-Grb2 Myc (wild type) and PSVEGrb2 Myc49L/203R, carrying a proline to lysine substitution at residue 49 in the amino-terminal SH3 domain and a glycine to arginine substitution at residue 203 in the COOH-terminal SH3 domain of Grb2(25) , were generously provided by Dr. R. A. Weinberg (Whitehead Institute, Cambridge, MA). The BamHI-XbaI fragment containing Grb2 myc49L/203R in the latter plasmid was subcloned into BamHI-XbaI-digested PGEX-20T vector as described earlier(25) . Both fusion proteins were purified by binding to glutathione-agarose and eluted with reduced glutathione.

Cell Culture, Stimulation, and Lysis

All cell lines were grown in RPMI 1640 medium supplemented with 10% fetal calf serum and antibiotics (complete medium). The cell lines used in this study included the Jurkat clone, 6.1 E (CD2, CD3, CD4), and Ramos cells, a human mIgM B cell line established from a patient with Burkitt's lymphoma. In all experiments, aliquots of 2 10^7 cells were used unless otherwise stated. All stimulations were conducted in a total of 1 ml of complete medium. Jurkat cells were treated with 1 µg/ml OKT3 (anti-CD3) or 100 nM PMA for the indicated time periods at 37 °C. Ramos cells were preincubated with 2 µg/ml goat-anti-human IgM for 2 min at 37 °C and then treated with 50 µg/ml avidin for 1-5 min. Cell pellets (12,000 g) were lysed in 0.2 ml of an appropriate lysis buffer (see below) by sonication. Particulates were removed at 12,000 g, and the supernatants were used for immunoprecipitation and Western blotting.

Immunoprecipitation

For Grb2, hSos1, and ZAP-70 immunoprecipitations, cells were lysed in buffer A (50 mM Tris, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 1 mM sodium vanadate, 10 mM NaF, 2 mM PMSF, 10 µg/ml leupeptin, and 0.009 TIU/ml aprotinin), and the lysates were incubated with l µg of anti-Grb2, 1 µg of anti-hSos1, or 5 µl of anti-ZAP-70 for 1 h at 4 °C. Thereupon, 30 µl of protein A-Sepharose beads were added for 1 h at 4 °C. The immunoprecipitates were washed three times with 0.5 ml of buffer B (20 mM Tris, pH 7.4, 150 mM NaCl, 0.1% Nonidet P-40, 1 mM sodium vanadate, 5 mM NaF, 2 mM PMSF, 10 µg/ml leupeptin, and 0.009 TIU/ml aprotinin) and then boiled in 60 µl of 1 SDS sample buffer for 5 min. The proteins were resolved by 10% SDS-PAGE and subsequently transferred to Immobilon-P. For -chain immunoprecipitation, cell extracts were precleared and incubated with 10 µl of polyclonal N-40 antiserum for 1 h at 4 °C, followed by incubation with 30 µl of protein A-Sepharose beads for 1 h at 4 °C(37) . The immunoprecipitates were washed three times with 0.5 ml of buffer C (20 mM Hepes, pH 7.5, 150 mM NaCl, 0.1% Nonidet P-40, 0.1 mM sodium vanadate, 0.1 mM PMSF, 1 µg/ml leupeptin, 0.009 TIU/ml aprotinin) and boiled in 60 µl of 1 SDS sample buffer. The immunoprecipitates were resolved by 15% SDS-PAGE under reducing conditions and subsequently transferred to Immobilon-P.

GST-Grb2 and GST-Grb249L/203R Affinity Association

Cells were lysed in 0.2 ml of buffer D (20 mM Tris, pH 7.4, 1% Triton X-100, 50 mM NaCl, 1 mM sodium vanadate, 10 mM sodium pyrophosphate, 5 mM EGTA, 0.1% bovine serum albumin, 10 µg/ml leupeptin, 10 mM NaF, 0.009 TIU/ml aprotinin). Supernatants were incubated with 10 µg each of recombinant GST, GST-Grb2 Myc or GST-Grb2 Myc49L/203R and added to 30 µl of glutathione-agarose beads for 1 h at 4 °C(25) . The beads were washed three times with 0.5 ml of buffer D lacking bovine serum albumin and boiled in 60 µl of 1 SDS sample buffer. Proteins were resolved by 10% SDS-PAGE and transferred to Immobilon-P.

PLC-1 SH2/Ig Affinity Association

Lysates were prepared in 0.2 ml of buffer A containing 5 mM dithiothreitol and incubated with 50 µl of protein A-Sepharose beads precoated with 30 µg of PLC-1 SH2-Ig fusion protein for 1 h at 4 °C(19) . The beads were washed three times with 0.5 ml of buffer B containing 5 mM dithiothreitol and then boiled in 60 µl of 1 SDS sample buffer. The proteins were resolved by 10% SDS-PAGE and transferred to Immobilon-P.

Subcellular Fractionation

The pellet of 10^8 cells was resuspended in 1 ml of chilled hypotonic buffer E (10 mM Tris, pH 7.4, 10 mM KCl, 1.5 mM MgCl(2), 0.2 mM PMSF, 1 mM sodium vanadate, 10 µg/ml leupeptin and 0.009 TIU/ml aprotinin) for 15 min and then lysed in a Dounce homogenizer by 50 mechanical strokes. All subsequent steps were performed at 4 °C. Lysates were spun for 30 min at 100,000 g in a Beckman TL-100 table top centrifuge, and the supernatant, designated cytosol, was collected. The pellet was rinsed with 2 ml of buffer E, recentrifuged at 100,000 g, and subsequently resuspended in 0.2 ml of buffer E plus 1% Nonidet P-40 for 15 min. Following recentrifugation at 100,000 g for 30 min, the supernatant, designated detergent-soluble material, was recovered. The pellet was washed with 0.2 ml of buffer E containing Nonidet P-40 and recentrifuged for 10 min at 100,000 g. The pellet was solubilized by sonication in 0.2 ml of RIPA buffer (20 mM Tris, pH 8.0, 0.15 M NaCl, 0.1% SDS, 1% Nonidet P-40, 0.5% deoxycholate, 1 mM EDTA, 1 mM EGTA, 1 mM sodium vanadate, 10 mM NaF, 1 mM PMSF, 10 µg/ml leupeptin, and 0.009 TIU/ml aprotinin) and centrifuged for 30 min at 100,000 g. The supernatant, designated detergent-insoluble material, was recovered. We confirmed by enzymatic assay that the detergent-soluble and detergent-insoluble fractions were substantially free from lactate dehydrogenase activity.

Western Blotting

SDS-PAGE gels were transferred to Immobilon-P at 0.35 mA for 16 h. The membrane was blocked with 6% bovine serum albumin, pH 7.6, for 2 h and incubated with the desired antibody, diluted in blocking solution plus 0.1% Tween-20 and 0.01% azide for 1 h at room temperature(2) . Detection was performed either by using the enhanced chemiluminescence (ECL) method or by overlaying the blot with I-protein A (1 µCi/ml) in blocking solution for 1 h, followed by four 10-min washes in Tris-buffered saline containing 0.1% Tween 20 and 0.1% bovine serum albumin(2) . The blots were visualized by autoradiography.


RESULTS

TCR Ligation Induces the Association of Grb2 with a Select Range of Tyrosine Phosphoproteins

Jurkat cells, either untreated or stimulated with anti-CD3 mAb, were lysed, and anti-Grb2 immunoprecipitates were prepared. These proteins were transferred to Immobilon-P and immunoblotted with anti-phosphotyrosine mAb. Fig. 1shows that anti-CD3 stimulation induced the association of Grb2 with a number of tyrosine phosphoproteins, including p110, p95, p75, p60-p70, p54, as well as a p36/p38 doublet (lanes4 and 5). Longer exposure of the blot also revealed other less prominent phosphoproteins, including p145. P36/p38 was consistently the most prominent tyrosine phosphoprotein in the immunoprecipitate. In contrast to ligation of the CD3 receptor, PMA treatment did not induce complexing of any of the above tyrosine phosphoproteins with Grb2 (Fig. 1, lane6). Similar findings were made upon anti-CD3 stimulation of additional T-cell lines, HPB-ALL and Hut-78 (not shown).


Figure 1: TCR ligation induces the selective association of Grb2 with specific tyrosine phosphoproteins. Jurkat cells (2 10^7) were left untreated or were treated with 1 µg/ml OKT3 for 1 or 5 min or with PMA for 5 min and then lysed. Lysates, representing detergent-soluble protein, were incubated with 1 µg of polyclonal anti-Grb2 antibody immobilized on protein A-Sepharose or with 10 µg of either GST-Grb2 myc or GST-Grb2 Myc49L/203R bound to glutathione-agarose beads as indicated. Precipitation with normal rabbit serum (NIS) or GST bound to glutathione beads was used as a negative control. Proteins bound by the antibody or fusion protein were resolved by 10% SDS-PAGE and electrophoretically transferred to an Immobilon-P membrane. The membrane was overlaid with 1 µg/ml anti-phosphotyrosine mAb (4G10) and developed by ECL.



We compared the profile of Grb2-associated phosphoproteins in the immune precipitates with phosphoproteins exhibiting binding affinity for a GST-Grb2 fusion protein. Cellular lysates were incubated with GST-Grb2 immobilized on glutathione-agarose beads, and the adsorbed proteins were analyzed on the same anti-phosphotyrosine immunoblot (Fig. 1). In lysates from anti-CD3-treated cells, GST-Grb2 complexed with p145, p110, p95, p60-70, and p54 (Fig. 1, lanes8 and 9). To study the contribution of SH2 and SH3 domains of Grb2 in these interactions, we conducted parallel affinity binding experiments with GST-Grb2(49L/203R), in which both the amino- and COOH-terminal SH3 domains have been mutagenized to render them non-functional, while the SH2 domain remains intact(25) . Interaction of the mutant fusion protein with lysates from anti-CD3-treated cells showed strong affinity for p36/p38 as well as additional tyrosine phosphoproteins in the range of 70-75 kDa (Fig. 1, lanes12 and 13). PMA treatment did not induce the binding of any tyrosine phosphoproteins with either GST-Grb2 or GST-Grb2(49L/203R) (Fig. 1, lanes10 and 14). Taken together, these results suggest that both the SH2 as well as the SH3 domains of Grb2 participate in binding of anti-CD3-induced tyrosine phosphoproteins.

TCR Ligation Induces Grb2 Association with hSos1 without Involving Shc

Since hSos1 is not a tyrosine phosphoprotein, we determined whether this protein can coprecipitate with Grb2 as part of the Grb2 complex. First, we showed that anti-Grb2 immunoprecipitates contain immune detectable hSos1 (Fig. 2A). hSos1/Grb2 association was extremely rapid, and hSos1 could be visualized within 30 s of CD3 receptor engagement (Fig. 2A, lanes3-8). Reciprocally, we demonstrated that anti-hSos1 coimmunoprecipitated Grb2 in anti-CD3-treated but not unstimulated Jurkat cells (Fig. 2B). In an anti-hSos1 immunoblot of a parallel immunoprecipitate (Fig. 2B, bottom), we showed that equal amounts of hSos1 protein were being precipitated from control (lane2) and stimulated cells (lanes3 and 4).


Figure 2: TCR ligation induces Grb2 association with hSos1. A, anti-hSos1 immunoblot. Jurkat cells (2 10^7) were untreated (Ø) or treated with 1 µg/ml OKT3 for the indicated time periods. Lysates were subjected to immunoprecipitation using 1 µg of polyclonal anti-Grb2 antibody. NIS and 100 µg of crude Jurkat cell lysate were included as controls. The immunoprecipitates (IP) were resolved by 10% SDS-PAGE. After transfer to Immobilon-P, the membrane was sequentially overlaid with 1 µg/ml polyclonal anti-hSos1 and 50 ng/ml horseradish peroxidase-conjugated goat anti-rabbit secondary antibody. The blot was developed by ECL. B, anti-Grb2 and anti-hSos1 immunoblot of an anti-hSos1 immunoprecipitate. Stimulated or unstimulated cell lysates were subjected to immunoprecipitation using 1 µg of polyclonal anti-hSos1 antibody. NIS and 100 µg of crude lysate were included as controls. The proteins were resolved by 10% SDS-PAGE and transferred to Immobilon-P. The blot was overlaid with 1 µg/ml anti-Grb2 mAb and then developed by ECL. The 185-kDa region of the blot is shown. The blot was stripped and overlaid with 1 µg/ml anti-hSos1 polyclonal antibody (bottom). Lanes1-4 are the same as lanes1-4 in the upperpanel.



When hSos1 immunoprecipitates from anti-CD3-treated cells were probed by anti-phosphotyrosine immunoblotting, we observed that these precipitates included a major 36-38-kDa phosphoprotein as well as phosphoproteins of 54, 70, and 95 kDa (Fig. 3A, lanes3, 4, 6, 7). Taken together with the results in Fig. 2, this finding is compatible with a CD3-induced assembly of Grb2, hSos, p36/p38, p70, p54, and p95.


Figure 3: Anti-hSos1 immunoprecipitates (IP) phosphoproteins of the same molecular weight as anti-Grb2, but Shc does not coprecipitate with Grb2. A, anti-phosphotyrosine immunoblot of anti-hSos1 immunoprecipitate. Jurkat cells (2 10^7) were left untreated (Ø) or treated with 1 µg/ml OKT3 for 1 or 5 min and then lysed. Lysates were immunoprecipitated with two different types of anti-hSos(1) antibody (Ab 1, UBI; Ab 2, Santa Cruz Biotechnology) as described in Fig. 2A. NIS was included as control. Immunoprecipitates were resolved by 10% SDS-PAGE. Anti-phosphotyrosine immunoblotting was performed as in Fig. 1. B, anti-Shc immunoblot of Grb2 immunoprecipitates. Jurkat cells (2 10^7) were either left untreated (Ø) or treated with 1 µg/ml OKT3 for 1 or 5 min or 100 nM PMA for 5 min. Ramos cells (2 10^7) were either left untreated (Ø) or treated with 2 µg/ml biotinylated goat anti-human IgM + 50 µg/ml avidin for 1 or 5 min or 100 nM PMA for 5 min. Lysates were immunoprecipitated with anti-Grb2 antibody as described in Fig. 1. NIS and crude lysates served as controls. Proteins were resolved by 10% SDS-PAGE and transferred to Immobilon-P. The blot was developed with 1 µg/ml polyclonal anti-Shc antibody, followed by 0.1 µCi/ml I-protein A. The 40-66-kDa region of the autoradiogram is shown.



The 54-kDa phosphoprotein (p54) shown in lanes3, 4, 6. and 7 (Fig. 3A) is of the approximate size of p52(24, 25, 26) . Immunoblotting for Shc failed, however, to identify p54 as Shc (not shown). Moreover, anti-Grb2 antibodies did not coprecipitate Shc in anti-CD3-treated Jurkat cells (Fig. 3B, lanes2-5). By contrast, cross-linking of the antigen receptor (mIgM) of a human B-cell line, Ramos, with anti-IgM showed receptor-induced association of p52 with Grb2 (Fig. 3B, lanes9 and 10). Among T-cells, these findings were not unique for the Jurkat cell line, as we have also been unable to show Shc complexing to Grb2 in HPB-ALL, Hut-78, and peripheral blood PHA blasts during CD3 ligation (not shown).

The 95-kDa phosphoprotein in lanes3, 4, 6, and 7 is the same size as p95, which undergoes enhanced tyrosine phosphorylation during anti-CD3 treatment in Jurkat cells(2, 29) . Since Vav is a putative GNEF in T-cells(29) , its coprecipitation with a definitive GNEF, hSos1, may explain this controversial aspect of Vav function (38) . Overlay of the blot in Fig. 3A failed to identify p95 as Vav (not shown). Moreover, we have previously shown that Vav immunocomplexes do not include p36/p38 (2) or Grb2 (not shown). Although anti-Vav leads to coprecipitation of a 70-kDa tyrosine phosphoprotein(2) , this protein does not share immune identity with ZAP-70 (not shown) and is likely the so-called Vav-associated protein (2, 39) . Taken together, these findings exclude participation of Vav in Grb2-hSos1 interactions but do not exclude Vav participation in TCR signaling.

PLC-1 Interacts with the Grb2 and p36/p38 via Its SH2 Domain

Previous reports have shown that TCR ligation induces tyrosine phosphorylation of PLC-1, which coprecipitates with a 36-38-kDa tyrosine phosphoprotein(19, 20) . Parallel immunoprecipitation of PLC-1 and Grb2, followed by anti-phosphotyrosine immunoblotting, demonstrated complexing of p36/p38 to PLC-1 as well as Grb2 (Fig. 4A). This is in agreement with what we and others have shown during TCR ligation (19, 20, 21) . Moreover, in anti-CD3-treated cells, PLC-1 and Grb2 coprecipitated with p110, p95, p75, p70, and p54 (Fig. 4A, lanes3 and 5). Anti-PLC-1 immunoblotting confirmed that p145 (Fig. 4A, lanes2 and 4) is identical to PLC-1 (not shown). In a separate experiment, Grb2 immunoprecipitates were analyzed by anti-PLC-1 immunoblotting. Fig. 4B shows that anti-CD3 treatment induced rapid (<30 s) association of PLC-1 with Grb2 (lane3) and that the association was sustained for at least 30 min (lane8).


Figure 4: PLC-1 associates with Grb2 complexes via its SH2 domain. A, anti-phosphotyrosine immunoblot of PLC-1 and Grb2 immunoprecipitates. Jurkat cells (2 10^7) were left untreated (Ø) or treated with 1 µg/ml OKT3 for 1 min or 5 min and then lysed. Lysates were immunoprecipitated with 5 µl of anti-PLC-1 antiserum or 1 µg of polyclonal anti-Grb2 antibody. NIS was included as control. The immunoprecipitates were resolved by 10% SDS-PAGE, and anti-phosphotyrosine immunoblotting was done as in Fig. 1. This blot was overexposed relative to that in Fig. 1. B, anti-PLC-1 immunoblot of a Grb2 immunoprecipitate. Anti-Grb2 immunoprecipitation was performed as in Fig. 1. NIS and crude lysate were included as controls. Proteins were resolved by 10% SDS-PAGE. After transfer to Immobilon-P, the membrane was overlaid with 1 µg/ml anti-PLC-1 mAb. The blot was developed by ECL. The 185-110-kDa region of the autoradiogram is shown. C, anti-phosphotyrosine and anti-Grb2 immunoblot of PLC-1 SH2/Ig-binding proteins. Lysates were incubated together with protein A-Sepharose beads coated with 30 µg of PLC1 SH2-Ig fusion protein in the presence of 5 mM dithiothreitol. Beads were extensively washed and boiled in 1 SDS sample buffer. Proteins were resolved by 10% SDS-PAGE and transferred to Immobilon-P, and anti-phophotyrosine immunoblotting was performed as in Fig. 1. For anti-Grb2 immunoblotting, duplicate samples were resolved by 12% SDS-PAGE, and immunoblotting for Grb2 was performed as in Fig. 1. Lanes1-4 (bottom) are the same as lanes1-4 in the anti-PY blot.



Since PLC-1 is a tyrosine phosphoprotein, its association with Grb2 may involve the SH2 domain of Grb2. Alternatively, PLC-1 may use its SH2 domains to bind a common tyrosine phosphoprotein, such as p36/p38, to which Grb2 is also complexed. To discriminate between the possibilities, we looked at T-cell tyrosine phosphoproteins that had affinity for PLC-1 SH2-Ig fusion protein. This fusion protein, which contains both the amino- and COOH-terminal SH2 domains of PLC-1 (19) , was immobilized on protein A-Sepharose beads and incubated with control and stimulated cell lysates. Anti-phosphotyrosine immunoblotting of the eluted proteins from stimulated cell lysates showed binding of p36/p38, p54, p70, p75, and p110 to the fusion protein (Fig. 4C, lanes3 and 4). No specific phosphoproteins bound to PLC-1 SH2/Ig when lysates from untreated cells were used (Fig. 4C, lane1), while some p75 binding was seen with PMA treatment (lane4). An anti-Grb2 blot of a parallel PLC-1-SH2 association experiment showed the presence of Grb2 in anti-CD3-treated (lanes2 and 3) but not control (lane1) or PMA-treated (lane4) samples (Fig. 4C, bottom). Since the PLC-1 SH2 fusion protein does not contain phosphotyrosine residues with which Grb2 can interact, it is likely that Grb2 is bound to p36/p38 or some other tyrosine phosphoprotein in the complex. Because p36/p38 is the most prominent phosphoprotein and is associated with PLC-1 and Grb2, it is a likely candidate.

ZAP-70 Associates with the PLC1-Grb2-hSos1-p36/p38 Complex

Since the anti-phosphotyrosine immunoblots in Figs. 1A, 3A, 4A, and 4C show a p70 substrate, we explored the possibility that the kinase ZAP-70 may be included in these complexes. An anti-ZAP-70 immunoblot of Grb2 immunoprecipitates showed the presence of ZAP-70 in anti-CD3-treated but not in control or PMA-treated samples (Fig. 5A, lanes3 and 4). Reciprocally, anti-ZAP-70 coprecipitated Grb2 in anti-CD3-treated but not control or PMA-treated cells (Fig. 5B, lanes6-9). Compared with the amount of Grb2 protein present in parallel Grb2 immunoprecipitates (Fig. 5B, lanes2-5), we estimated that about 5% of cellular Grb2 was ZAP-70 associated after anti-CD3 treatment.


Figure 5: ZAP-70 associates with the PLC1-Grb2-hSos1-p36/p38 complex. A, anti-ZAP-70 immunoblot of a Grb2 immunoprecipitate. Jurkat cells (2 10^7) were treated with 1 µg/ml OKT3 or PMA as described in Fig. 1. Anti-Grb2 immunoprecipitation was done as in Fig. 1, and proteins were resolved by 10% SDS-PAGE. The membrane was overlaid with 1:250 dilution of polyclonal anti-ZAP-70(833) antiserum, followed by 1 µCi/ml I-protein A and visualized by autoradiography. B, anti-Grb2 immunoblot of Grb2 and ZAP-70 immunoprecipitates. Lysates were immunoprecipitated with anti-Grb2 as above or with 5 µl of anti-ZAP-70(833) anti-serum. Proteins were resolved by 10% SDS-PAGE and immunoblotted for Grb2 as described in Fig. 1. The 23-kDa region of the autoradiogram is shown. C, anti-PLC-1 immunoblot of Grb2 and anti-ZAP-70 immunoprecipitates. Grb2 and ZAP-70 immunoprecipitation were performed as above. The proteins were resolved by 10% SDS-PAGE and anti-PLC-1 immunoblotting performed as described in Fig. 4B. D, anti-ZAP-70 immunoblot of ZAP-70 immunoprecipitates. Anti-ZAP-70 immunoprecipitation was performed as in B, and the blot was overlaid with 1:1000 dilution of anti-ZAP-70 antiserum, followed by 1 µCi/ml I-protein A.



To confirm that ZAP-70 associates with the pool of Grb2, which is complexed to PLC-1, we looked for the presence of PLC-1 in ZAP-70 immunoprecipitates (Fig. 5C). Anti-PLC-1 immunoblotting showed rapid association of PLC-1 with ZAP-70 in anti-CD3-treated cells (Fig. 5C, lanes7 and 8). A parallel anti-Grb2 immunoprecipitate to show PLC-1 coprecipitation was included for comparison (Fig. 5C, lanes2-4). This result is not due to quantitative differences in the amount of ZAP-70 being precipitated because anti-ZAP-70 overlay showed equal amounts of protein in stimulated and unstimulated samples (Fig. 5D). This blot also shows that ZAP-70 underwent hypomobility shift in anti-CD3- and PMA-treated cells (Fig. 5D, lanes3-5). In a separate experiment, we also determined that hSos1 was present in anti-ZAP-70 immunoprecipitates (not shown). Taken together, these results indicate that ZAP-70 is a member of the PLC-1-Grb2-hSos1-p36/p38 complex, where it may contribute to the phosphorylation of one or more components.

Grb2 Redistributes to the Detergent (Nonidet P-40)-soluble and -insoluble Cellular Fractions in Association with p36/p38, ZAP-70, and the -Chain of the TCR

Grb2 undergoes intracellular translocation in response to complexing of the TCR(20) . Following treatment with anti-CD3 mAb for 1 and 5 min, Grb2 protein increased 2.1- and 2.9-fold, respectively, in Nonidet P-40 extracts of the particulate material (i.e. the pellet remaining after hypotonic lysis and 100,000 g centrifugation) (Fig. 6A). There was also increased association of Grb2 (approximately 2- and 3-fold after 1 and 5 min anti-CD3 treatment, respectively) with the particulate material, which remains after Nonidet P-40 extraction (Fig. 6A). Grb2 was released from the so-called Nonidet P-40-insoluble fraction by treatment of the pellet with RIPA buffer. The detergent-soluble and -insoluble fractions differ insofar as the insoluble fraction includes an abundance of 220-240-kDa spectrin dimers, while the detergent-soluble material contained very little spectrin (Fig. 6A). The cytosol included a small amount of 220-kDa spectrin (Fig. 6A). Because spectrin dimers are predominantly associated with the Nonidet P-40-insoluble fraction, it suggests that this fraction may be relatively enriched for select cytoskeletal components compared to the Nonidet P-40-soluble fraction. Because Grb2 has been shown in Fig. 4and 5 to associate with ZAP-70 and PLC-1, we looked for redistribution of these components upon anti-CD3 treatment. Fig. 6A confirms their relocation from the cytosol to the Nonidet P-40-soluble and -insoluble fractions upon cellular activation (Fig. 6A).


Figure 6: Grb2, ZAP-70, and PLC-1 redistributes to detergent-soluble and -insoluble cellular compartments where Grb2 is associated with the -chain of the TCR. A, immunoblot autoradiograms. Jurkat cells (1 10^8) were untreated (Ø) or treated with 1 µg/ml OKT3 for 1 or 5 min before lysis in 1 ml of hypotonic buffer. After centrifugation at 100,000 g for 1 h, the supernatant was collected as cytosol, and the particulate material was washed twice in hypotonic buffer and extracted in 200 µl of a buffer containing 1% Nonidet P-40. Nonidet P-40-soluble material was collected after recentrifugation. The detergent-insoluble pellet was washed and then homogenized in 200 µl of RIPA buffer. After centrifugation at 100,000 g, the supernatants, designated Nonidet P-40-insoluble material, were recovered. Equal amounts (100 µg) of Nonidet P-40-soluble and -insoluble protein along with 80 µl of cytosol were resolved by 10 or 12% SDS-PAGE. Gels were transferred to Immobilon-P and overlaid with antibodies to spectrin (1:500 dilution), Grb2, ZAP-70, and anti-PLC-1 as described under ``Experimental Procedures.'' Immunoblots were developed by ECL or I-protein A overlay as described in Fig. 1, 4, and 5. The autoradiographic density of the blotted proteins were read in a Micro-Tech densitometer, and their relative density is displayed below each lane (with the exception of spectrin, which appeared not to change upon stimulation). B, anti-phosphotyrosine immunoblot of Grb2 immunoprecipitates (IP). Anti-Grb2 immunoprecipitation was conducted using 200 µg of cytosol and Nonidet P-40-soluble and -insoluble material, as described in Fig. 1. Proteins were resolved by 12% SDS-PAGE and transferred to Immobilon-P, and anti-phosphotyrosine immunoblotting was done as described in Fig. 1. The same result was obtained in two repeat experiments and is identical to a result obtained with PLC-1 SH2-Ig fusion protein as affinity matrix. C, anti--immunoblot of a Grb2 immunoprecipitate. Anti-Grb2 immunoprecipitation was performed on 100 µl of Nonidet P-40-soluble and -insoluble material obtained as described above. Proteins were resolved by non-reduced 12% SDS-PAGE. The blot was overlaid with 1:1000 dilution of polyclonal anti--antibody and detected by 1 µCi/ml I-protein A. The blot was stripped and reprobed with 4G10, which confirmed that this -species is tyrosine phosphorylated (not shown). NIS, non-immune serum control precipitate from the Nonidet P-40-insoluble fraction of unstimulated cells. D, anti-Grb2 immunoblot of anti--immunoprecipitate. 10 µl of polyclonal anti--antiserum(N-40) were used to immunoprecipitate 100 µl of detergent-insoluble material extracted with RIPA buffer. Proteins were resolved on reducing 12% SDS-PAGE. The blot was overlaid with anti-Grb2 and developed as described in Fig. 2.



To study the intracellular distribution of p36/p38, the cytosolic as well as Nonidet P-40-soluble and -insoluble fractions were subjected to anti-Grb2 immunoprecipitation followed by anti-phosphotyrosine immunoblotting (Fig. 6B). While anti-CD3 induced association of p110, p75, p70, p54, p21, and p18 with Grb2 in Nonidet P-40-soluble and -insoluble fractions, there was an interesting difference in p36/p38 association with Grb2 in these fractions (Fig. 6B, lanes4-9). p36/p38 was inducibly associated with Grb2 in Nonidet P-40-soluble fractions (Fig. 6B, lanes4-6) but was constitutively associated with Grb2 in Nonidet P-40-insoluble extracts (lanes7-9). The reason for the constitutive phosphorylation and association of p36/p38 with Grb2 in detergent-insoluble material is not known. Identical results to that shown in Fig. 6B were obtained when cytosol and detergent-soluble and -insoluble fractions were affinity interacted with PLC-1 SH2-Ig fusion protein (not shown). Although some phosphoproteins in the cytosol coprecipitated with Grb2, no anti-CD3-induced changes in their association occurred (Fig. 6B, lanes1-3). Immunoblotting of Grb2 immunocomplexes with antisera to ZAP-70 and PLC-1 confirmed the association of these molecules with Grb2 localized to the Nonidet P-40-insoluble fraction (not shown). This confirms that the coordinate redistribution of Grb2, ZAP-70, and PLC-1 in Fig. 6A is due to inclusion in a multi-molecular complex that undergoes intracellular shift.

The p18 and p21 phosphoproteins seen at the bottom of Fig. 6B are of the same molecular weight as the TCR--species, which are phosphorylated when TCR is ligated by an agonist peptide (40) . This suggested that Grb2 may be -associated. This notion was strengthened by the ability of polyclonal anti--antiserum to precipitate a phosphoprotein complex from stimulated cells, which include p145, p110, p95, p70, p36/p38, and p21 (not shown). Moreover, immunoblotting for -chain following anti-Grb2 immunoprecipitation showed coprecipitation in both Nonidet P-40-soluble and -insoluble extracts (Fig. 6C). While the amount of associated with Grb2 increased in Nonidet P-40-insoluble material upon anti-CD3 treatment, this increase was not seen in the Nonidet P-40-soluble fraction (Fig. 6C). Reciprocally, we found that -chain immunoprecipitation leads to coprecipitation of Grb2 as shown by immunoblotting (Fig. 6D).


DISCUSSION

In this paper, we show that Grb2 plays a role in signaling via the TCR by recruiting hSos1 to a signaling complex that includes PLC-1, the PTK ZAP-70, and a 36-38-kDa tyrosine phosphoprotein. The complex excluded Shc. The association of Grb2 with this complex is dependent on its SH2 domain. Grb2 redistributed from a predominant cytosolic location in unstimulated cells to the particulate compartment in anti-CD3-treated cells. Moreover, Grb2 associated with -chain of the TCR in this compartment.

Grb2 plays a critical role in signaling by RTKs and receptor-associated PTKs(25, 26) . Previous studies have highlighted the potential importance of Grb2 in signaling by the TCR(20, 21, 28, 32) . Sieh et al.(20) demonstrated a prominent p36/p38 phosphoprotein that associated with the SH2 domain of Grb2 and formed a stable complex with Grb2, hSos1, and PLC-1 upon TCR stimulation. Buday et al.(21) also showed that Grb2-SH2 binds p36 and hSos1. Motto et al.(32) and Reif et al.(28) found a 75-kDa phosphoprotein that associates with the carboxyl-terminal SH3 domain of Grb2 in anti-CD3-treated T-cells. In addition, Motto et al.(32) also found a 116-kDa phosphoprotein in anti-CD3-treated cell lysates that binds to the amino-terminal SH3 domain of Grb2. Our results are in agreement with these findings and show, in addition, that ZAP-70 and the -chain of the TCR also bind to Grb2 ( Fig. 5and 6). Although the aggregate effects of Grb2 in TCR signaling still require to be elucidated, an important role for Grb2 in T-cells appears to be the activation of p21(20, 28) . To this end, several groups have shown that Ras plays an important role in TCR signaling(6, 30, 31) . We have also recently highlighted the role of a Ras-dependent serine/threonine kinase, Raf-1, in the mitogen-activated protein kinase cascade in Jurkat cells(2) . Although a role for GAP proteins has been suggested, there is a rapidly developing consensus that Ras activation in T-lymphocytes is dependent on a GNEF that exchanges GDP for GTP(20, 21, 28, 30, 31, 32) . T-lymphocytes are unusual in that they express two potential GNEFs, hSos1 as well as p95(29) . Although it was shown that the TCR in Jurkat cells controls tyrosine phosphorylation of p95, the data implicating Vav as a GNEF are controversial (38) . We provide evidence in this paper that hSos is involved in TCR responses through association with Grb2 (Fig. 2). Moreover, this reaction is extremely fast (<30 s) and agrees with the rapid kinetics of Ras, Raf-1, MEK-1, and ERK2 activation in Jurkat cells(2, 41) . Although anti-phosphotyrosine immunoblotting of Grb2 and hSos1 immunoprecipitates ( Fig. 1and Fig. 3A) show a 95-kDa Grb2-associated phosphoprotein, this protein was not recognized by anti-Vav antisera (not shown). These findings suggest that Vav does not function as a component of the Grb2 complex that is induced after TCR ligation. Our findings do not exclude the possibility, however, that Vav is associated with the complex in low stoichiometric amounts or involved in TCR signaling in some other way.

Gauged from the number of proteins that are complexed to Grb2 (Fig. 1), it is quite plausible that Grb2 plays an additional role in TCR signaling. Of particular interest is the presence of PLC-1 and p36/p38 in the Grb2 complex ( Fig. 1and Fig. 4). Similar proteins were noticed in Grb2 complexes by Sieh et al.(20) . Our study shows, in addition, that the SH2 domains of PLC-1 and Grb2 are involved in complexing to p36/p38 (Fig. 4C). This suggests that Grb2 and PLC-1 are independently complexed to p36/p38 or one of the associated phosphoproteins. It is likely that p36/p38 is a novel adaptor protein that interacts with Grb2 as well as PLC-1. Although PLC-1 activation requires tyrosine phosphorylation by a TCR-associated PTK, the relationship between phosphorylation and activation is not simple. Recently, Yang et al.(42) demonstrated that EGF-induced activation of PLC-1 in hepatocytes requires both tyrosine phosphorylation and association of PLC-1 with the cytoskeleton. We suggest that the shift of PLC-1 to the detergent-insoluble compartment upon CD3 ligation is required for its activation (Fig. 6A). However, the accurate assessment of PLC-1 activation and kinetics has been hindered by the complex regulatory mechanisms that are involved in its stimulation, including its tyrosine phosphorylation, its putative interaction with the actin-associated protein, profilin(43) , and the translocation of PLC-1 from the cellular cytoplasm to the plasma membrane(44) . Whatever the explanation for PLC-1 activation in T-cells, its inclusion in a complex that also involves hSos1 strongly suggests that Ras activation and inositol/phospholipid turnover are regulated in the same TCR-associated molecular complex.

The presence of ZAP-70 in the Grb2-associated complex and its redistribution to the non-cytosolic cellular compartment are likely of considerable importance both in the formation of the complex as well as for ZAP-70 function. ZAP-70 can associate with one or more of the ARH motifs present in the CD3 complex (, , ) as well as the -chain of the TCR(1, 13, 14) . These motifs contain the amino acid sequence YXXL-X-YXXL and are phosphorylated by the PTK p56(1, 13, 14) . The added tyrosine phosphate groups are recognized by the SH2 domain of ZAP-70. One scenario, therefore, is that ZAP-70 is an early participant that assists in the formation of the Grb2-associated complex by providing the phosphorylation sites that are necessary to recruit Grb2, p36/p38, and PLC-1. ZAP-70 may then phosphorylate PLC-1 or other specific substrates. Noteworthy for its absence from the lysates of T-cells obtained from patients suffering from ZAP-70 deficiency is an inducible p36 substrate present in lysates from normal human T-cells(45) . It needs to be confirmed, however, that this phosphoprotein is the same as p36 in Fig. 1or a substrate for ZAP-70. Although we have been able to show during in vitro kinase assays that the relative abundance of ZAP-70 autophosphorylation (not shown) is in accordance with the actual amount of ZAP-70 protein in each fraction (Fig. 6B), it is difficult to determine whether the kinase is actually activated. This question awaits the identification of ZAP-70 substrates.

Another interesting finding of this study is the redistribution of Grb2-associated components from the cytosol to detergent-soluble and -insoluble fractions upon anti-CD3 treatment (Fig. 6A). Utilizing a detergent-soluble fraction, Sieh et al.(20) and Buday et al.(21) have shown that p36/p38 has a predilection for the particulate fraction of the cell. However, these authors did not investigate the detergent-insoluble fraction, which appears to show changes equally dramatic to that of the detergent-soluble fraction (Fig. 6A). Although it remains to be proven, the relatively high spectrin content in the detergent-insoluble fraction suggests that this cell fraction is enriched for select cytoskeletal proteins (Fig. 6A). It is possible that many of the TCR-associated signaling events take place in the interface between the cell membrane and the cytoskeleton. To this end, it is interesting that among the TCR subunits, the -chain in particular has been found to localize in a detergent-insoluble T-cell fraction, which may include the cytoskeleton (46) . The participation of the cytoskeleton could be important toward the activation of PLC-1 (see above) as well as the activation of Raf-1(47) .

Although Sieh et al.(20) detected low levels of tyrosine phosphorylation of Shc in anti-CD3-treated Jurkat cells, in a previous study we failed to detect similar phosphorylation. Moreover, in this study we found no evidence for TCR-induced Shc/Grb2 association in four different T-cell types tested (Fig. 3B). The results in T-cells were in direct contrast with prominent Shc/Grb2 association in a B-cell line, Ramos, during antigen receptor ligation (Fig. 3B). Although this could indicate that Shc does not play an important role in TCR signaling, Ravichandran et al.(48) showed Shc binding to TCR- in an antigen-responsive T-cell clone. While we do not understand the reason for these differences, we suggest that the method of TCR ligation and differentiation state of the T-cell may determine the involvement of Shc. The alternative use of Shc or another hypothetical adaptor protein such as p36/p38 may determine signaling specificity of TCR.


FOOTNOTES

*
This work was supported by U. S. Public Health Service Grant GM41576 and the Concern Foundation, Los Angeles. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Medicine/School of Medicine, 10833 Le Conte Ave., 52-175 Center for Health Sciences, Los Angeles, CA 90024-1680. Tel.: 310-825-3718; Fax: 310-206-8107.

^1
The abbreviations used are: TCR, T-cell antigen receptor; PLC-1, phospholipase C-1; PTK, protein tyrosine kinase; RTK, receptor tyrosine protein kinases; GNEF, guanine nucleotide exchange factor; mAb, monoclonal antibody; PMSF, phenylmethylsulfonyl fluoride; PAGE, polyacrylamide gel electrophoresis; NIS, non-immune serum; PMA, phorbol 12-myristate 13-acetate; GST, glutathione S-transferase; TIU, trypsin inhibitory units.


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