©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The SH3 Domain-binding T Cell Tyrosyl Phosphoprotein p120
DEMONSTRATION OF ITS IDENTITY WITH THE c-cbl PROTOONCOGENE PRODUCT AND IN VIVO COMPLEXES WITH Fyn, Grb2, AND PHOSPHATIDYLINOSITOL 3-KINASE (*)

(Received for publication, March 16, 1995; and in revised form, May 11, 1995)

Toru Fukazawa (1)(§),   Kris A. Reedquist (1)(§),  (¶),   Thomas Trub (2) Stephen Soltoff (3) Govindaswamy Panchamoorthy (1)(**),   Brian Druker (4) Lewis Cantley (3) Steven E. Shoelson (2) Hamid Band (1)(§§)

From the (1)Lymphocyte Biology Section, Department of Rheumatology and Immunology, Brigham and Women's Hospital and the (2)Research Division, Joslin Diabetes Center, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, the (3)Division of Signal Transduction, Beth Israel Hospital, and Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, and the (4)Division of Hematology and Medical Oncology, Oregon Health Sciences University, Portland, Oregon 97201

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Previously, we have identified p120 as a Fyn/Lck SH3 and SH2 domain-binding protein that is tyrosine phosphorylated rapidly after T cell receptor triggering. Here, we used direct protein purification, amino acid sequence analysis, reactivity with antibodies, and two-dimensional gel analyses to identify p120 as the human c-cbl protooncogene product. We demonstrate in vivo complexes of p120 with Fyn tyrosine kinase, the adaptor protein Grb2, and the p85 subunit of phosphatidylinositol (PI) 3-kinase. The association of p120 with Fyn and the p85 subunit of PI 3-kinase (together with PI 3-kinase activity) was markedly increased by T cell activation, consistent with in vitro binding of p120 to their SH2 as well as SH3 domains. In contrast, a large fraction of p120 was associated with Grb2 prior to activation, and this association did not change upon T cell activation. In vitro, p120interacted with Grb2 exclusively through its SH3 domains. These results demonstrate a novel Grb2-p120 signaling complex in T cells, distinct from the previously analyzed Grb2-Sos complex. The association of p120 with ubiquitous signaling proteins strongly suggests a general signal transducing function for this enigmatic protooncogene with established leukemogenic potential but unknown physiological function.


INTRODUCTION

The engagement of the T cell receptor (TCR) (^1)by major histocompatibility complex-bound antigenic peptides leads to T cell activation, a prerequisite for effective immune responses. One of the earliest and obligatory biochemical steps in T cell activation is the tyrosyl phosphorylation of cellular proteins including the receptor components themselves(1, 2) . Unlike growth factor receptors with intrinsic tyrosine kinase domains(3) , the TCR components signal through noncovalently associated cytoplasmic tyrosine kinases(1, 2) . In particular two Src family kinases, p59 (Fyn) and p56 (Lck), have been demonstrated to play critical and apparently nonoverlapping roles in T cell activation. Fyn interacts physically with the cytoplasmic signaling domains of the TCR / and CD3 and chains(4) , whereas Lck interacts with the cytoplasmic tails of the CD4 and CD8 coreceptors(5, 6) . In addition, Lck plays a role in T cell activation apparently independent of its CD4/8 association(1, 2) . A distinct cytoplasmic tyrosine kinase ZAP-70 has also been demonstrated to be critical for T cell activation and apparently functions downstream of the Src family kinases(1) .

Similar to the TCR, Src kinase-mediated tyrosine phosphorylation is an early and a critical event downstream of other surface receptors that lack intrinsic tyrosine kinase domains, such as the B cell antigen receptor(1) , Fc receptors(7) , and certain cytokine receptors(8) . Thus, elucidation of tyrosine phosphorylation-dependent signaling events downstream of the TCR is likely to provide insights of general significance.

The mechanisms by which early phosphorylation substrates are recruited to TCR-coupled tyrosine kinases are poorly understood. Studies with receptor tyrosine kinases such as epidermal growth factor receptor have elucidated the critical roles of the Src homology domains (SH2 and SH3) in assembling signaling complexes(3, 9, 10) . The SH2 domains bind to phosphotyrosyl (pY) peptide motifs (11, 12) and mediate activation-induced phosphorylation-dependent interactions between signaling proteins(3, 9, 10) . In contrast, the SH3 domains bind to small proline-rich peptide motifs(13, 14, 15, 16, 17, 18) , thus providing a basis for protein-protein interactions prior to receptor activation.

In an attempt to define the role of SH3 domain-mediated binding to recruit cellular proteins to T cell tyrosine kinases, we recently identified a Fyn/Lck SH3 domain-binding protein p120(19) . Notably, p120 was one of the earliest tyrosine phosphorylation substrates upon triggering through the TCR. Preliminary evidence indicated that p120 was present in vivo as preformed complexes with Fyn and Lck and served as a substrate for these tyrosine kinases in vitro(19) . Subsequently, an unidentified protein of similar size (116 kDa), which was also tyrosine phosphorylated rapidly by stimulation of Jurkat T cells through their TCR, was shown to interact in vitro with Grb2-SH3 fusion proteins and to bind to Grb2-SH2-specific phosphopeptide matrices, indirectly suggesting that this protein was present as an in vivo complex with Grb2 (20) . More recently, it was reported that a 120-kDa TCR-induced tyrosine-phosphorylated protein of Jurkat T cells was reactive with antibodies to the c-cbl protooncogene product, and in vitro binding experiments demonstrated that Cbl protein in cell lysates was able to bind to Grb2 fusion proteins(21) .

In the present study we have characterized further the in vivo interactions of the Fyn/Lck SH3 domain-binding protein, p120, with other T cell signaling proteins that possess SH2 and SH3 domains. Unlike earlier studies(20, 21) , we use coimmunoprecipitation analyses to show in vivo complexes of p120 with SH2/SH3-bearing T cell signaling proteins, the Src family tyrosine kinase Fyn and adaptor protein Grb2; in addition, we demonstrate that the Grb2-associated fraction of p120 is a target of early TCR-elicited tyrosine phosphorylation. Significantly, we also demonstrate a predominantly SH2 domaindependent interaction of p120 with the PI 3-kinase p85, which results in the recruitment of this enzyme into p120-containing protein complexes in an activation-dependent manner.

We used the in vivo Grb2-p120 association to immunoaffinity purify the p120 polypeptide. The determined amino acid sequences of three distinct tryptic peptides revealed that p120 is identical to the human p120 protooncogene(22) , and this was established further by immunochemical and two-dimensional gel analyses. These analyses independently confirm and extend the recent results showing that the Cbl protein is a target of TCR-mediated tyrosine phosphorylation in Jurkat cells(21) . Together, these results strongly suggest that p120 serves as a multifunctional SH2 and SH3 domain-binding protein in the tyrosine kinase-mediated signal transduction cascade downstream of the TCR. Given the preferential expression of c-Cbl in hematopoietic cells(23) , induction of pre-B and myeloid cell leukemias by its viral form(24) , and its in vivo complexes with ubiquitous signaling proteins (this study), it is likely that c-Cbl also participates in signaling downstream of other hematopoietic cell receptors.


MATERIALS AND METHODS

Peptides

The following pY peptides were synthesized and HPLC purified, as described(25) : the pYEEI motif peptide corresponding to Tyr-324 of the hamster medium-sized tumor antigen (hmtY324) (EPQpYEEIPIYL; bold is SH2-binding motif) and its unphosphorylated version (YEEI); the pYVNV motif peptide corresponding to Tyr-317 of Shc protein (PSpYVNVQNL) and its mutant version pYVAV (PSpYVAVQNL); and the pYMNM motif peptide corresponding to CD28 Tyr-191 (HSDpYMNMTPR)(11, 12) . The following proline-rich peptides were synthesized on an Applied Biosystems peptide synthesizer: p85 (PI 3-kinase p85alpha amino acids 83-101, ISPPTPKPRPPRPLPVAPG)(26) ; Sos (human son-of-sevenless amino acids 1147-1162; VPVPPPVPPRRRPESA)(27) ; and dynamin (rat brain dynamin amino acids 786-806; PAVPPARPGSRGPAPGPPPAG)(28) .

Antibodies

The monoclonal antibodies (mAb) used in this work were: anti-TCR1 (anti-human TCR chain; IgG1)(29) ; OKT8 (anti-human CD8alpha; IgG2a) (ATCC); 4G10 (anti-pY; IgG2a)(30) ; 2Ad2 (anti-CD3 ; IgM) (from Ellis Reinherz, Dana-Farber Cancer Institute, Boston); SPV-T3b (anti-CD3 ; IgG2a) (from Hergen Spits, Netherlands Cancer Institute, Amsterdam); TiA2 (anti-TCR ; IgG1) (from Paul Anderson, Dana-Farber Cancer Institute); anti-phospholipase C1 (a mixture of mAbs, Upstate Biotechnology Inc.); 9E10 (anti-Myc epitope tag; IgG1) (31) (hybridoma provided by J. Michael Bishop, University of California, San Francisco); anti-Sos (against amino acids 1-109; IgG3; reacts with Sos1 and 2) (Transduction Laboratories, Lexington, KY). The polyclonal rabbit antibodies used were: anti-PI 3-kinase p85 (alpha and beta) (against the C-terminal 19-kDa fragment) (Transduction Laboratories); anti-Grb2 (against amino acids 195-217 of human Grb2) and anti-p120 (against a 15-amino acid synthetic peptide corresponding to the C terminus of Cbl) (both from Santa Cruz Biotechnology, Inc., Santa Cruz, CA); anti-Lck (against amino acids 22-51) (Upstate Biotechnology Inc.); and anti-Fyn (against amino acids 35-51) (from Chris Rudd, Dana-Farber Cancer Institute). Normal rabbit serum from young adult unimmunized rabbits was used as a negative control.

The second-step reagents for immunoblotting were: I-labeled protein A (Amersham Corp.); horseradish peroxidase (HRPO)-conjugated protein A and sheep anti-mouse IgG (Cappel-Organon Technika, Durham, NC); and donkey anti-rabbit Ig-HRPO (Amersham Corp.).

Glutathione S-Transferase (GST) Fusion Proteins

Expression of human Lck-SH3, Fyn-SH3, mutant Fyn-SH3(W119K), Fyn-SH2, and mutant Fyn-SH2(R176K) proteins as C-terminal fusions with GST in pGEX-2T.K vector has been described(19, 32) . Murine c-Abl type IV SH3 (33) in pGEX-2T vector (Pharmacia Biotech Inc.) was provided by Bruce Mayer (Children's Hospital, Boston). Wild type murine Grb2 (34) (provided by Mike Moran, University of Toronto) or its mutants were expressed in pGEX-3X vector (Pharmacia). Mutations were introduced by polymerase chain reaction and verified by DNA sequencing. (^2)The mutants are depicted as 3-2-3 (reflecting the N-terminal SH3, SH2, and C-terminal SH3 domains) with an asterisk on the mutated domain: 3*-2-3 (P49L); 3-2-3* (P206L); 3-2*-3 (R86K); and 3*-2-3* (P49L,P206L). The following GST fusion constructs of the PI 3-kinase p85alpha (26) (provided by Joseph Schlessinger, New York University Medical Center) were expressed in pGEX-3X vector: p85-SH2(N) (amino acids 321-440); p85-SH3/SH2(N) (amino acids 1-440); p85-SH2(N+C) (amino acids 321-725). p85-SH3 (amino acids 1-80) in pGEX-2T has been described(35) .

Fusion proteins were affinity purified on glutathione-Sepharose beads (Pharmacia) using a Triton X-100-soluble fraction of the isopropyl-1-thio-beta--galactopyranoside-induced Escherichia coli (DH5alpha strain), as described(19, 32) . Proteins were quantitated by Bradford assay (Bio-Rad) against a bovine serum albumin standard and analyzed on Coomassie gels to confirm quantitation and to assess purity (usually more than 95%).

Generation of Myc Epitope-tagged Grb2 (Grb2myc) Transfectants

Retroviral supernatants were obtained from PA317 packaging cells transiently transfected (by the Ca(2)PO(4) method) with pBabepuro (vector) or pBabepuroGrb2mycA (pBabepuro with mouse Grb2 tagged at the C terminus with anti-Myc antibody epitope) (from Robert Weinberg, Whitehead Institute, Cambridge, MA)(36) . Eight 10^6 HSB2 cells were infected for 16 h with 4 ml of viral supernatant in 20 ml of medium containing 4 µg/ml Polybrene. Forty-eight h later selection was initiated in 1 µg/ml puromycin (Sigma). Vector transfectants were used as pooled populations. Grb2myc transfectants were cloned at limiting dilution, analyzed for Grb2myc expression by Western blotting, and clones with high protein expression were used further.

Activation of Jurkat Cells

Cells were washed and resuspended in RPMI 1640 with 20 mM HEPES and 0.2 mM vanadate at 1-2 10^8/ml and then incubated either without (control) or with anti-CD3 mAb (SPV-T3b or 2Ad2; 1:200 ascites) for 2 min or as specified. Cells were lysed at 5 10^7/ml in cold lysis buffer (0.5% Triton X-100 (Fluka), 50 mM Tris, pH 7.5 (at room temperature), 150 mM sodium chloride, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin and pepstatin, 1 mM vanadate, and 10 mM sodium fluoride).

Binding of Cellular Polypeptides to GST Fusion Proteins

10-20 µg of purified GST fusion proteins noncovalently coupled to glutathione-Sepharose beads were rocked with lysate from 2.5-5 10^7 cells for 1 h at 4 °C and washed six times in lysis buffer. Bound proteins were solubilized in sample buffer, resolved by SDS-polyacrylamide gel electrophoresis (PAGE) under reducing conditions, and subjected to immunoblotting with the indicated antibodies.

Immunoprecipitations and Gel Electrophoresis of Proteins

Antibodies were added to lysates (5 10^7 cell equivalents) precleared with Staphylococcus aureus Cowan I strain (Pansorbin, Calbiochem). After 1-2 h of rocking at 4 °C, 10-20 µl of protein A-Sepharose 4B (Pharmacia) was added and incubation continued for 45-60 min. Beads were washed six times in lysis buffer, and bound proteins were solubilized in Laemmli sample buffer with 2-mercaptoethanol, resolved by SDS-PAGE, and subjected to immunoblotting with the indicated antibodies. For two-dimensional gel analysis, immunoprecipitations or fusion protein binding reactions were performed as above, and proteins were eluted from beads at 90 °C for 2 min in isoelectric focusing (IEF) buffer with SDS(37) . Samples were mixed with IEF standards (Bio-Rad) and run on IEF tube gels with pH 3.5-10 Ampholine (Pharmacia) followed by SDS-PAGE. Immunoblotting was carried out as described below. IEF standards on filters were visualized by Coomassie staining and immunoblotting with anti-actin and anti-myoglobin (from Sigma).

Immunoblotting

Polypeptides were transferred to PVDF membranes (Immobilon-P, Millipore, Bedford, MA) which were blocked at room temperature with 2% gelatin (Bio-Rad) in TBS-T (10 mM Tris, pH 8, 150 mMNaCl(2), 0.05% Tween 20 (Bio-Rad)) for 1 h to overnight, incubated with optimal concentrations of primary antibodies in TBS-T for 1 h at room temperature, and washed six times in TBS-T. Filters were then incubated with 10 ng/ml (0.3 µCi/ml) I-labeled protein A for 1 h at room temperature, washed six times, and dried, and autoradiograms were developed with Kodak XAR-5 film at -80 °C. Alternatively, HRPO-conjugated second-step reagents (protein A, sheep anti-mouse Ig, or donkey anti-rabbit Ig) were used, followed by visualization by enhanced chemiluminescence (ECL) using autoradiographic film (Reflection®, DuPont NEN). Chemiluminescence reagents were from Amersham Corp. (ECL kit) or DuPont NEN (Renaissance chemiluminescence reagent) and were used according to the supplier's recommendations. For repeated immunoblotting, membranes were stripped in 62.5 mM Tris-HCl, pH 6.7, 2% SDS, and 0.1 M 2-mercaptoethanol for 30-45 min at 50 °C, rinsed in TBS-T, and blocked with gelatin/TBS-T prior to reprobing with antibodies.

p120 Protein Purification

Affinity matrix was prepared by coupling antibodies to protein A-Sepharose CL-4B beads using dimethyl pimelimidate (Pierce)(38) . 2.5 g of HSB2-Grb2myc transfectant cells were washed in phosphate-buffered saline (137 mM NaCl, 15.7 mM NaH(2)PO(4), 1.47 mM KH(2)PO(4), 2.68 mM KCl, pH 7.4), lysed in 200 ml of Triton X-100 lysis buffer (see above), and passed over 3-ml preclearing columns of OKT8 (anti-CD8), W6/32 (anti-HLA class I), and anti-TCR1 (anti-TCR ), and then applied to a 3-ml column of 9E10 (anti-Myc epitope). The column was washed with 10 volumes each of lysis buffer and phosphate-buffered saline. Bound polypeptides were eluted in 1-ml fractions with 0.1 M glycine HCl, pH 3, 150 mM NaCl and immediately neutralized with Tris, pH 8 (0.1 M final concentration). 0.3% of each fraction was resolved on SDS-PAGE and analyzed by silver staining (39) and anti-pY immunoblotting. The remainder of the fractions was precipitated with trichloroacetic acid, washed with cold acetone, and solubilized in sample buffer. Fractions with p120 were pooled, run on SDS, 7.5% PAGE, and transferred to PVDF membrane (Immobilon-P). A concurrently resolved Fyn SH3 binding reaction and a part of the preparative sample were visualized by anti-pY immunoblotting for alignment with the p120 band on the PVDF membrane, which was visualized by Ponceau S staining. The p120 band was excised and submitted to Harvard Microchemistry Facility where it was digested in situ with trypsin, and resulting peptides were separated by HPLC and sequenced by collisionally activated dissociation on a Finningon TSQ700 triple-quadruple mass spectrometer. Determined sequences were compared with protein sequence data bases using the FASTA program.

Measurement of PI 3-Kinase Activity

The immunoprecipitates (see above) were washed four times in phosphate-buffered saline plus 1% (v/v) Nonidet P-40, followed by three washes in TNE (10 mM Tris, pH 7.5, 100 mM NaCl, 1 mM EDTA). All wash solutions contained 0.2 mM sodium orthovanadate. To assay the PI 3-kinase activity, crude brain phosphoinositides (Sigma, P-6023) plus [-P]ATP (10 µCi/sample) were added to the immunoprecipitates for 10 min at room temperature. The phosphoinositides were suspended in 10 mM HEPES, pH 7.5, 1 mM EGTA, sonicated prior to use, and added at a final concentration of 0.2 mg/ml. The [-P]ATP was added in a mixture that provided a final concentration of 50 µM ATP, 5 mM MgCl(2), and 1 mM HEPES. The lipid kinase assay was terminated by adding 1 N HCl, and lipids were extracted into a chloroform/methanol mixture (1:1, v/v). The lipid-containing organic phase was resolved on oxalate-coated thin layer chromatography (TLC) plates (Silica Gel 60; MCB reagents, Merck), developed in 1-propanol/acetic acid (2 M) (65:35 mixture), and lipid species were visualized by autoradiography.


RESULTS

Strategy for Purification of p120

To facilitate anti-pY mAb detection of p120 during purification, we sought a cell line with constitutively tyrosine-phosphorylated p120. Based on our previous finding that p120 served as a substrate for Lck in vitro(19) , we reasoned that p120 may be hyperphosphorylated in the human T cell line HSB2, which expresses a constitutively activated mutant Lck(40) . Anti-pY immunoblotting of the whole cell lysates showed that the 120-kDa polypeptide in HSB2 cells was markedly hyperphosphorylated compared with that in anti-CD3-stimulated Jurkat cells (data not shown). This polypeptide showed binding to SH3 domains of the Fyn and Lck but not Abl, a pattern identical to that observed with p120 in Jurkat T cells(19) .

To purify p120 from HSB2 cells, we took advantage of its ability to form stable in vivo complexes with Grb2 adaptor protein (described below). To facilitate immunoaffinity purification of p120, we generated a transfectant cell line (HSB2-Grb2myc) that expressed high levels of a Myc epitope-tagged Grb2 protein. This is seen as a 28-kDa species in anti-Grb2 and anti-Myc immunoprecipitates of HSB2-Grb2myc cells but not in the HSB2-puro^r control cells (Fig.1A). Anti-pY immunoblotting revealed the association of p120 with transfected Grb2myc protein (Fig.1B). Thus, HSB2-Grb2myc transfectant provided a suitable reagent for immunoaffinity purification of p120.


Figure 1: Expression of the Myc epitope-tagged Grb2 in HSB2 cells and its interaction with p120. Transfectants were derived by retroviral infection with a control vector (HSB2-puro^r) or a Grb2myc vector (HSB2-Grb2myc). Antibodies: anti-TCR1, control (Cont.), anti-Myc epitope 9E10 (alphamyc), or rabbit anti-Grb2 (alphaGrb2). Panel A, Grb2myc expression in HSB2-Grb2myc cells. Triton X-100 lysates of 2 10^6 cells, metabolically labeled with [S]methionine and cysteine, were immunoprecipitated with the indicated antibodies (shown on top), and immunoprecipitated species (see arrows) were resolved by SDS-PAGE and visualized by fluorography. Panel B, association of Grb2myc with p120. Whole cell lysate (10^6 cells) or immunoprecipitations from 2 10^7 cells with indicated antibodies (I.P., shown on top) were subjected to immunoblotting with anti-pY mAb (upper panel) followed by sheep anti-mouse-HRPO and ECL detection. The lower portion of the same filter was probed with anti-Grb2 followed by donkey anti-rabbit-HRPO and ECL detection.



p120 Is Identical to the c-cbl Protooncogene Product

Triton X-100 lysate of HSB2-Grb2myc cells was applied to a 9E10 anti-Myc mAb column, and eluates containing Grb2myc and associated polypeptides were analyzed by silver staining and anti-pY immunoblotting; each analysis revealed a discrete 120-kDa species (not shown). Fractions with a detectable p120 signal in silver staining were pooled, resolved by SDS-PAGE, and transferred to Immobilon-P membrane. The excised p120 band was cleaved with trypsin, and individual peptides were sequenced.

The determined sequences of three distinct peptides are shown in Table1. The eight determined amino acids of peptide 1 showed complete identity with amino acids 117-124 of the human c-cbl protooncogene which are preceded by a potential trypsin cleavage site (Arg-116)(22) . All of the six determined residues of peptide 2 matched amino acids 129-134 of the human c-Cbl protein; a glutamine at c-Cbl position 128 corresponded to the unidentified first residue in peptide 2 and was preceded by a tryptic cleavage site (lysine). Seven of 10 unambiguous and 3 of 4 probable residues of peptide 3 matched those within amino acids 837-850 of the human c-Cbl without any gaps. Again, a tryptic cleavage site (lysine) preceded this region in c-Cbl. The four mismatches included three unambiguous residues; the reason(s) for this discrepancy is not known. Since a single functional c-cbl gene has been identified in the human genome(22, 41, 42) , the determined peptide sequences indicate that p120 is identical to the human c-Cbl protein.



To confirm further the identity between p120 and c-Cbl, we examined whether p120 (operationally defined as the Fyn/Lck SH3 domain-binding protein) (19) was recognized by an anti-Cbl antibody. Anti-pY immunoblotting demonstrated expected binding of p120 to wild type Fyn SH2 and SH3 domains but not to their nonbinding point mutants or to Abl SH3 (Fig.2). Anti-Cbl antibody selectively immunoblotted the fusion protein-bound p120. Furthermore, p120 detected in fusion protein binding reactions and anti-pY immunoprecipitates comigrated with directly immunoprecipitated p120 (data not shown).


Figure 2: p120 is recognized by anti-Cbl antibodies. Whole cell lysates (10^6 cells) or GST fusion protein binding reactions from anti-CD3 (2Ad2)-stimulated Jurkat T cells (5 10^7 cells) were resolved by SDS-PAGE and subjected to immunoblotting with anti-pY antibody 4G10 followed by detection with I-protein A (top). The filter was reprobed with anti-Cbl antibody and detected with HRPO-conjugated anti-rabbit antibody using ECL (bottom).



Finally, we compared p120 and c-Cbl proteins by two-dimensional gel analysis to demonstrate their biochemical identity. Fyn SH3-bound material, anti-Cbl immunoprecipitates or their mixture was resolved by IEF followed by SDS-PAGE, and resolved species were visualized by anti-pY immunoblotting (Fig.3). The cell lysates were derived from anti-CD3-stimulated Jurkat cells. Anti-pY immunoprecipitates resolved into a number of distinct spots or arrays of spots, corresponding to major pY proteins observed in one-dimensional analysis (run on the side). Only one array of these spots was observed in Fyn-SH3-bound material and corresponded to p120 band in the SH3 binding reaction subjected to direct SDS-PAGE on the same gel; an additional spot near the origin of the IEF gel may reflect incomplete entry of the protein. An identical series of spots was observed in anti-Cbl immunoprecipitate, and a mixture of Fyn-SH3-bound material and anti-Cbl immunoprecipitate produced a pattern identical to each of the individual components. Similar two-dimensional gel analysis of Grb2-associated p120 also demonstrated it to be identical to c-Cbl protein (not shown). Together, the peptide sequences, reactivity with anti-Cbl antibody, and identical two-dimensional gel patterns demonstrate unambiguously that p120 is identical to the human c-cbl protooncogene product. Comparison of anti-pY with other two-dimensional gel patterns (Fig.3) suggests that additional tyrosine-phosphorylated polypeptides in the 120-kDa size range exist in T cells, but these are distinct from c-Cbl.


Figure 3: Biochemical identity between Fyn SH3-binding p120 and c-Cbl proteins demonstrated by two-dimensional gel analyses. GST Fyn-SH3 binding reactions or immunoprecipitations (alpha-pY or alpha-Cbl) were carried out from Triton X-100 lysates of 10^8 anti-CD3 (2Ad2)-stimulated Jurkat cells and proteins were eluted into IEF sample buffer with SDS. Individual samples or their mixtures (indicated on top) were subjected to IEF on a pH 3.5-10 Ampholine in the first dimension (IEF; from alkali to acid) and SDS-PAGE in the second dimension. The resolved proteins were transferred to PVDF membrane and subjected to anti-pY immunoblotting, followed by HRPO-protein A and ECL detection. An aliquot of anti-pY immunoprecipitate and Fyn-SH3 binding reaction was resolved during SDS-PAGE on each gel to identify p120 unambiguously. Two-dimensional standards were included with samples and visualized by Coomassie staining of filters or immunoblotting with anti-actin and anti-myoglobin antibodies. The identity of p120 and c-Cbl is revealed by their identical migration when resolved as a mixture (middle panel on left). NRS, normal rabbit serum.



p120 Interacts in Vivo with Multiple SH3 Domain-containing T Cell Signaling Proteins

Our previous analyses indicated that only a fraction of p120 was associated with Fyn and Lck proteins in vivo compared with that available for Fyn SH3 binding in vitro(19) . In addition, in vitro binding of Cbl protein to Grb2 fusion proteins has been reported(21) . These findings raised the possibility that p120 may interact with additional SH3 domain-containing proteins in T cells. To test this possibility, immunoprecipitates of various SH3 domain-containing proteins from unstimulated or anti-CD3-stimulated Jurkat cells were subjected to anti-pY and anti-Cbl immunoblotting (Fig.4).


Figure 4: In vivo association of p120 with Fyn, Grb2, and PI 3-kinase p85 in Jurkat T cells. Immunoprecipitations from Triton X-100 lysates of 5 10^7 unstimulated(-) or anti-CD3 (2Ad2)-stimulated (+) Jurkat cells with indicated antibodies (I.P., shown on top) or whole cell lysates (from 10^6 cells) were resolved on SDS, 10% PAGE, transferred to PVDF membranes, and subjected to immunoblotting with antibodies indicated on the right, followed by HRPO-conjugates (anti-mouse-HRPO for anti-pY blot; anti-rabbit-HRPO for anti-Cbl blot; and HRPO-protein A for anti-PI 3-kinase p85 and anti-Grb2 blots) and ECL detection. Immunoprecipitated species are indicated by arrows on the left; Ig, immunoglobulin heavy chain. Two unmarked solid arrows indicate the positions of 100- and 75-kDa polypeptides (referred to as p100 and p75 in the text). The open arrow indicates a 90-kDa phospholipase C1-associated polypeptide. Normal rabbit serum (NRS) and alphaCD8 are used as negative controls for polyclonal rabbit and monoclonal mouse antibodies, respectively. Each immunoblot represents a reprobing of the same filter.



Anti-pY immunoblotting demonstrated an activation-dependent increase in pY content on p120 (lanes 9 and 10). Conversely, anti-Cbl immunoblotting showed that the amount of p120 in anti-pY immunoprecipitates increased with anti-CD3 stimulation (lanes 13 and 14). Anti-Fyn immunoprecipitates revealed an associated 120-kDa protein reactive with both the anti-pY and anti-Cbl antibodies (lane 7), and a higher signal was detected by each antibody in anti-CD3-stimulated cells (lane 8); the enhancement of Fyn-p120association upon T cell activation extends our previous results that p120 can concurrently bind to Fyn SH3 and SH2 domains in vitro(19) .

Significantly, a similar amount of p120 was associated with Grb2 in unstimulated and anti-CD3-stimulated Jurkat cells; however, Grb2-associated Cbl showed an increased pY signal after anti-CD3 stimulation (lanes 3 and 4; also see Fig.5below for time course). p120 was also detected in immunoprecipitations of the PI 3-kinase p85; increased p120signals were observed after anti-CD3 stimulation using anti-Cbl as well as anti-pY immunoblotting (lanes 5 and 6).


Figure 5: Time course of tyrosine phosphorylation of Grb2-associated p120 upon T cell activation. Anti-Grb2 immunoprecipitations from 5 10^7 unstimulated (-) or anti-CD3 (2Ad2)-stimulated (+) Jurkat cells were resolved by SDS-PAGE and subjected to anti-pY (top panel) or anti-Grb2 (bottom panel) immunoblotting, followed by detection with I-protein A. The time of anti-CD3 stimulation is shown in seconds (s) or minutes (m). The top and bottom panels represent upper and lower parts of a single filter.



In contrast to its association with Grb2 and PI 3-kinase p85, p120 was not detected in immunoprecipitates of phospholipase C1, although phospholipase C1 was immunoprecipitated efficiently (confirmed by immunoblotting; data not shown) and showed activation-dependent tyrosine phosphorylation (lanes 11 and 12). Thus, p120 forms in vivo complexes with certain SH2/SH3 domain-containing T cell signaling proteins but not with others. Each of these interactions was confirmed by stable (Myc-tagged Grb2) or transient (HA-tagged p85 and untagged Fyn) transfection into Jurkat T cells (data not shown). The identity of Grb2- and p85-associated p120 protein as Cbl was also confirmed by two-dimensional gel analysis (data not shown).

The above analyses also yielded two additional findings. First, anti-PI 3-kinase p85 immunoprecipitations revealed a coimmunoprecipitated 36-38-kDa inducibly tyrosine-phosphorylated polypeptide identical in mobility to p36/38 which associates with Grb2 and phospholipase C1 (compare lanes 4, 6, and 12)(43, 44) . Second, both Grb2 and PI 3-kinase p85 coimmunoprecipitated unidentified polypeptides of 100 and 75 kDa which underwent activation-dependent tyrosine phosphorylation. These polypeptides are likely to mediate interactions between Grb2 and p85, both of which lack tyrosine phosphorylation (data not shown), yet coimmunoprecipitate with each other (Fig.4, lanes 3-6) and are incorporated into pY complexes (Fig.4, lane 13 versus 14) in an activation-dependent manner. It is likely that the 75-kDa polypeptide corresponds to the recently identified Grb2 SH3 domain-binding SLYP-76 protein(45) .

Grb2-associated p120 Is Tyrosine Phosphorylated Rapidly upon T cell Activation

The previously identified SH3 domain-binding proteins that interact with Grb2 in vivo (Sos and dynamin) are not tyrosine phosphorylated; in contrast, the Grb2-associated p120 is (Fig.4). To characterize this complex further, we specifically examined the kinetics of tyrosine phosphorylation on Grb2-associated p120upon triggering through the TCR (Fig.5). Anti-pY immunoblotting of whole cell lysates (not shown) confirmed earlier analyses that p120 phosphorylation was maximal by 30 s(19) . Importantly, tyrosine phosphorylation of Grb2-associated p120 followed an essentially identical time course. Anti-Grb2 immunoblotting of immunoprecipitates demonstrated equal amounts of Grb2. Thus, the Grb2-p120 complex is readily accessible to the TCR-coupled tyrosine kinases for very early phosphorylation.

Association of the PI 3-Kinase Enzymatic Activity with p120

The interaction of PI 3-kinase p85 with p120 and other rapidly tyrosine-phosphorylated proteins (e.g. p36/38, p100, and p75) suggested a mechanism to recruit PI 3-kinase enzymatic activity into pY signaling complexes. To examine if this is the case, we measured the PI 3-kinase activity associated with various immunoprecipitates from unstimulated and anti-CD3-stimulated Jurkat T cell lysates. As expected, a high level of PI 3-kinase activity was observed in anti-PI 3-kinase p85 immunoprecipitates, and the level of this activity showed little change upon stimulation of cells (Fig.6). Significantly, a substantial level of PI 3-kinase activity was detected in anti-Cbl immunoprecipitates and increased further upon TCR stimulation. The relative levels of Cbl-associated PI 3-kinase activity correlated with the amount of coimmunoprecipitated PI 3-kinase p85 protein (data not shown). Notably, a small amount of PI 3-kinase activity was observed in anti-pY immunoprecipitations of unstimulated cells and increased substantially upon T cell activation. Interestingly, the amounts of the PI 3-kinase activity associated with anti-TCR or anti-Fyn immunoprecipitates were substantially lower than those associated with the anti-Cbl immunoprecipitates. These data suggest that PI 3-kinase p85-p120 interaction may provide a major mechanism to recruit PI 3-kinase into TCR activation-induced signaling complexes.


Figure 6: Association of the PI 3-kinase activity with p120. Immunoprecipitations carried out with the indicated antibodies (I.P., shown on top) from 5 10^7 unstimulated(-) or anti-CD3 (2Ad2)-stimulated (+) Jurkat cell lysates were subjected to lipid kinase assays as described under ``Materials and Methods.'' The reaction products were subjected to TLC and visualized by autoradiography. Lane 11 shows PI products generated with anti-p85 immunoprecipitates from A431 cells which overexpress epidermal growth factor receptor. Note that PI species in the middle lanes migrated slower for technical reasons; the phosphatidylinositol trisphosphate species (PIP3) is the third major spot after the origin.



p120 Selectively Binds to SH3 Domains of Grb2

To assess the contribution of the SH2 and SH3 domains of Grb2 toward p120 binding, lysates of anti-CD3-stimulated Jurkat T cells were incubated with GST fusion proteins of wild type Grb2 or its mutants with point mutations in the pY-binding pocket of the SH2 or proline peptide-binding pouches of the SH3 domains. Bound proteins were detected by anti-pY and anti-Cbl immunoblotting.

The major tyrosine-phosphorylated species associated with Grb2 in vivo, namely p120, p100, p75, and p36/38, were observed in wild type GST-Grb2 binding reactions (Fig.7A). Binding to p120 was essentially abolished when the N-terminal SH3 domain alone was mutated and was decreased but still substantial when the C-terminal SH3 domain alone was mutated. p120 did not bind to double SH3 mutants of Grb2. Anti-Cbl blots revealed an identical binding pattern. In contrast to p120 binding, binding to p36/38 was retained in single or double SH3 domain mutants, whereas it was abolished by the SH2 domain mutation. In addition, a fusion protein with only the Grb2-SH2 domain failed to bind to p120, whereas it did bind to p36/38. These results demonstrate that p120 binds to Grb2 exclusively through its SH3 domains; the exclusive binding of p36/38 to SH2 domain confirms previous reports(43, 44) .


Figure 7: Binding of p120 to GST fusion proteins of Grb2 is mediated through SH3-proline peptide interactions. Panel A, mutations in Grb2 SH3 domains abrogate p120 binding, but the SH2 mutation does not. Cell lysates were incubated with GST fusion proteins noncovalently immobilized on glutathione-Sepharose beads (5-µl packed beads; total volume 1 ml) for 1 h, and bound proteins were solubilized in sample buffer. Whole cell lysate (10^6 cells) or binding reactions of the indicated GST fusion proteins (10 µg each; shown on top) with lysates of 2.5 10^7 anti-CD3 (SPV-T3b)-stimulated Jurkat cells were subjected to anti-pY immunoblotting, followed by HRPO-protein A and ECL detection. 3-2-3 refers to N-terminal SH3, SH2, and C-terminal SH3 domains of Grb2. Asterisks denote mutated domains. Mutated residues were: N-terminal SH3, P49L; SH2, R86K; C-terminal SH3, P206L. Lanes 11 and 12 are from a separate experiment. The filter was stripped and immunoblotted with anti-Cbl antibody (lower panel). Panel B, the p120binding to GST-Grb2 is abrogated by competing proline-rich peptides, whereas p36/38 binding is retained. Competing peptides were added separately to bead-bound fusion proteins and cell lysate at the indicated concentrations (shown in µM). After 15 min, beads and lysate were mixed, and binding reactions and immunoblotting were carried out as in panel A. Peptides were: PI 3-kinase p85alpha amino acids 83-101, Sos1 amino acids 1147-1162, and dynamin amino acids 786-806. -, no peptide. Panel C, the p120 binding is retained in the presence of competing pY peptides, whereas the p36/38 binding is abrogated. Binding reactions and immunoblotting were as in panel B. Peptides were: EPQpYEEIPIYL (pYEEI); EPQYEEIPIYL (YEEI); PSpYVNVQNL (pYVNV); PSpYVAVQNL (pYVA*V). -, no peptide.



p120 Binding to Grb2 Is Abolished Selectively by Proline-rich Peptides

To address further the mechanism of Grb2-p120 interaction, we performed competition experiments with Grb2 SH2- and SH3-specific peptides. For SH3-mediated binding, we used three proline-rich peptides that bind to Grb2 with a range of affinities^2: Sos (mSos-1 amino acids 1147- 1162; ED for Grb2 binding, 125 µM), p85 (p85alpha amino acids 83-101; ED for Grb2 binding 540 µM), and dynamin (dynamin amino acids 786-806; ED for Grb2 binding, >3000 µM). As expected(19) , p120 binding to the Fyn SH3 domain was abolished by p85 peptide and decreased by Sos peptide (100 µM) but was little affected by dynamin-derived peptide (Fig.7B). In contrast, p120 binding to Grb2 fusion protein was inhibited by Sos-N16 in a dose-related manner with complete inhibition at 100 µM; inhibition with p85 peptide was partial, and dynamin peptide had little effect. Binding of Grb2 to p75 was also inhibited by Sos-N16 albeit less efficiently. In contrast to p120 and p75, proline-rich peptides did not affect Grb2-binding to p36/38.

For phosphopeptide competition, we used a Grb2 SH2-specific Shc-derived peptide (pYVNV motif), a mutant Shc peptide in which the critical Arg has been changed to Ala, and a Src family SH2-specific peptide (pYEEI motif)(11, 12) . Twenty µM pYEEI (but not YEEI) markedly reduced pY protein binding to Fyn SH2, with nearly complete inhibition of p120 binding; pYVNV had relatively little effect (Fig.7C). Whereas pYVNV produced a dose-dependent inhibition of Grb2 binding to p36/38, binding of p120 was retained; the decrease in p120 (and p75) signal seen even at the lowest concentration (0.8 µM) was not dose-related. The specificity of the pYVNV peptide was demonstrated by the inability of either the pYVAV or the pYEEI peptide to inhibit pY protein binding to Grb2. Together, the peptide competition and mutational data demonstrate that p120 interacts with Grb2 exclusively through the SH3 domain.

Both the SH2 and SH3 Domains of the PI 3-Kinase p85 Bind to p120

To determine the role of PI 3-kinase p85 SH2 and SH3 domains toward p120 binding, we incubated the PI 3-kinase p85-derived GST fusion proteins with lysates of anti-CD3-stimulated Jurkat T cells and detected the bound proteins by anti-pY immunoblotting. p85-SH2(N) showed binding to p120and other tyrosine-phosphorylated proteins as did fusion proteins that incorporated the SH2(N) and SH3 domains, or the two SH2 (N+C) domains (Fig.8A). In contrast, p120 was the major polypeptide detected in p85-SH3 binding reactions. Thus, in vitro both the SH2 and SH3 domains of PI 3-kinase p85 are capable of binding to p120.


Figure 8: Binding of p120 to GST fusion proteins of the PI 3-kinase p85. Panel A, both the SH2 domains and the SH3 domain of PI 3-kinase p85 are capable of binding to p120. Binding reactions and anti-pY immunoblotting were carried out as in Fig.7A. p120 is indicated. The particular domains incorporated in GST fusion proteins are shown on top. Panel B, binding of p120 to GST-p85-SH2(N)/SH3 is mediated predominantly through SH2-pY peptide interactions. Peptide competitions were performed as in Fig.7B. Peptides were: pYEEI; HSDpYMNMTPR (pYMNM); HSDYMNMTPR (YMNM); PI 3-kinase p85alpha amino acids 83-101 (p85). -, no peptide. Note that pYMNM alone nearly completely competes out the p120 binding to p85-SH2(N)/SH3 fusion protein.



Specific Peptide Inhibition of p120 Binding to PI 3-Kinase p85 Fusion Proteins

To delineate further the mechanism of p120-PI 3-kinase p85 interaction, we determined the effects of SH2- and SH3-specific peptides on p120binding to p85-SH2(N), p85-SH3, or p85-SH3/SH2(N) GST fusion proteins (Fig.8B). Whereas binding of p85-SH2(N) to pY proteins was little affected by the Src SH2-specific pYEEI peptide, a dose-related inhibition was observed with the p85 SH2-specific pYMNM peptide corresponding to the CD28 cytoplasmic tail, with complete inhibition at a 20 µM concentration; the unphosphorylated peptide had no effect. Binding of p120 to the p85-SH3 fusion protein was inhibited completely by the p85-derived proline-rich peptide even at 20 µM peptide; 100 µM Sos peptide also inhibited p120binding.

Interestingly, 20 µM phosphopeptide markedly reduced the binding of p120 to SH3/SH2(N) fusion protein. Consistent with this result, 100 µM p85 peptide produced a relatively minor inhibition of p120 binding. A combination of phosphopeptide and proline-rich peptide produced an essentially complete inhibition. Thus, although both the SH2 and SH3 domains of PI 3-kinase p85 can bind to p120, SH2 binding predominates when both domains are present in a single fusion protein.

p120 Does Not Form Stable Complexes with m-Sos

Since Grb2 is known to complex with the m-Sos protein through its SH3 domains, it was important to determine whether Grb2 concurrently binds to Sos and p120, resulting in an Sos-p120 complex. This possibility appeared unlikely since the requirements for the SH3 domains of Grb2 for in vitro binding to p120 were similar to those for Sos binding(27, 36, 46) . However, to assess this possibility directly, we carried out anti-Sos immunoblotting of immunoprecipitations shown in Fig.4. As expected, Sos was detected in anti-Grb2 immunoprecipitations (Fig.9). In contrast, no Sos was detected in anti-Cbl immunoprecipitations, indicating that the two SH3 domains of Grb2 do not bind concurrently to Sos and p120. Unexpectedly, the amount of Sos associated with Grb2 increased substantially after T cell activation (Fig.9). Concurrently, substantial coimmunoprecipitation of Sos1 was observed in anti-pY immunoprecipitations. Thus, both p120 and Sos appear to associate independently with Grb2 and are recruited into pY protein complexes upon T cell activation.


Figure 9: Lack of complex formation between p120 and Sos proteins. Immunoprecipitations with indicated antibodies (I.P., shown on top) were subjected to immunoblotting with anti-Sos antibody followed by sheep anti-mouse Ig-HRPO and ECL detection. This blot represents a reprobing of the membrane used in Fig.4.




DISCUSSION

Identification of Fyn/Lck SH3 Domain-binding Protein p120 as the c-Cbl Protooncogene Product

p120 was initially identi-fied as a T cell pY polypeptide that bound to the SH3 domains of the Src family tyrosine kinases Fyn and Lck in vitro and formed complexes with these proteins in T cells prior to activation(19) . Given its rapid tyrosine phosphorylation upon TCR stimulation(19) , biochemical identification of p120 was of significant interest. Several previously identified polypeptides represented candidates for p120. These include the Src substrate p110 and p125 tyrosine kinase which interact with Src and Fyn SH3 domains(47, 48) ; a Fyn-associated T cell phosphoprotein, p120/p130, which reacts with antibodies to Src substrate p130(49) ; and p120, recently shown to undergo TCR-dependent tyrosine phosphorylation and to bind to Grb2 fusion proteins in vitro(21) .

We employed a direct protein purification approach to determine the identity of p120. Two of the three peptides showed a complete match with the human p120 sequences(22, 41, 42) . The third peptide, whose sequence had some ambiguities, also corresponded to a Cbl sequence but with four mismatches out of 14. Although the reasons for mismatch (e.g. incorrect sequence, signals derived from a comigrating contaminant peptide, or an alternative mRNA transcript in HSB2 cells) are unknown, collectively the sequence data provided direct evidence for the identity of p120 with the c-cbl protooncogene product. Further support for this conclusion was provided by immunochemical cross-reactivity and demonstration of identical two-dimensional gel profiles of c-Cbl and p120. Given that only a single functional c-cbl gene is known to be present in the human genome(22, 41, 42) , these results establish conclusively the identity of the SH3 domain-binding p120 polypeptide (19) as the human c-cbl protooncogene product. Our direct biochemical analyses confirm and extend the recent finding that c-Cbl protein is tyrosine phosphorylated upon TCR stimulation (21) . Interestingly, c-Cbl was also identified by expression cloning with GST fusion proteins of Nck protein, which contains three SH3 domains(50) .

Interaction of p120 with Src Family Tyrosine Kinases in Vivo

Fyn-p120 interaction was demonstrated using parental Jurkat T cells as well as transfectants of an SV40 T antigen-expressing Jurkat line, which allowed easier detection of Fyn-p120 complexes due to higher Fyn expression (not shown). Interestingly, overexpression of Fyn led to an increase in basal tyrosine phosphorylation of p120 (data not shown), suggesting that p120 is an in vivo substrate for Src family tyrosine kinases. Consistent with this notion, p120 was also found to associate with Lck albeit at low levels (19) (and data not shown), and a marked hyperphosphorylation of p120 was seen in HSB2 cells that express a constitutively active Lck(40) . These data extend our previous results which showed that immunoprecipitated Fyn and Lck phosphorylated the associated p120 (19) and strongly suggest that p120 is an in vivo target for the TCR-coupled Src family tyrosine kinases. It is likely that SH3 domain binding recruits p120 as a substrate, analogous to SH3-mediated recruitment of ras-GTPase activating protein-associated p62 to Fyn, as demonstrated recently in a HeLa cell overexpression system(51) .

In Vivo Grb2-p120 Association Defines a Novel T Cell Signaling Complex Distinct from Grb2-Sos

Findings reported here demonstrate that p120 forms in vivo complexes with the adaptor protein Grb2 that is thought to play key signaling roles downstream of many cell surface receptors including the TCR(3, 9, 10, 43, 44, 52) . In vitro binding experiments with SH2 or SH3 domain mutants as well as peptide competition data demonstrated that p120 interacts with Grb2 only via its SH3 domains, leaving its SH2 domain available for interaction with other tyrosyl phosphoproteins, such as Shc(52) , p36/38 (43, 44) ( Fig.4and 5), and other unidentified polypeptides (e.g. p100 and p75) that are tyrosine phosphorylation upon T cell activation(20, 43, 53) (and Fig.4and Fig. 5). Thus, Grb2 is likely to tether p120 to a number of T cell signaling molecules. These complexes may serve to propagate signals originating from the receptor or alter the subcellular location of key signaling proteins upon T cell activation.

It is noteworthy that the only well characterized protein known to interact with Grb2 in T cells is the Ras guanine nucleotide exchanger Sos(43, 44, 52, 53) . The Ras pathway is essential for T cell activation(54, 55) , underscoring the importance of Grb2-Sos complexes. Our studies demonstrate that in T cells, Grb2 also forms a stable complex with a non-Sos protooncogene, p120, and that the Grb2-associated Cbl is an early target of TCR-coupled tyrosine kinases. These findings define a distinct Grb2-mediated signaling pathway downstream of the TCR. It is unlikely that Grb2 concurrently binds to Sos and p120, linking these proteins in series in a single signaling cascade. First, the structural requirements for Grb2 binding to p120 and Sos were identical, with an essential role of the N-terminal SH3 domain and a smaller contribution of the C-terminal SH3 domain(27, 36, 46) . Second, although both p120 and Sos were readily detectable in Grb2 immunoprecipitates, no Sos-p120 complexes could be detected ( Fig.4and Fig. 9). Since Grb2 is known to form Shc-mediated complexes with phosphorylated TCR , a Grb2-p120 complex may also interact with TCR , establishing a signaling pathway parallel to Grb2-Sos.

In Vivo Interaction of p120 with the PI 3-Kinase

Using both the parental and transfected Jurkat T cells (data not shown), we demonstrate in vivo association between p120 and PI 3-kinase p85. In addition, we demonstrate a substantial level of PI 3-kinase activity associated with p120. The PI 3-kinase-Cbl interaction was observed in unstimulated cells but showed a marked increase following TCR stimulation. Consistent with these results, in vitro binding and peptide competition experiments demonstrated that PI 3-kinase p85-Cbl interactions were primarily SH2-mediated.

Significantly, the amount of PI 3-kinase activity in anti-Cbl immunoprecipitates far exceeded that in anti-Fyn or anti-TCR immunoprecipitates. In addition, PI 3-kinase p85 protein was difficult to detect in association with Src family kinases (data not shown), whereas PI 3-kinase p85-Cbl interaction was readily demonstrable. These data lead us to suggest that p120 may play a prominent role in coupling the PI 3-kinase enzyme with the TCR signaling machinery. It is likely that other tyrosyl phosphoproteins found to associate with PI 3-kinase p85 (e.g. p36/38, p75, p100) also contribute to this pathway. The p120-mediated PI 3-kinase recruitment may complement other previously described mechanisms, such as the Fyn SH3 binding to proline-rich regions of the PI 3-kinase p85(35, 56) .

Potential Linkage of p120 to Ras Signaling Pathway

Whereas p120 and Sos represent alternate SH3 ligands for Grb2, given the well documented role of Ras in cell proliferation, and oncogenic activity of mutant forms of cbl and ras, it is intriguing to suggest that the Grb2-p120 complex may participate as a positive or negative modifier of the Ras pathway. Interestingly, Ras activation following the TCR stimulation is only partly mediated by an increase in guanine nucleotide exchange(57, 58) , suggesting that decreased GTPase activating protein activity or other mechanisms may exist. It is possible that tyrosine phosphorylation of p120 decreases its interaction with Grb2 allowing Grb2 to bind to Sos. Although T cell activation resulted in an increase in the amount of Sos associated with Grb2 (Fig.8), we did not notice any activation-dependent change in the amount of Grb2-associated p120. Alternatively, the ternary complex of Grb2, p120, and PI 3-kinase p85 may serve to bring p120 in the proximity of Ras through direct interaction of the PI 3-kinase with the effector domain of Ras, as has been demonstrated recently(59) .

Implications for Mechanisms of c-Cbl Function and Oncogenicity

p120 was identified by virtue of its homology to the v-cbl oncogene, which represented a C-terminally truncated protein and produced pre-B and myeloid leukemias in mice(22, 41) . Interestingly, v-Cbl sequences retained a putative nuclear localization signal and showed both nuclear and cytoplasmic localization; c-Cbl also possesses highly basic and acidic regions, a proline-rich region, and a putative leucine zipper, suggesting that c-Cbl may be a transcription factor(22, 41) . Demonstration of basal (phosphorylation-independent) and activation-induced (phosphorylation-dependent) in vivo complexes of p120 with cytoplasmic and membrane-anchored T cell signaling proteins suggests that Cbl is likely to function within the cytoplasm. Indeed, recent studies have demonstrated that Cbl derivatives with small deletions within the putative ring finger region are highly oncogenic yet entirely cytoplasmic(42) .

Given the widespread signaling roles of the proteins that we have shown to interact with Cbl, together with its interaction with another SH3 domain-containing adaptor protein Nck(50) , our findings suggest that p120 is likely to function in signal transduction downstream of TCR as well as other related receptors. Consistent with this suggestion, tyrosine phosphorylation of the Cbl protein was observed recently in HL60 myelomonocytic cell line upon triggering through Fc receptors(60) . Interestingly, v-cbl is known to induce pre-B and myeloid leukemias in mice, and human c-cbl on chromosome 11q23 is closely linked to breakpoints involved in translocations [t(4;11) or t(11;14)] found in B cell, myeloid, and T cell leukemias(24, 61) . Notably, we have observed p120 to be one of the earliest tyrosine phosphorylation substrates upon triggering through the B cell receptor and have detected Grb2-Cbl and PI 3-kinase p85-Cbl complexes in B cells.^3 Thus, it is likely that oncogenicity of the aberrant forms of cbl results from a constitutive activation of the signaling machinery in which it physiologically participates. Consistent with this suggestion, recent analyses have shown that oncogenic point mutants of p120 are constitutively tyrosine phosphorylated, and that Cbl protein interacts with the BCR-abl and v-abl oncogenes (42 and data not shown).

Demonstration of p120 as an SH3-binding protein strongly implicates its proline-rich region in binding to SH3 domains. Examination of the proline-rich regions of p120 reveals multiple potential binding motifs, including consensus sequences preferred by Src family and PI 3-kinase p85 SH3 domains (e.g. RPLPCTP (amino acids 563-569) and RPIPKVP (amino acids 593-599))(14, 15, 16, 17, 18) . Given the substantial length of this region and multiplicity of potential SH3-binding motifs, it will be of interest to determine whether SH3 domains of different signaling proteins bind the same or distinct sites. The latter would suggest the interesting possibility that p120 may concurrently tether multiple SH3 domain-containing signaling proteins.

In conclusion, we demonstrate that the Src family SH3 domain-binding protein p120 is identical to the c-cbl protooncogene product and forms in vivo complexes with the Fyn tyrosine kinase, Grb2 adaptor protein, and PI 3-kinase p85. These studies identify p120 as a multifunctional SH2 and SH3 domain-binding protein and strongly suggest signal transduction functions for this protooncogene with known oncogenic potential but with no previously known physiological function.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants R29-AI28508 and AR36308 and a Geyer Foundation grant (to H. B.) and by a National Science Foundation grant (to S. E. S.). 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.

§
The first two authors contributed equally to this work.

Predoctoral fellow of the Howard Hughes Medical Institute and an honorary fellow of the Ryan Foundation.

**
Fellow of the Charles A. King Trust/Medical Foundation of Boston.

§§
To whom correspondence should be addressed: Lymphocyte Biology Section, Dept. of Rheumatology and Immunology, Brigham and Women's Hospital, Harvard Medical School, Seeley G. Mudd Building, Rm. 514, 250 Longwood Ave., Boston, MA 02115. Tel.: 617-432-1557; Fax: 617-432-2799.

^1
The abbreviations used are: TCR, T cell receptor; pY, phosphotyrosyl; PI, phosphatidylinositol; HPLC, high performance liquid chromatography; mAb, monoclonal antibody; HRPO, horseradish peroxidase; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; IEF, isoelectric focusing; PVDF, polyvinylidene difluoride; ECL, enhanced chemiluminescence.

^2
T. Trub and S. E. Shoelson, manuscript in preparation.

^3
G. Panchamoorthy and H. Band, unpublished results.


ACKNOWLEDGEMENTS

We thank Drs. Paul Anderson, Vimla Band, J. Michael Bishop, Bruce Mayer, Mike Moran, Ellis Reinherz, Roger Perlmutter, Chris Rudd, Joseph Schlessinger, and Hergen Spits for critical reagents; Rob Littlefield for artwork; Bill Lane for peptide sequencing; and Mike Brenner for encouragement.


REFERENCES

  1. Weiss, A., and Littman, D. R. (1994) Cell 76,263-274 [Medline] [Order article via Infotrieve]
  2. Perlmutter, R. M., Levin, S. D., Appleby, M. W., Anderson, S. J., and Alberola-Ila, J. (1993) Ann. Rev. Immunol. 11,451-499 [CrossRef][Medline] [Order article via Infotrieve]
  3. Schlessinger, J. (1994) Curr. Opin. Genet. & Dev. 4,25-30 [Medline] [Order article via Infotrieve]
  4. Timson Gauen, L. K., Kong, A. N., Samelson, L. E., and Shaw, A. S. (1992) Mol. Cell. Biol. 12,5438-5446 [Abstract]
  5. Rudd, C. E., Trevillyan, J. M., Dasgupta, J. D., Wong, L. L., and Schlossman, S. F. (1988) Proc. Natl. Acad. Sci. U. S. A. 85,5190-5194 [Abstract]
  6. Veillette, A., Bookman, M. A., Horak, E. M., and Bolen, J. B. (1988) Cell 55,301-308 [Medline] [Order article via Infotrieve]
  7. Eiseman, E., and Bolen, J. B. (1992) Nature 355,78-80 [CrossRef][Medline] [Order article via Infotrieve]
  8. Kobayashi, N., Kono, T., Hatakeyama, M., Minami, Y., Miyazaki, T., Perlmutter, R. M., and Taniguchi, T. (1993) Proc. Natl. Acad. Sci. U. S. A. 90,4201-4205 [Abstract]
  9. Pawson, T., and Gish, G. D. (1992) Cell 71,359-362 [Medline] [Order article via Infotrieve]
  10. Downward, J. (1994) FEBS Lett. 338,113-117 [CrossRef][Medline] [Order article via Infotrieve]
  11. Songyang, Z., Shoelson, S. E., Chaudhuri, M., Gish, G., Pawson, T., Haser, W. G., King, F., Roberts, T., Ratnofsky, S., Lechleider, R. J., Neel, B. G., Birge, R. B., Fajardo, J. E., Chou, M. M., Hanafusa, H., Schaffhausen, B., and Cantley, L. C. (1993) Cell 72,767-778 [Medline] [Order article via Infotrieve]
  12. Songyang, Z., Shoelson, S. E., McGlade, J., Olivier, P., Pawson, T., Bustelo, X. R., Barbacid, M., Sabe, H., Hanafusa, H., Yi, T., Ren, R., Baltimore, D., Ratnofsky, S., Feldman, R. A., and Cantley, L. C. (1994) Mol. Cell. Biol. 14,2777-2785 [Abstract]
  13. Ren, R., Mayer, B. J., Cicchetti, P., and Baltimore, D. (1993) Science 259,1157-1161 [Medline] [Order article via Infotrieve]
  14. Lim, W. A., Richards, F. M., and Fox, R. O. (1994) Nature 372,375-379 [CrossRef][Medline] [Order article via Infotrieve]
  15. Feng, S. B., Chen, J. K., Yu, H. T., Simon, J. A., and Schreiber, S. L. (1994) Science 266,1241-1247 [Medline] [Order article via Infotrieve]
  16. Rickles, R. J., Botfield, M. C., Weng, Z. G., Taylor, J. A., Green, O. M., Brugge, J. S., and Zoller, M. J. (1994) EMBO J. 13,5598-5604 [Abstract]
  17. Cheadle, C., Ivashchenko, Y., South, V., Searfoss, G. H., French, S., Howk, R., Ricca, G. A., and Jaye, M. (1994) J. Biol. Chem. 269,24034-24039 [Abstract/Free Full Text]
  18. Sparks, A. B., Quilliam, L. A., Thorn, J. M., Der, C. J., and Kay, B. K. (1994) J. Biol. Chem. 269,23853-23856 [Abstract/Free Full Text]
  19. Reedquist, K. A., Fukazawa, T., Druker, B., Panchamoorthy, G., Shoelson, S. E., and Band, H. (1994) Proc. Natl. Acad. Sci. U. S. A. 91,4135-4139 [Abstract]
  20. Motto, D. G., Ross, S. E., Jackman, J. K., Sun, Q., Olson, A. L., Findell, P. R., and Koretzky, G. A. (1994) J. Biol. Chem. 269,21608-21613 [Abstract/Free Full Text]
  21. Donovan, J. A., Wange, R. L., Langdon, W. Y., and Samelson, L. E. (1994) J. Biol. Chem. 269,22921-22924 [Abstract/Free Full Text]
  22. Blake, T. J., Shapiro, M., Morse, H. C., and Langdon, W. Y. (1991) Oncogene 6,653-657 [Medline] [Order article via Infotrieve]
  23. Langdon, W. Y., Hyland, C. D., Grumont, R. J., and Morse, H. C. (1989) J. Virol. 63,5420-5424 [Medline] [Order article via Infotrieve]
  24. Langdon, W. Y., Hartley, J. W., Klinken, S. P., Ruscetti, S. K., and Morse, H. C. (1989) Proc. Natl. Acad. Sci. U. S. A. 86,1168-1172 [Abstract]
  25. Domchek, S. M., Auger, K. R., Chatterjee, S., Burke, T. R., Jr., and Shoelson, S. E. (1992) Biochemistry 31,9865-9870 [Medline] [Order article via Infotrieve]
  26. Skolnik, E. Y., Margolis, B., Mohammadi, M., Lowenstein, E., Fischer, R., Drepps, A., Ullrich, A., and Schlessinger, J. (1991) Cell 65,83-90 [Medline] [Order article via Infotrieve]
  27. Chardin, P., Camonis, J. H., Gale, N. W., van Aelst, L., Schlessinger, J., Wigler, M. H., and Bar-Sagi, D. (1993) Science 260,1338-1343 [Medline] [Order article via Infotrieve]
  28. Gout, I., Dhand, R., Hiles, I. D., Fry, M. J., Panayotou, G., Das, P., Truong, O., Totty, N. F., Hsuan, J., Booker, G. W., Campbell, I. D., and Waterfield, M. D. (1993) Cell 75,25-36 [Medline] [Order article via Infotrieve]
  29. Band, H., Hochstenbach, F., McLean, J., Hata, S., Krangel, M. S., and Brenner, M. B. (1987) Science 238,682-684 [Medline] [Order article via Infotrieve]
  30. Druker, B., Mamon, T., and Roberts, T. (1989) N. Engl. J. Med. 321,1383-1391 [Medline] [Order article via Infotrieve]
  31. Evan, G. I., Lewis, G. K., Ramsay, G., and Bishop, J. M. (1985) Mol. Cell. Biol. 5,3610-3616 [Medline] [Order article via Infotrieve]
  32. Panchamoorthy, G., Fukazawa, T., Stolz, L., Payne, G., Reedquist, K., Shoelson, S., Songyang, Z., Cantley, L., Walsh, C., and Band, H. (1994) Mol. Cell. Biol. 14,6372-6385 [Abstract]
  33. Maroney, A. C., Qureshi, S. A., Foster, D. A., and Brugge, J. S. (1992) Oncogene 7,1207-1214 [Medline] [Order article via Infotrieve]
  34. Suen, K. L., Bustelo, X. R., Pawson, T., and Barbacid, M. (1993) Mol. Cell. Biol. 13,5500-5512 [Abstract]
  35. Kapeller, R., Prasad, K. V. S., Janssen, O., Hou, W., Schaffhausen, B. S., Rudd, C. E., and Cantley, L. C. (1994) J. Biol. Chem. 269,1927-1933 [Abstract/Free Full Text]
  36. Egan, S. E., Giddings, B. W., Brooks, M. W., Buday, L., Sizeland, A. M., and Weinberg, R. A. (1993) Nature 363,45-51 [CrossRef][Medline] [Order article via Infotrieve]
  37. Jones, P. A. (1984) Methods Enzymol. 108,452-466 [Medline] [Order article via Infotrieve]
  38. Harlow, E., and Lane, D. (1988) Antibodies , pp. 521-523, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  39. Morrissey, J. H. (1981) Anal. Biochem. 117,307-310 [Medline] [Order article via Infotrieve]
  40. Wright, D. D., Sefton, B. M., and Kamps, M. P. (1994) Mol. Cell. Biol. 14,2429-2437 [Abstract]
  41. Blake, T. J., Heath, K. G., and Langdon, W. Y. (1993) EMBO J. 12,2017-2026 [Abstract]
  42. Andoniou, C. E., Thien, C. B. F., and Langdon, W. Y. (1994) EMBO J. 13,4515-4523 [Abstract]
  43. Buday, L., Egan, S. E., Rodriguez Viciana, P., Cantrell, D. A., and Downward, J. (1994) J. Biol. Chem. 269,9019-9023 [Abstract/Free Full Text]
  44. Sieh, M., Batzer, A., Schlessinger, J., and Weiss, A. (1994) Mol. Cell. Biol. 14,4435-4442 [Abstract]
  45. Jackman, J. K., Motto, D. G., Sun, Q. M., Tanemoto, M., Turck, C. W., Pelz, G. A., Koretzky, G. A., and Findell, P. R. (1995) J. Biol. Chem. 270,7029-7032 [Abstract/Free Full Text]
  46. Rozakis-Adcock, M., Fernley, R., Wade, J., Pawson, T., and Bowtell, D. (1993) Nature 363,83-85 [CrossRef][Medline] [Order article via Infotrieve]
  47. Flynn, D. C., Leu, T.-H., Reynolds, A. B., and Parsons, J. T. (1993) Mol. Cell. Biol. 13,7892-7898 [Abstract]
  48. Cobb, B. S., Schaller, M. D., Leu, T. H., and Parsons, J. T. (1994) Mol. Cell. Biol. 14,147-155 [Abstract]
  49. Dasilva, A. J., Janssen, O., and Rudd, C. E. (1993) J. Exp. Med. 178,2107-2113 [Abstract]
  50. Rivero-Lezcano, O. M., Sameshima, J. H., Marcilla, A., and Robbins, K. C. (1994) J. Biol. Chem. 269,17363-17366 [Abstract/Free Full Text]
  51. Richard, S., Yu, D., Blumer, K. J., Hausladen, D., Olszowy, M. W., Connelly, P. A., and Shaw, A. S. (1995) Mol. Cell. Biol. 15,186-197 [Abstract]
  52. Ravichandran, K. S., Lee, K. K., Songyang, Z., Cantley, L. C., Burn, P., and Burakoff, S. J. (1993) Science 262,902-905 [Medline] [Order article via Infotrieve]
  53. Reif, K., Buday, L., Downward, J., and Cantrell, D. A. (1994) J. Biol. Chem. 269,14081-14087 [Abstract/Free Full Text]
  54. Izquierdo, M., Leevers, S. J., Marshall, C. J., and Cantrell, D. (1993) J. Exp. Med. 178,1199-1208 [Abstract]
  55. Woodrow, M., Clipstone, N. A., and Cantrell, D. (1993) J. Exp. Med. 178,1517-1522 [Abstract]
  56. Prasad, K. V., Janssen, O., Kapeller, R., Raab, M., Cantley, L. C., and Rudd, C. E. (1993) Proc. Natl. Acad. Sci. U. S. A. 90,7366-7370 [Abstract]
  57. Izquierdo, M., Downward, J., Graves, J. D., and Cantrell, D. A. (1992) Mol. Cell. Biol. 12,3305-3312 [Abstract]
  58. Downward, J., Graves, J. D., Warne, P. H., Rayter, S., and Cantrell, D. A. (1990) Nature 346,719-723 [CrossRef][Medline] [Order article via Infotrieve]
  59. Rodriguez-Viciana, P., Warne, P. H., Dhand, R., Vanhaesebroeck, B., Gout, I., Fry, M. J., Waterfield, M. D., and Downward, J. (1994) Nature 370,527-532 [CrossRef][Medline] [Order article via Infotrieve]
  60. Marcilla, A., Rivero-Lezcano, O. M., Agarwal, A., and Robbins, K. C. (1995) J. Biol. Chem. 270,9115-9120 [Abstract/Free Full Text]
  61. Savage, P. D., Shapiro, M., Langdon, W. Y., Geurts van Kessel, A. D., Seuanez, H. N., Akao, Y., Croce, C., Morse, H. C., and Kersey, J. H. (1991) Cytogenet. Cell Genet. 56,112-115 [Medline] [Order article via Infotrieve]

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