Advertisement

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 *
  • Toru Fukazawa
    Footnotes
    Affiliations
    From the Lymphocyte Biology Section, Department of Rheumatology and Immunology, Brigham and Women's Hospital Harvard Medical School, Boston, Massachusetts 02115
    Search for articles by this author
  • Kris A. Reedquist
    Footnotes
    Affiliations
    From the Lymphocyte Biology Section, Department of Rheumatology and Immunology, Brigham and Women's Hospital Harvard Medical School, Boston, Massachusetts 02115
    Search for articles by this author
  • Thomas Trub
    Affiliations
    Research Division, Joslin Diabetes Center, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115
    Search for articles by this author
  • Stephen Soltoff
    Affiliations
    Division of Signal Transduction, Beth Israel Hospital, and Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115
    Search for articles by this author
  • Govindaswamy Panchamoorthy
    Footnotes
    Affiliations
    From the Lymphocyte Biology Section, Department of Rheumatology and Immunology, Brigham and Women's Hospital Harvard Medical School, Boston, Massachusetts 02115
    Search for articles by this author
  • Brian Druker
    Affiliations
    Division of Hematology and Medical Oncology, Oregon Health Sciences University, Portland, Oregon 97201
    Search for articles by this author
  • Lewis Cantley
    Affiliations
    Division of Signal Transduction, Beth Israel Hospital, and Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115
    Search for articles by this author
  • Steven E. Shoelson
    Affiliations
    Research Division, Joslin Diabetes Center, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115
    Search for articles by this author
  • Hamid Band
    Correspondence
    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
    Affiliations
    From the Lymphocyte Biology Section, Department of Rheumatology and Immunology, Brigham and Women's Hospital Harvard Medical School, Boston, Massachusetts 02115
    Search for articles by this author
  • Author 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.
Open AccessPublished:August 11, 1995DOI:https://doi.org/10.1074/jbc.270.32.19141
      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 p120cbl with Fyn tyrosine kinase, the adaptor protein Grb2, and the p85 subunit of phosphatidylinositol (PI) 3-kinase. The association of p120cbl 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 p120cbl to their SH2 as well as SH3 domains. In contrast, a large fraction of p120cbl was associated with Grb2 prior to activation, and this association did not change upon T cell activation. In vitro, p120cblinteracted with Grb2 exclusively through its SH3 domains. These results demonstrate a novel Grb2-p120cbl signaling complex in T cells, distinct from the previously analyzed Grb2-Sos complex. The association of p120cbl with ubiquitous signaling proteins strongly suggests a general signal transducing function for this enigmatic protooncogene with established leukemogenic potential but unknown physiological function.
      The engagement of the T cell receptor (TCR)
      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.
      1The 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.
      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(
      • Weiss A.
      • Littman D.R.
      ,
      • Perlmutter R.M.
      • Levin S.D.
      • Appleby M.W.
      • Anderson S.J.
      • Alberola-Ila J.
      ). Unlike growth factor receptors with intrinsic tyrosine kinase domains(
      • Schlessinger J.
      ), the TCR components signal through noncovalently associated cytoplasmic tyrosine kinases(
      • Weiss A.
      • Littman D.R.
      ,
      • Perlmutter R.M.
      • Levin S.D.
      • Appleby M.W.
      • Anderson S.J.
      • Alberola-Ila J.
      ). In particular two Src family kinases, p59fyn (Fyn) and p56lck (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 ζ/η and CD3 γ and ϵ chains(
      • Timson Gauen L.K.
      • Kong A.N.
      • Samelson L.E.
      • Shaw A.S.
      ), whereas Lck interacts with the cytoplasmic tails of the CD4 and CD8 coreceptors(
      • Rudd C.E.
      • Trevillyan J.M.
      • Dasgupta J.D.
      • Wong L.L.
      • Schlossman S.F.
      ,
      • Veillette A.
      • Bookman M.A.
      • Horak E.M.
      • Bolen J.B.
      ). In addition, Lck plays a role in T cell activation apparently independent of its CD4/8 association(
      • Weiss A.
      • Littman D.R.
      ,
      • Perlmutter R.M.
      • Levin S.D.
      • Appleby M.W.
      • Anderson S.J.
      • Alberola-Ila J.
      ). 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(
      • Weiss A.
      • Littman D.R.
      ).
      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(
      • Weiss A.
      • Littman D.R.
      ), Fc receptors(
      • Eiseman E.
      • Bolen J.B.
      ), and certain cytokine receptors(
      • Kobayashi N.
      • Kono T.
      • Hatakeyama M.
      • Minami Y.
      • Miyazaki T.
      • Perlmutter R.M.
      • Taniguchi T.
      ). 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(
      • Schlessinger J.
      ,
      • Pawson T.
      • Gish G.D.
      ,
      • Downward J.
      ). The SH2 domains bind to phosphotyrosyl (pY) peptide motifs (
      • 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.
      • Cantley L.C.
      ,
      • 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.
      • Cantley L.C.
      ) and mediate activation-induced phosphorylation-dependent interactions between signaling proteins(
      • Schlessinger J.
      ,
      • Pawson T.
      • Gish G.D.
      ,
      • Downward J.
      ). In contrast, the SH3 domains bind to small proline-rich peptide motifs(
      • Ren R.
      • Mayer B.J.
      • Cicchetti P.
      • Baltimore D.
      ,
      • Lim W.A.
      • Richards F.M.
      • Fox R.O.
      ,
      • Feng S.B.
      • Chen J.K.
      • Yu H.T.
      • Simon J.A.
      • Schreiber S.L.
      ,
      • Rickles R.J.
      • Botfield M.C.
      • Weng Z.G.
      • Taylor J.A.
      • Green O.M.
      • Brugge J.S.
      • Zoller M.J.
      ,
      • Cheadle C.
      • Ivashchenko Y.
      • South V.
      • Searfoss G.H.
      • French S.
      • Howk R.
      • Ricca G.A.
      • Jaye M.
      ,
      • Sparks A.B.
      • Quilliam L.A.
      • Thorn J.M.
      • Der C.J.
      • Kay B.K.
      ), 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(
      • Reedquist K.A.
      • Fukazawa T.
      • Druker B.
      • Panchamoorthy G.
      • Shoelson S.E.
      • Band H.
      ). 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(
      • Reedquist K.A.
      • Fukazawa T.
      • Druker B.
      • Panchamoorthy G.
      • Shoelson S.E.
      • Band H.
      ). 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 (
      • Motto D.G.
      • Ross S.E.
      • Jackman J.K.
      • Sun Q.
      • Olson A.L.
      • Findell P.R.
      • Koretzky G.A.
      ). 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(
      • Donovan J.A.
      • Wange R.L.
      • Langdon W.Y.
      • Samelson L.E.
      ).
      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(
      • Motto D.G.
      • Ross S.E.
      • Jackman J.K.
      • Sun Q.
      • Olson A.L.
      • Findell P.R.
      • Koretzky G.A.
      ,
      • Donovan J.A.
      • Wange R.L.
      • Langdon W.Y.
      • Samelson L.E.
      ), 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 p120cbl protooncogene(
      • Blake T.J.
      • Shapiro M.
      • Morse H.C.
      • Langdon W.Y.
      ), 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(
      • Donovan J.A.
      • Wange R.L.
      • Langdon W.Y.
      • Samelson L.E.
      ). Together, these results strongly suggest that p120cbl 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(
      • Langdon W.Y.
      • Hyland C.D.
      • Grumont R.J.
      • Morse H.C.
      ), induction of pre-B and myeloid cell leukemias by its viral form(
      • Langdon W.Y.
      • Hartley J.W.
      • Klinken S.P.
      • Ruscetti S.K.
      • Morse H.C.
      ), 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.

      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(
      • Reedquist K.A.
      • Fukazawa T.
      • Druker B.
      • Panchamoorthy G.
      • Shoelson S.E.
      • Band H.
      ). Given its rapid tyrosine phosphorylation upon TCR stimulation(
      • Reedquist K.A.
      • Fukazawa T.
      • Druker B.
      • Panchamoorthy G.
      • Shoelson S.E.
      • Band H.
      ), biochemical identification of p120 was of significant interest. Several previously identified polypeptides represented candidates for p120. These include the Src substrate p110 and p125FAK tyrosine kinase which interact with Src and Fyn SH3 domains(
      • Flynn D.C.
      • Leu T.-H.
      • Reynolds A.B.
      • Parsons J.T.
      ,
      • Cobb B.S.
      • Schaller M.D.
      • Leu T.H.
      • Parsons J.T.
      ); a Fyn-associated T cell phosphoprotein, p120/p130, which reacts with antibodies to Src substrate p130(
      • Dasilva A.J.
      • Janssen O.
      • Rudd C.E.
      ); and p120cbl, recently shown to undergo TCR-dependent tyrosine phosphorylation and to bind to Grb2 fusion proteins in vitro(
      • Donovan J.A.
      • Wange R.L.
      • Langdon W.Y.
      • Samelson L.E.
      ).
      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 p120cbl sequences(
      • Blake T.J.
      • Shapiro M.
      • Morse H.C.
      • Langdon W.Y.
      ,
      • Blake T.J.
      • Heath K.G.
      • Langdon W.Y.
      ,
      • Andoniou C.E.
      • Thien C.B.F.
      • Langdon W.Y.
      ). 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(
      • Blake T.J.
      • Shapiro M.
      • Morse H.C.
      • Langdon W.Y.
      ,
      • Blake T.J.
      • Heath K.G.
      • Langdon W.Y.
      ,
      • Andoniou C.E.
      • Thien C.B.F.
      • Langdon W.Y.
      ), these results establish conclusively the identity of the SH3 domain-binding p120 polypeptide (
      • Reedquist K.A.
      • Fukazawa T.
      • Druker B.
      • Panchamoorthy G.
      • Shoelson S.E.
      • Band H.
      ) 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 (
      • Donovan J.A.
      • Wange R.L.
      • Langdon W.Y.
      • Samelson L.E.
      ). Interestingly, c-Cbl was also identified by expression cloning with GST fusion proteins of Nck protein, which contains three SH3 domains(
      • Rivero-Lezcano O.M.
      • Sameshima J.H.
      • Marcilla A.
      • Robbins K.C.
      ).

      Interaction of p120cbl with Src Family Tyrosine Kinases in Vivo

      Fyn-p120cbl 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-p120cbl complexes due to higher Fyn expression (not shown). Interestingly, overexpression of Fyn led to an increase in basal tyrosine phosphorylation of p120cbl (data not shown), suggesting that p120cbl is an in vivo substrate for Src family tyrosine kinases. Consistent with this notion, p120cbl was also found to associate with Lck albeit at low levels (
      • Reedquist K.A.
      • Fukazawa T.
      • Druker B.
      • Panchamoorthy G.
      • Shoelson S.E.
      • Band H.
      ) (and data not shown), and a marked hyperphosphorylation of p120cbl was seen in HSB2 cells that express a constitutively active Lck(
      • Wright D.D.
      • Sefton B.M.
      • Kamps M.P.
      ). These data extend our previous results which showed that immunoprecipitated Fyn and Lck phosphorylated the associated p120 (
      • Reedquist K.A.
      • Fukazawa T.
      • Druker B.
      • Panchamoorthy G.
      • Shoelson S.E.
      • Band H.
      ) and strongly suggest that p120cbl is an in vivo target for the TCR-coupled Src family tyrosine kinases. It is likely that SH3 domain binding recruits p120cbl 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(
      • Richard S.
      • Yu D.
      • Blumer K.J.
      • Hausladen D.
      • Olszowy M.W.
      • Connelly P.A.
      • Shaw A.S.
      ).

      In Vivo Grb2-p120cbl Association Defines a Novel T Cell Signaling Complex Distinct from Grb2-Sos

      Findings reported here demonstrate that p120cbl 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(
      • Schlessinger J.
      ,
      • Pawson T.
      • Gish G.D.
      ,
      • Downward J.
      ,
      • Buday L.
      • Egan S.E.
      • Rodriguez Viciana P.
      • Cantrell D.A.
      • Downward J.
      ,
      • Sieh M.
      • Batzer A.
      • Schlessinger J.
      • Weiss A.
      ,
      • Ravichandran K.S.
      • Lee K.K.
      • Songyang Z.
      • Cantley L.C.
      • Burn P.
      • Burakoff S.J.
      ). In vitro binding experiments with SH2 or SH3 domain mutants as well as peptide competition data demonstrated that p120cbl interacts with Grb2 only via its SH3 domains, leaving its SH2 domain available for interaction with other tyrosyl phosphoproteins, such as Shc(
      • Ravichandran K.S.
      • Lee K.K.
      • Songyang Z.
      • Cantley L.C.
      • Burn P.
      • Burakoff S.J.
      ), p36/38 (
      • Buday L.
      • Egan S.E.
      • Rodriguez Viciana P.
      • Cantrell D.A.
      • Downward J.
      ,
      • Sieh M.
      • Batzer A.
      • Schlessinger J.
      • Weiss A.
      ) (Fig. 4 and 5), and other unidentified polypeptides (e.g. p100 and p75) that are tyrosine phosphorylation upon T cell activation(
      • Motto D.G.
      • Ross S.E.
      • Jackman J.K.
      • Sun Q.
      • Olson A.L.
      • Findell P.R.
      • Koretzky G.A.
      ,
      • Buday L.
      • Egan S.E.
      • Rodriguez Viciana P.
      • Cantrell D.A.
      • Downward J.
      ,
      • Reif K.
      • Buday L.
      • Downward J.
      • Cantrell D.A.
      ) (and Figure 4:, Figure 5:). Thus, Grb2 is likely to tether p120cbl 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(
      • Buday L.
      • Egan S.E.
      • Rodriguez Viciana P.
      • Cantrell D.A.
      • Downward J.
      ,
      • Sieh M.
      • Batzer A.
      • Schlessinger J.
      • Weiss A.
      ,
      • Ravichandran K.S.
      • Lee K.K.
      • Songyang Z.
      • Cantley L.C.
      • Burn P.
      • Burakoff S.J.
      ,
      • Reif K.
      • Buday L.
      • Downward J.
      • Cantrell D.A.
      ). The Ras pathway is essential for T cell activation(
      • Izquierdo M.
      • Leevers S.J.
      • Marshall C.J.
      • Cantrell D.
      ,
      • Woodrow M.
      • Clipstone N.A.
      • Cantrell D.
      ), 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, p120cbl, 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 p120cbl, linking these proteins in series in a single signaling cascade. First, the structural requirements for Grb2 binding to p120cbl and Sos were identical, with an essential role of the N-terminal SH3 domain and a smaller contribution of the C-terminal SH3 domain(
      • Chardin P.
      • Camonis J.H.
      • Gale N.W.
      • van Aelst L.
      • Schlessinger J.
      • Wigler M.H.
      • Bar-Sagi D.
      ,
      • Egan S.E.
      • Giddings B.W.
      • Brooks M.W.
      • Buday L.
      • Sizeland A.M.
      • Weinberg R.A.
      ,
      • Rozakis-Adcock M.
      • Fernley R.
      • Wade J.
      • Pawson T.
      • Bowtell D.
      ). Second, although both p120cbl and Sos were readily detectable in Grb2 immunoprecipitates, no Sos-p120cbl complexes could be detected (Figure 4:, Figure 5:, Figure 6:, Figure 7:, Figure 8:, Figure 9:). Since Grb2 is known to form Shc-mediated complexes with phosphorylated TCR ζ, a Grb2-p120cbl complex may also interact with TCR ζ, establishing a signaling pathway parallel to Grb2-Sos.

      In Vivo Interaction of p120cbl with the PI 3-Kinase

      Using both the parental and transfected Jurkat T cells (data not shown), we demonstrate in vivo association between p120cbl and PI 3-kinase p85. In addition, we demonstrate a substantial level of PI 3-kinase activity associated with p120cbl. 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 ζ 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 p120cbl 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 p120cbl-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(
      • Kapeller R.
      • Prasad K.V.S.
      • Janssen O.
      • Hou W.
      • Schaffhausen B.S.
      • Rudd C.E.
      • Cantley L.C.
      ,
      • Prasad K.V.
      • Janssen O.
      • Kapeller R.
      • Raab M.
      • Cantley L.C.
      • Rudd C.E.
      ).

      Potential Linkage of p120cbl to Ras Signaling Pathway

      Whereas p120cbl 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-p120cbl 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(
      • Izquierdo M.
      • Downward J.
      • Graves J.D.
      • Cantrell D.A.
      ,
      • Downward J.
      • Graves J.D.
      • Warne P.H.
      • Rayter S.
      • Cantrell D.A.
      ), suggesting that decreased GTPase activating protein activity or other mechanisms may exist. It is possible that tyrosine phosphorylation of p120cbl 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 p120cbl. Alternatively, the ternary complex of Grb2, p120cbl, and PI 3-kinase p85 may serve to bring p120cbl in the proximity of Ras through direct interaction of the PI 3-kinase with the effector domain of Ras, as has been demonstrated recently(
      • Rodriguez-Viciana P.
      • Warne P.H.
      • Dhand R.
      • Vanhaesebroeck B.
      • Gout I.
      • Fry M.J.
      • Waterfield M.D.
      • Downward J.
      ).

      Implications for Mechanisms of c-Cbl Function and Oncogenicity

      p120cbl 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(
      • Blake T.J.
      • Shapiro M.
      • Morse H.C.
      • Langdon W.Y.
      ,
      • Blake T.J.
      • Heath K.G.
      • Langdon W.Y.
      ). 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(
      • Blake T.J.
      • Shapiro M.
      • Morse H.C.
      • Langdon W.Y.
      ,
      • Blake T.J.
      • Heath K.G.
      • Langdon W.Y.
      ). Demonstration of basal (phosphorylation-independent) and activation-induced (phosphorylation-dependent) in vivo complexes of p120cbl 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(
      • Andoniou C.E.
      • Thien C.B.F.
      • Langdon W.Y.
      ).
      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(
      • Rivero-Lezcano O.M.
      • Sameshima J.H.
      • Marcilla A.
      • Robbins K.C.
      ), our findings suggest that p120cbl 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 Fcγ receptors(
      • Marcilla A.
      • Rivero-Lezcano O.M.
      • Agarwal A.
      • Robbins K.C.
      ). 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(
      • Langdon W.Y.
      • Hartley J.W.
      • Klinken S.P.
      • Ruscetti S.K.
      • Morse H.C.
      ,
      • Savage P.D.
      • Shapiro M.
      • Langdon W.Y.
      • Geurts van Kessel A.D.
      • Seuanez H.N.
      • Akao Y.
      • Croce C.
      • Morse H.C.
      • Kersey J.H.
      ). Notably, we have observed p120cbl 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.
      G. Panchamoorthy and H. Band, unpublished results.
      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 p120cbl are constitutively tyrosine phosphorylated, and that Cbl protein interacts with the BCR-abl and v-abl oncogenes (42 and data not shown).
      Demonstration of p120cbl as an SH3-binding protein strongly implicates its proline-rich region in binding to SH3 domains. Examination of the proline-rich regions of p120cbl 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))(
      • Lim W.A.
      • Richards F.M.
      • Fox R.O.
      ,
      • Feng S.B.
      • Chen J.K.
      • Yu H.T.
      • Simon J.A.
      • Schreiber S.L.
      ,
      • Rickles R.J.
      • Botfield M.C.
      • Weng Z.G.
      • Taylor J.A.
      • Green O.M.
      • Brugge J.S.
      • Zoller M.J.
      ,
      • Cheadle C.
      • Ivashchenko Y.
      • South V.
      • Searfoss G.H.
      • French S.
      • Howk R.
      • Ricca G.A.
      • Jaye M.
      ,
      • Sparks A.B.
      • Quilliam L.A.
      • Thorn J.M.
      • Der C.J.
      • Kay B.K.
      ). 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 p120cbl 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 p120cbl 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.

      Acknowledgments

      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

        • Weiss A.
        • Littman D.R.
        Cell. 1994; 76: 263-274
        • Perlmutter R.M.
        • Levin S.D.
        • Appleby M.W.
        • Anderson S.J.
        • Alberola-Ila J.
        Ann. Rev. Immunol. 1993; 11: 451-499
        • Schlessinger J.
        Curr. Opin. Genet. & Dev. 1994; 4: 25-30
        • Timson Gauen L.K.
        • Kong A.N.
        • Samelson L.E.
        • Shaw A.S.
        Mol. Cell. Biol. 1992; 12: 5438-5446
        • Rudd C.E.
        • Trevillyan J.M.
        • Dasgupta J.D.
        • Wong L.L.
        • Schlossman S.F.
        Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 5190-5194
        • Veillette A.
        • Bookman M.A.
        • Horak E.M.
        • Bolen J.B.
        Cell. 1988; 55: 301-308
        • Eiseman E.
        • Bolen J.B.
        Nature. 1992; 355: 78-80
        • Kobayashi N.
        • Kono T.
        • Hatakeyama M.
        • Minami Y.
        • Miyazaki T.
        • Perlmutter R.M.
        • Taniguchi T.
        Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4201-4205
        • Pawson T.
        • Gish G.D.
        Cell. 1992; 71: 359-362
        • Downward J.
        FEBS Lett. 1994; 338: 113-117
        • 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.
        • Cantley L.C.
        Cell. 1993; 72: 767-778
        • 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.
        • Cantley L.C.
        Mol. Cell. Biol. 1994; 14: 2777-2785
        • Ren R.
        • Mayer B.J.
        • Cicchetti P.
        • Baltimore D.
        Science. 1993; 259: 1157-1161
        • Lim W.A.
        • Richards F.M.
        • Fox R.O.
        Nature. 1994; 372: 375-379
        • Feng S.B.
        • Chen J.K.
        • Yu H.T.
        • Simon J.A.
        • Schreiber S.L.
        Science. 1994; 266: 1241-1247
        • Rickles R.J.
        • Botfield M.C.
        • Weng Z.G.
        • Taylor J.A.
        • Green O.M.
        • Brugge J.S.
        • Zoller M.J.
        EMBO J. 1994; 13: 5598-5604
        • Cheadle C.
        • Ivashchenko Y.
        • South V.
        • Searfoss G.H.
        • French S.
        • Howk R.
        • Ricca G.A.
        • Jaye M.
        J. Biol. Chem. 1994; 269: 24034-24039
        • Sparks A.B.
        • Quilliam L.A.
        • Thorn J.M.
        • Der C.J.
        • Kay B.K.
        J. Biol. Chem. 1994; 269: 23853-23856
        • Reedquist K.A.
        • Fukazawa T.
        • Druker B.
        • Panchamoorthy G.
        • Shoelson S.E.
        • Band H.
        Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4135-4139
        • Motto D.G.
        • Ross S.E.
        • Jackman J.K.
        • Sun Q.
        • Olson A.L.
        • Findell P.R.
        • Koretzky G.A.
        J. Biol. Chem. 1994; 269: 21608-21613
        • Donovan J.A.
        • Wange R.L.
        • Langdon W.Y.
        • Samelson L.E.
        J. Biol. Chem. 1994; 269: 22921-22924
        • Blake T.J.
        • Shapiro M.
        • Morse H.C.
        • Langdon W.Y.
        Oncogene. 1991; 6: 653-657
        • Langdon W.Y.
        • Hyland C.D.
        • Grumont R.J.
        • Morse H.C.
        J. Virol. 1989; 63: 5420-5424
        • Langdon W.Y.
        • Hartley J.W.
        • Klinken S.P.
        • Ruscetti S.K.
        • Morse H.C.
        Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 1168-1172
        • Domchek S.M.
        • Auger K.R.
        • Chatterjee S.
        • Burke Jr., T.R.
        • Shoelson S.E.
        Biochemistry. 1992; 31: 9865-9870
        • Skolnik E.Y.
        • Margolis B.
        • Mohammadi M.
        • Lowenstein E.
        • Fischer R.
        • Drepps A.
        • Ullrich A.
        • Schlessinger J.
        Cell. 1991; 65: 83-90
        • Chardin P.
        • Camonis J.H.
        • Gale N.W.
        • van Aelst L.
        • Schlessinger J.
        • Wigler M.H.
        • Bar-Sagi D.
        Science. 1993; 260: 1338-1343
        • 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.
        • Waterfield M.D.
        Cell. 1993; 75: 25-36
        • Band H.
        • Hochstenbach F.
        • McLean J.
        • Hata S.
        • Krangel M.S.
        • Brenner M.B.
        Science. 1987; 238: 682-684
        • Druker B.
        • Mamon T.
        • Roberts T.
        N. Engl. J. Med. 1989; 321: 1383-1391
        • Evan G.I.
        • Lewis G.K.
        • Ramsay G.
        • Bishop J.M.
        Mol. Cell. Biol. 1985; 5: 3610-3616
        • Panchamoorthy G.
        • Fukazawa T.
        • Stolz L.
        • Payne G.
        • Reedquist K.
        • Shoelson S.
        • Songyang Z.
        • Cantley L.
        • Walsh C.
        • Band H.
        Mol. Cell. Biol. 1994; 14: 6372-6385
        • Maroney A.C.
        • Qureshi S.A.
        • Foster D.A.
        • Brugge J.S.
        Oncogene. 1992; 7: 1207-1214
        • Suen K.L.
        • Bustelo X.R.
        • Pawson T.
        • Barbacid M.
        Mol. Cell. Biol. 1993; 13: 5500-5512
        • Kapeller R.
        • Prasad K.V.S.
        • Janssen O.
        • Hou W.
        • Schaffhausen B.S.
        • Rudd C.E.
        • Cantley L.C.
        J. Biol. Chem. 1994; 269: 1927-1933
        • Egan S.E.
        • Giddings B.W.
        • Brooks M.W.
        • Buday L.
        • Sizeland A.M.
        • Weinberg R.A.
        Nature. 1993; 363: 45-51
        • Jones P.A.
        Methods Enzymol. 1984; 108: 452-466
        • Harlow E.
        • Lane D.
        Antibodies. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1988: 521-523
        • Morrissey J.H.
        Anal. Biochem. 1981; 117: 307-310
        • Wright D.D.
        • Sefton B.M.
        • Kamps M.P.
        Mol. Cell. Biol. 1994; 14: 2429-2437
        • Blake T.J.
        • Heath K.G.
        • Langdon W.Y.
        EMBO J. 1993; 12: 2017-2026
        • Andoniou C.E.
        • Thien C.B.F.
        • Langdon W.Y.
        EMBO J. 1994; 13: 4515-4523
        • Buday L.
        • Egan S.E.
        • Rodriguez Viciana P.
        • Cantrell D.A.
        • Downward J.
        J. Biol. Chem. 1994; 269: 9019-9023
        • Sieh M.
        • Batzer A.
        • Schlessinger J.
        • Weiss A.
        Mol. Cell. Biol. 1994; 14: 4435-4442
        • Jackman J.K.
        • Motto D.G.
        • Sun Q.M.
        • Tanemoto M.
        • Turck C.W.
        • Pelz G.A.
        • Koretzky G.A.
        • Findell P.R.
        J. Biol. Chem. 1995; 270: 7029-7032
        • Rozakis-Adcock M.
        • Fernley R.
        • Wade J.
        • Pawson T.
        • Bowtell D.
        Nature. 1993; 363: 83-85
        • Flynn D.C.
        • Leu T.-H.
        • Reynolds A.B.
        • Parsons J.T.
        Mol. Cell. Biol. 1993; 13: 7892-7898
        • Cobb B.S.
        • Schaller M.D.
        • Leu T.H.
        • Parsons J.T.
        Mol. Cell. Biol. 1994; 14: 147-155
        • Dasilva A.J.
        • Janssen O.
        • Rudd C.E.
        J. Exp. Med. 1993; 178: 2107-2113
        • Rivero-Lezcano O.M.
        • Sameshima J.H.
        • Marcilla A.
        • Robbins K.C.
        J. Biol. Chem. 1994; 269: 17363-17366
        • Richard S.
        • Yu D.
        • Blumer K.J.
        • Hausladen D.
        • Olszowy M.W.
        • Connelly P.A.
        • Shaw A.S.
        Mol. Cell. Biol. 1995; 15: 186-197
        • Ravichandran K.S.
        • Lee K.K.
        • Songyang Z.
        • Cantley L.C.
        • Burn P.
        • Burakoff S.J.
        Science. 1993; 262: 902-905
        • Reif K.
        • Buday L.
        • Downward J.
        • Cantrell D.A.
        J. Biol. Chem. 1994; 269: 14081-14087
        • Izquierdo M.
        • Leevers S.J.
        • Marshall C.J.
        • Cantrell D.
        J. Exp. Med. 1993; 178: 1199-1208
        • Woodrow M.
        • Clipstone N.A.
        • Cantrell D.
        J. Exp. Med. 1993; 178: 1517-1522
        • Prasad K.V.
        • Janssen O.
        • Kapeller R.
        • Raab M.
        • Cantley L.C.
        • Rudd C.E.
        Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7366-7370
        • Izquierdo M.
        • Downward J.
        • Graves J.D.
        • Cantrell D.A.
        Mol. Cell. Biol. 1992; 12: 3305-3312
        • Downward J.
        • Graves J.D.
        • Warne P.H.
        • Rayter S.
        • Cantrell D.A.
        Nature. 1990; 346: 719-723
        • Rodriguez-Viciana P.
        • Warne P.H.
        • Dhand R.
        • Vanhaesebroeck B.
        • Gout I.
        • Fry M.J.
        • Waterfield M.D.
        • Downward J.
        Nature. 1994; 370: 527-532
        • Marcilla A.
        • Rivero-Lezcano O.M.
        • Agarwal A.
        • Robbins K.C.
        J. Biol. Chem. 1995; 270: 9115-9120
        • Savage P.D.
        • Shapiro M.
        • Langdon W.Y.
        • Geurts van Kessel A.D.
        • Seuanez H.N.
        • Akao Y.
        • Croce C.
        • Morse H.C.
        • Kersey J.H.
        Cytogenet. Cell Genet. 1991; 56: 112-115