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Modification of Phosphatidylinositol 3-Kinase SH2 Domain Binding Properties by Abl- or Lck-mediated Tyrosine Phosphorylation at Tyr-688*

  • Maria von Willebrand
    Affiliations
    Divisions of Cell Biology and Cellular Immunology, La Jolla Institute for Allergy and Immunology, San Diego, California 92121
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  • Scott Williams
    Affiliations
    Divisions of Cell Biology and Cellular Immunology, La Jolla Institute for Allergy and Immunology, San Diego, California 92121
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  • Manju Saxena
    Affiliations
    Divisions of Cell Biology and Cellular Immunology, La Jolla Institute for Allergy and Immunology, San Diego, California 92121
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  • Jennifer Gilman
    Affiliations
    Divisions of Cell Biology and Cellular Immunology, La Jolla Institute for Allergy and Immunology, San Diego, California 92121
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  • Pankaj Tailor
    Affiliations
    Divisions of Cell Biology and Cellular Immunology, La Jolla Institute for Allergy and Immunology, San Diego, California 92121
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  • Thomas Jascur
    Affiliations
    Divisions of Cell Biology and Cellular Immunology, La Jolla Institute for Allergy and Immunology, San Diego, California 92121
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  • Gustavo P. Amarante-Mendes
    Footnotes
    Affiliations
    Divisions of Cell Biology and Cellular Immunology, La Jolla Institute for Allergy and Immunology, San Diego, California 92121
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  • Douglas R. Green
    Affiliations
    Divisions of Cell Biology and Cellular Immunology, La Jolla Institute for Allergy and Immunology, San Diego, California 92121
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  • Tomas Mustelin
    Correspondence
    To whom correspondence should be addressed. Tel.: 619-558-3547; Fax: 619-558-3526
    Affiliations
    Divisions of Cell Biology and Cellular Immunology, La Jolla Institute for Allergy and Immunology, San Diego, California 92121
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  • Author Footnotes
    * This work was supported by Finska Läkaresällskapet, the Finnish Cancer Organizations, National Institutes of Health Grant GM52735 and American Cancer Society Grant CB-82 (to D. R. G.), and National Institutes of Health Grants GM48960 and AI35603 (to T. M.). This is publication 185 from the La Jolla Institute for Allergy and Immunology.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
    ‡ Brazilian Research Council (CNPq) Fellow.
Open AccessPublished:February 13, 1998DOI:https://doi.org/10.1074/jbc.273.7.3994
      In cells expressing the oncogenic Bcr-Abl tyrosine kinase, the regulatory p85 subunit of phosphatidylinositol 3-kinase is phosphorylated on tyrosine residues. We report that this phosphorylation event is readily catalyzed by the Abl and Lck protein-tyrosine kinases in vitro, by Bcr-Abl or a catalytically activated Lck-Y505F in co-transfected COS cells, and by endogenous kinases in transfected Jurkat T cells upon triggering of their T cell antigen receptor. Using these systems, we have mapped a major phosphorylation site to Tyr-688 in the C-terminal SH2 domain of p85. Tyrosine phosphorylation of p85 in vitro or in vivo was not associated with detectable change in the enzymatic activity of the phosphatidylinositol 3-kinase heterodimer, but correlated with a strong reduction in the binding of some, but not all, phosphoproteins to the SH2 domains of p85. This provides an additional candidate to the list of SH2 domains regulated by tyrosine phosphorylation and may explain why association of phosphatidylinositol 3-kinase with some cellular ligands is transient or of lower stoichiometry than anticipated.
      Phosphatidylinositol 3-kinases (PI3Ks)
      The abbreviations used are: PI3K, phosphatidylinositol 3-kinase; HA, hemagglutinin; Tyr(P), phosphotyrosine; SH2, Src homology 2 region; SH3, Src homology 3 region; mAb, monoclonal antibody; TPCK,l-1-tosylamido-2-phenylethyl chloromethyl ketone; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis.
      1The abbreviations used are: PI3K, phosphatidylinositol 3-kinase; HA, hemagglutinin; Tyr(P), phosphotyrosine; SH2, Src homology 2 region; SH3, Src homology 3 region; mAb, monoclonal antibody; TPCK,l-1-tosylamido-2-phenylethyl chloromethyl ketone; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis.
      are a family of enzymes involved in a multiplicity of cellular functions, including cell proliferation and transformation (
      • Auger K.R.
      • Cantley L.C.
      ,
      • Coughlin S.R.
      • Escobedo J.A.
      • Williams L.T.
      ,
      • Kaplan D.R.
      • Whitman M.
      • Schaffhausen B.
      • Pallas D.C.
      • White M.
      • Cantley L.C.
      • Roberts T.M.
      ), lymphocyte activation (
      • Gold M.R.
      • Chan V.W.-F.
      • Turck C.
      • DeFranco A.L.
      ,
      • Jascur T.
      • Gilman J.
      • Mustelin T.
      ,
      • von Willebrand M.
      • Jascur T.
      • Bonnefoy-Bérard N.
      • Yano H.
      • Altman A.
      • Matsuda Y.
      • Mustelin T.
      ,
      • Ward S.G.
      • Ley S.C.
      • MacPhee C.
      • Cantrell D.A.
      ), G protein signaling (
      • Stoyanov B.
      • Volinia S.
      • Hanck T.
      • Rubio I.
      • Loubtchenkov M.
      • Malek D.
      • Stoyanova S.
      • Vanhaesebroeck B.
      • Dhand R.
      • Nürnberg B.
      • Gierschik P.
      • Seedorf K.
      • Hsuan J.J.
      • Waterfield M.D.
      • Wetzker R.
      ), DNA repair (
      • Savitsky K.
      • Bar-Shira A.
      • Gilad S.
      • Rotman G.
      • Ziv Y.
      • Vanagaite L.
      • Tagle D.A.
      • Smith S.
      • Uziel T.
      • Sfez S.
      • Ashkenazi M.
      • Pecker I.
      • Frydman M.
      • Harnik R.
      • Patanjali S.R.
      • Simmons A.
      • Clines G.A.
      • Sartiel A.
      • Gatti R.A.
      • Chessa L.
      • Sanal O.
      • Lavin M.F.
      • Jaspers N.G.J.
      • Taylor A.M.R.
      • Arlett C.F.
      • Miki T.
      • Weissman S.M.
      • Lovett M.
      • Collins F.S.
      • Shiloh Y.
      ), intracellular vesicle trafficking (
      • Schu P.V.
      • Takegawa K.
      • Fry M.J.
      • Stack J.H.
      • Waterfield M.D.
      • Emr S.D.
      ,
      • Volinia S.
      • Dhand R.
      • Vanhaesebroeck B.
      • MacDougall L.K.
      • Stein R.
      • Zvelebil M.J.
      • Domin J.
      • Panaretou C.
      • Waterfield M.D.
      ), and inhibition of programmed cell death (
      • Minshall C.
      • Arkins S.
      • Freund G.G.
      • Kelley K.W.
      ,
      • Yao R.
      • Cooper G.M.
      ). The currently best characterized type of PI3K is the heterodimeric enzymes that consist of a 110-kDa catalytic subunit (p110α or p110β; Refs.
      • Hiles I.
      • Otsu M.
      • Volinia S.
      • Fry M.J.
      • Gout I.
      • Dhand R.
      • Panayotou G.
      • Ruiz-Larrea F.
      • Thompson A.
      • Totty N.
      • Hsuan J.
      • Courtneidge S.A.
      • Parker P.J.
      • Waterfield M.D.
      and
      • Hu P.
      • Mondino A.
      • Skolnik E.Y.
      • Schlessinger J.
      ) and a 85-kDa regulatory subunit (p85α or p85β; Refs.
      • Escobedo J.A.
      • Navankasattusas S.
      • Kavanaugh W.M.
      • Milfay D.
      • Fried V.A.
      • Williams L.T.
      ,
      • Otsu M.
      • Hiles I.
      • Gout I.
      • Fry M.J.
      • Ruiz-Larrea F.
      • Panayotou G.
      • Thompson A.
      • Dhand R.
      • Hsuan J.
      • Totty N.
      • Smith A.D.
      • Morgan S.J.
      • Courtneidge S.A.
      • Parker P.J.
      • Waterfield M.D.
      ,
      • Skolnik E.Y.
      • Margolis B.
      • Mohammadi M.
      • Lowenstein E.
      • Fischer R.
      • Drepps A.
      • Ullrich A.
      • Schlessinger J.
      ), and that are utilized for signaling by activated growth factor, cytokine, and antigen receptors. In these heterodimeric PI3Ks, the p85 subunit also functions as an adaptor protein that mediates protein-protein interactions through its two Src homology 2 (SH2) domains, one SH3 domain, two proline-rich sequences, and a region with similarity to the breakpoint cluster region gene (
      • Escobedo J.A.
      • Navankasattusas S.
      • Kavanaugh W.M.
      • Milfay D.
      • Fried V.A.
      • Williams L.T.
      ,
      • Otsu M.
      • Hiles I.
      • Gout I.
      • Fry M.J.
      • Ruiz-Larrea F.
      • Panayotou G.
      • Thompson A.
      • Dhand R.
      • Hsuan J.
      • Totty N.
      • Smith A.D.
      • Morgan S.J.
      • Courtneidge S.A.
      • Parker P.J.
      • Waterfield M.D.
      ,
      • Skolnik E.Y.
      • Margolis B.
      • Mohammadi M.
      • Lowenstein E.
      • Fischer R.
      • Drepps A.
      • Ullrich A.
      • Schlessinger J.
      ). The two SH2 domains of p85 are involved in recruitment of PI3K to activated growth factor receptors (
      • Kaplan D.R.
      • Whitman M.
      • Schaffhausen B.
      • Pallas D.C.
      • White M.
      • Cantley L.C.
      • Roberts T.M.
      ,
      • Escobedo J.A.
      • Navankasattusas S.
      • Kavanaugh W.M.
      • Milfay D.
      • Fried V.A.
      • Williams L.T.
      ) or other proteins having the general motif phosphotyrosine Tyr(P)-X-X-methionine (
      • Songyang Z.
      • Shoelson S.E.
      • Chaudhuri M.
      • Gish G.
      • Pawson T.
      • Haser W.G.
      • King F.
      • Roberts T.
      • Ratnofsky S.
      • Leichleider R.J.
      • Neel B.G.
      • Birge R.B.
      • Fajardo J.E.
      • Chou M.M.
      • Hanafusa H.
      • Schaffhausen B.
      • Cantley L.C.
      ), or, in some cases, Tyr(P)-X-X-leucine (
      • Exley M.
      • Varticovski L.
      • Peter M.
      • Sancho J.
      • Terhorst C.
      ,
      • Zenner G.
      • Vorherr T.
      • Mustelin T.
      • Burn P.
      ). In T cells, the physiologically relevant ligands for p85 include tyrosine-phosphorylated CD28 (
      • Pages F.
      • Ragueneau M.
      • Rottapel R.
      • Truneh A.
      • Nunes J.
      • Imbert J.
      • Olive D.
      ), subunits of the T cell antigen receptor (
      • Exley M.
      • Varticovski L.
      • Peter M.
      • Sancho J.
      • Terhorst C.
      ,
      • Zenner G.
      • Vorherr T.
      • Mustelin T.
      • Burn P.
      ,
      • Carrera A.C.
      • Rodriguez-Borlado L.
      • Martinez-Alonso C.
      • Merida I.
      ), CD5 (
      • Dennehy K.M.
      • Broszeit R.
      • Garnett D.
      • Durrheim G.A.
      • Spruyt L.L.
      • Beyers A.D.
      ), CD7 (
      • Lee D.M.
      • Patel D.D.
      • Pendergast A.M.
      • Haynes B.F.
      ), and the c-Cbl proto-oncogene product (
      • Meisner H.
      • Conway B.R.
      • Hartley D.
      • Czech M.P.
      ). In addition to causing a subcellular relocation of PI3K, these SH2 ligands cause an allosteric activation of the catalytic p110 subunit, which is bound to the region between the two SH2 domains of p85 (
      • Hu P.
      • Mondino A.
      • Skolnik E.Y.
      • Schlessinger J.
      ,
      • Dhand R.
      • Hara K.
      • Hiles I.
      • Bax B.
      • Gout I.
      • Panayotou G.
      • Fry M.J.
      • Yonezawa K.
      • Kasuga M.
      • Waterfield M.D.
      ,
      • Holt K.H.
      • Olson A.L.
      • Moye-Rowley W.S.
      • Pessin J.E.
      ,
      • Klippel A.
      • Escobedo J.A.
      • Hu Q.
      • Williams L.T.
      ).
      Several additional modes of PI3K regulation have been demonstrated, and it is likely that they act in concert to regulate the production of 3-phosphorylated inositol phospholipids in response to a variety of stimuli. The catalytic p110 interacts with activated GTP-bound Ras proteins through a region adjacent to its p85-binding NH2terminus (
      • Rodriguez-Viciana P.
      • Warne P.H.
      • Dhand R.
      • Vanhaesebroeck B.
      • Gout I.
      • Fry M.J.
      • Waterfield M.D.
      • Downward J.
      ,
      • Rodriguez-Viciana P.
      • Warne P.H.
      • Vanhaesebroeck B.
      • Waterfield M.D.
      • Downward J.
      ). Active Ras enhances PI3K activity in intact cells (
      • Rodriguez-Viciana P.
      • Warne P.H.
      • Dhand R.
      • Vanhaesebroeck B.
      • Gout I.
      • Fry M.J.
      • Waterfield M.D.
      • Downward J.
      ,
      • Rodriguez-Viciana P.
      • Warne P.H.
      • Vanhaesebroeck B.
      • Waterfield M.D.
      • Downward J.
      ), but some data indicate that Ras also acts downstream of PI3K (
      • Hu Q.
      • Klippel A.
      • Muslin A.J.
      • Fantl W.J.
      • Williams L.T.
      ). In T cells, the two p85 isoforms have been shown to undergo phosphorylation on both serine and threonine (
      • Reif K.
      • Gout I.
      • Waterfield M.D.
      • Cantrell D.A.
      ,
      • Dhand R.
      • Hiles I.
      • Panayotou G.
      • Roche S.
      • Fry M.J.
      • Gout I.
      • Totty N.F.
      • Truong O.
      • Vicendo P.
      • Yonezawa K.
      • Kasuga M.
      • Courtneidge S.A.
      • Waterfield M.D.
      ). Tyrosine phosphorylation of the p85 subunit has been shown to occur in many different systems, such as in response to platelet-derived growth factor (
      • Kaplan D.R.
      • Whitman M.
      • Schaffhausen B.
      • Pallas D.C.
      • White M.
      • Cantley L.C.
      • Roberts T.M.
      ), insulin (
      • Hayashi H.
      • Nishioka Y.
      • Kamohara S.
      • Kanai F.
      • Ishii K.
      • Fukui Y.
      • Shibasaki F.
      • Takenawa T.
      • Kido H.
      • Katsunuma N.
      • Ebina Y.
      ), B cell antigen receptor ligation (
      • Gold M.R.
      • Chan V.W.-F.
      • Turck C.
      • DeFranco A.L.
      ), interleukin-2 (
      • Karnitz L.M.
      • Sutor S.L.
      • Abraham R.T.
      ), and in cells transformed by the Bcr-Abl fusion protein-tyrosine kinase (
      • Amarante-Mendes G.P.
      • Jascur T.
      • Nishioka W.K.
      • Mustelin T.
      • Green D.R.
      ,
      • Gotoh A.
      • Miyazawa K.
      • Ohyashiki K.
      • Toyama K.
      ,
      • Skorski T.
      • Kanakaraj P.
      • Nieborowska-Skorska M.
      • Ratajczak M.Z.
      • Wen S.-C.
      • Zon G.
      • Gewirtz A.M.
      • Perussia B.
      • Calabretta B.
      ,
      • Varticovski L.
      • Daley G.Q.
      • Jackson P.
      • Baltimore D.
      • Cantley L.C.
      ). The sites of phosphorylation in p85 have been mapped to tyrosines 368, 508, and 607 in insulin-stimulated cells (
      • Hayashi H.
      • Nishioka Y.
      • Kamohara S.
      • Kanai F.
      • Ishii K.
      • Fukui Y.
      • Shibasaki F.
      • Takenawa T.
      • Kido H.
      • Katsunuma N.
      • Ebina Y.
      ), but the physiological function of this phosphorylation has remained unknown. Tyrosine phosphorylation of p85 seems not to be required for the enzymatic activity of PI3K. Instead, tyrosine phosphorylation of p85 has been reported to correlate with the dissociation of PI3K from the activated insulin receptor kinase (
      • Zhang W.
      • Johnson J.D.
      • Rutter W.J.
      ).
      We have studied the tyrosine phosphorylation of p85 in hematopoietic cells, and report that phosphorylation occurs at least at Tyr-688 in the C-terminal SH2 domain. This event does not detectably affect the catalytic activity of PI3K per se, but causes a change in the binding properties of the SH2 domain. This change is likely to modify the function of PI3K in intact cells.

      DISCUSSION

      Taken together, our findings indicate that PI3K can be phosphorylated at Tyr-688 in the C-terminal SH2 domain of the p85 subunit in Bcr-Abl expressing HL-60 cells, by active Lck in COS cells, by an unidentified receptor-activated protein-tyrosine kinase in T cells, and by both Abl and Lck in vitro. This phosphorylation does not measurably affect the lipid kinase activity of PI3K (at least at physiological stoichiometry), but was found to change the ligand binding properties of the SH2 domain(s). These results are in agreement with previous observations showing that expression of Bcr-Abl in NIH 3T3 cells induces tyrosine phosphorylation of PI3K without any significant increase in PI3K products in vivo(
      • Varticovski L.
      • Daley G.Q.
      • Jackson P.
      • Baltimore D.
      • Cantley L.C.
      ).
      The analysis of SH2 domain function in p85 is complicated by the tandem arrangement of the two SH2 domains, resulting in their cooperative binding to many ligands. Our results indicate that tyrosine phosphorylation of the C-terminal SH2 domain reduced the affinity for some ligands, while the binding of others was unchanged. Two alternative explanations could be envisualized: either tyrosine phosphorylation changed the ligand selection from the classical Tyr(P)-X-X-methionine to something more or less different, or only the C-terminal SH2 domain was inhibited, while the N-terminal SH2 domain remained unchanged. In the latter case, only of those ligands that bind exclusively to the C-terminal SH2 domain or that require simultaneous binding to both domains will bind less strongly. We have previously observed that expression of the HA-tagged p85 proteins in T cells resulted in the co-immunoprecipitation of phospho-TCRζ only when both SH2 domains were present in the p85 protein. Such a requirement for two SH2 domains would explain why the TCRζ binds despite not having the optimal Tyr(P)-X-X-methionine (
      • Songyang Z.
      • Shoelson S.E.
      • Chaudhuri M.
      • Gish G.
      • Pawson T.
      • Haser W.G.
      • King F.
      • Roberts T.
      • Ratnofsky S.
      • Leichleider R.J.
      • Neel B.G.
      • Birge R.B.
      • Fajardo J.E.
      • Chou M.M.
      • Hanafusa H.
      • Schaffhausen B.
      • Cantley L.C.
      ) motif. Apparently, two Tyr(P)-X-X-leucine motifs in tandem in TCRζ can bind the two SH2 domains of p85 simultaneously and thereby increase the affinity to physiologically relevant levels. The binding of PI3K to TCRζ and CD3 subunits (
      • Jascur T.
      • Gilman J.
      • Mustelin T.
      ,
      • Exley M.
      • Varticovski L.
      • Peter M.
      • Sancho J.
      • Terhorst C.
      ,
      • Carrera A.C.
      • Rodriguez-Borlado L.
      • Martinez-Alonso C.
      • Merida I.
      ), as well as to isolated phosphopeptides derived from these proteins (
      • Zenner G.
      • Vorherr T.
      • Mustelin T.
      • Burn P.
      ), has been reported. The findings reported in the present paper may explain why p85 binding to these receptor subunits is of low stoichiometry when assessed by co-immunoprecipitation.
      In the Bcr-Abl expressing cells, p85 was phosphorylated on tyrosine, but also co-immunoprecipitated with several Tyr(P)-containing proteins. While this may seem conflicting, it is clear that only a fraction of p85 is tyrosine phosphorylated, and it is impossible to judge if any of the co-immunoprecipitating protein bound to the phosphorylated minority of p85 molecules or (more likely) to the unphosphorylated majority. The requirement for both SH2 domains for efficient phosphorylation of p85 proteins in Bcr-Abl expressing cells, suggests that both are involved directly or indirectly in association with the protein-tyrosine kinase responsible for this phosphorylation. The simplest model predicts that the p85 SH2 domains bind directly to Bcr-Abl, but dissociate from it upon phosphorylation of the C-terminal SH2 domain. This would explain why the co-immunoprecipitation of Bcr-Abl and PI3K is of very low stoichiometry.
      We recently reported that the Lck kinase is phosphorylated at Tyr-192 in the EF loop of its SH2 domain in activated T cells, and in COS-1 cells co-transfected with either Syk or Zap (
      • Couture C.
      • Baier G.
      • Oetken C.
      • Williams S.
      • Telford D.
      • Marie-Cardine A.
      • Baier-Bitterlich G.
      • Fischer S.
      • Burn P.
      • Altman A.
      • Mustelin T.
      ,
      • Couture C.
      • Songyang Z.
      • Jascur T.
      • Williams S.
      • Tailor P.
      • Cantley L.C.
      • Mustelin T.
      ). The phosphorylation of the SH2 domain, or the mutation of Tyr-192 to an acidic residue, caused a strong decline in the affinity of the domain for tyrosine-phosphorylated ligands (
      • Couture C.
      • Songyang Z.
      • Jascur T.
      • Williams S.
      • Tailor P.
      • Cantley L.C.
      • Mustelin T.
      ). A similar change was reported by Stover and co-workers (
      • Stover D.R.
      • Furet P.
      • Lydon N.B.
      ) for the c-Src SH2 domain upon its phosphorylation at Tyr-213 by the platelet-derived growth factor kinase. In this paper, we add a third example to the list of SH2 domains regulated by tyrosine phosphorylation, namely the C-terminal SH2 domain of PI3K p85. Interestingly, Tyr-688 resides in the same region of the SH2 domain as Tyr-192 in the Lck SH2 domain. Comparison of the amino acid sequences of SH2 domains from different proteins shows that many, but not all, contain tyrosine residues in the corresponding location in the EF loop (
      • Waksman G.
      • Kominos D.
      • Robertson S.C.
      • Pant N.
      • Baltimore D.
      • Birge R.B.
      • Cowburn D.
      • Hanafusa H.
      • Mayer B.J.
      • Overduin M.
      • Resh M.D.
      • Rios C.B.
      • Silverman L.
      • Kuriyan J.
      ). Notably, the N-terminal SH2 domain of p85 does not. Thus, it is tempting to speculate that the regulation of SH2 domains by tyrosine phosphorylation is a more general mechanism for the termination of SH2-ligand interactions, perhaps in part explaining their transient nature in intact cells.

      Acknowledgment

      We are grateful to Dr. Lewis C. Cantley for valuable discussions and advice.

      References

        • Auger K.R.
        • Cantley L.C.
        Cancer Cells. 1991; 3: 263-275
        • Coughlin S.R.
        • Escobedo J.A.
        • Williams L.T.
        Science. 1989; 243: 1191-1194
        • Kaplan D.R.
        • Whitman M.
        • Schaffhausen B.
        • Pallas D.C.
        • White M.
        • Cantley L.C.
        • Roberts T.M.
        Cell. 1987; 50: 1021-1029
        • Gold M.R.
        • Chan V.W.-F.
        • Turck C.
        • DeFranco A.L.
        J. Immunol. 1992; 148: 2012-2022
        • Jascur T.
        • Gilman J.
        • Mustelin T.
        J. Biol. Chem. 1997; 272: 14483-14488
        • von Willebrand M.
        • Jascur T.
        • Bonnefoy-Bérard N.
        • Yano H.
        • Altman A.
        • Matsuda Y.
        • Mustelin T.
        Eur. J. Biochem. 1996; 235: 828-835
        • Ward S.G.
        • Ley S.C.
        • MacPhee C.
        • Cantrell D.A.
        Eur. J. Immunol. 1992; 22: 45-49
        • Stoyanov B.
        • Volinia S.
        • Hanck T.
        • Rubio I.
        • Loubtchenkov M.
        • Malek D.
        • Stoyanova S.
        • Vanhaesebroeck B.
        • Dhand R.
        • Nürnberg B.
        • Gierschik P.
        • Seedorf K.
        • Hsuan J.J.
        • Waterfield M.D.
        • Wetzker R.
        Science. 1995; 269: 690-693
        • Savitsky K.
        • Bar-Shira A.
        • Gilad S.
        • Rotman G.
        • Ziv Y.
        • Vanagaite L.
        • Tagle D.A.
        • Smith S.
        • Uziel T.
        • Sfez S.
        • Ashkenazi M.
        • Pecker I.
        • Frydman M.
        • Harnik R.
        • Patanjali S.R.
        • Simmons A.
        • Clines G.A.
        • Sartiel A.
        • Gatti R.A.
        • Chessa L.
        • Sanal O.
        • Lavin M.F.
        • Jaspers N.G.J.
        • Taylor A.M.R.
        • Arlett C.F.
        • Miki T.
        • Weissman S.M.
        • Lovett M.
        • Collins F.S.
        • Shiloh Y.
        Science. 1995; 268: 1749-1753
        • Schu P.V.
        • Takegawa K.
        • Fry M.J.
        • Stack J.H.
        • Waterfield M.D.
        • Emr S.D.
        Science. 1993; 260: 88-91
        • Volinia S.
        • Dhand R.
        • Vanhaesebroeck B.
        • MacDougall L.K.
        • Stein R.
        • Zvelebil M.J.
        • Domin J.
        • Panaretou C.
        • Waterfield M.D.
        EMBO J. 1995; 14: 3339-3348
        • Minshall C.
        • Arkins S.
        • Freund G.G.
        • Kelley K.W.
        J. Immunol. 1996; 156: 939-947
        • Yao R.
        • Cooper G.M.
        Science. 1995; 267: 2003-2006
        • Hiles I.
        • Otsu M.
        • Volinia S.
        • Fry M.J.
        • Gout I.
        • Dhand R.
        • Panayotou G.
        • Ruiz-Larrea F.
        • Thompson A.
        • Totty N.
        • Hsuan J.
        • Courtneidge S.A.
        • Parker P.J.
        • Waterfield M.D.
        Cell. 1992; 70: 419-425
        • Hu P.
        • Mondino A.
        • Skolnik E.Y.
        • Schlessinger J.
        Mol. Cell. Biol. 1993; 13: 7677-7688
        • Escobedo J.A.
        • Navankasattusas S.
        • Kavanaugh W.M.
        • Milfay D.
        • Fried V.A.
        • Williams L.T.
        Cell. 1991; 65: 75-82
        • Otsu M.
        • Hiles I.
        • Gout I.
        • Fry M.J.
        • Ruiz-Larrea F.
        • Panayotou G.
        • Thompson A.
        • Dhand R.
        • Hsuan J.
        • Totty N.
        • Smith A.D.
        • Morgan S.J.
        • Courtneidge S.A.
        • Parker P.J.
        • Waterfield M.D.
        Cell. 1991; 65: 91-97
        • Skolnik E.Y.
        • Margolis B.
        • Mohammadi M.
        • Lowenstein E.
        • Fischer R.
        • Drepps A.
        • Ullrich A.
        • Schlessinger J.
        Cell. 1991; 65: 83-90
        • Songyang Z.
        • Shoelson S.E.
        • Chaudhuri M.
        • Gish G.
        • Pawson T.
        • Haser W.G.
        • King F.
        • Roberts T.
        • Ratnofsky S.
        • Leichleider 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
        • Exley M.
        • Varticovski L.
        • Peter M.
        • Sancho J.
        • Terhorst C.
        J. Biol. Chem. 1994; 269: 15140-15146
        • Zenner G.
        • Vorherr T.
        • Mustelin T.
        • Burn P.
        J. Cell. Biochem. 1996; 63: 94-103
        • Pages F.
        • Ragueneau M.
        • Rottapel R.
        • Truneh A.
        • Nunes J.
        • Imbert J.
        • Olive D.
        Nature. 1994; 369: 327-329
        • Carrera A.C.
        • Rodriguez-Borlado L.
        • Martinez-Alonso C.
        • Merida I.
        J. Biol. Chem. 1994; 269: 19435-19440
        • Dennehy K.M.
        • Broszeit R.
        • Garnett D.
        • Durrheim G.A.
        • Spruyt L.L.
        • Beyers A.D.
        Eur. J. Immunol. 1997; 27: 679-686
        • Lee D.M.
        • Patel D.D.
        • Pendergast A.M.
        • Haynes B.F.
        Int. Immunol. 1996; 8: 1195-1203
        • Meisner H.
        • Conway B.R.
        • Hartley D.
        • Czech M.P.
        Mol. Cell. Biol. 1995; 15: 3571-3578
        • Dhand R.
        • Hara K.
        • Hiles I.
        • Bax B.
        • Gout I.
        • Panayotou G.
        • Fry M.J.
        • Yonezawa K.
        • Kasuga M.
        • Waterfield M.D.
        EMBO J. 1994; 13: 511-521
        • Holt K.H.
        • Olson A.L.
        • Moye-Rowley W.S.
        • Pessin J.E.
        Mol. Cell. Biol. 1994; 14: 42-49
        • Klippel A.
        • Escobedo J.A.
        • Hu Q.
        • Williams L.T.
        Mol. Cell. Biol. 1993; 13: 5560-5566
        • 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
        • Rodriguez-Viciana P.
        • Warne P.H.
        • Vanhaesebroeck B.
        • Waterfield M.D.
        • Downward J.
        EMBO J. 1996; 15: 2442-2451
        • Hu Q.
        • Klippel A.
        • Muslin A.J.
        • Fantl W.J.
        • Williams L.T.
        Science. 1995; 268: 100-102
        • Reif K.
        • Gout I.
        • Waterfield M.D.
        • Cantrell D.A.
        J. Biol. Chem. 1993; 268: 10780-10788
        • Dhand R.
        • Hiles I.
        • Panayotou G.
        • Roche S.
        • Fry M.J.
        • Gout I.
        • Totty N.F.
        • Truong O.
        • Vicendo P.
        • Yonezawa K.
        • Kasuga M.
        • Courtneidge S.A.
        • Waterfield M.D.
        EMBO J. 1994; 13: 522-533
        • Hayashi H.
        • Nishioka Y.
        • Kamohara S.
        • Kanai F.
        • Ishii K.
        • Fukui Y.
        • Shibasaki F.
        • Takenawa T.
        • Kido H.
        • Katsunuma N.
        • Ebina Y.
        J. Biol. Chem. 1993; 268: 7107-7117
        • Karnitz L.M.
        • Sutor S.L.
        • Abraham R.T.
        J. Exp. Med. 1994; 179: 1799-1808
        • Amarante-Mendes G.P.
        • Jascur T.
        • Nishioka W.K.
        • Mustelin T.
        • Green D.R.
        Cell Death Differ. 1997; 4: 541-555
        • Gotoh A.
        • Miyazawa K.
        • Ohyashiki K.
        • Toyama K.
        Leukemia. 1994; 8: 115-120
        • Skorski T.
        • Kanakaraj P.
        • Nieborowska-Skorska M.
        • Ratajczak M.Z.
        • Wen S.-C.
        • Zon G.
        • Gewirtz A.M.
        • Perussia B.
        • Calabretta B.
        Blood. 1995; 86: 726-736
        • Varticovski L.
        • Daley G.Q.
        • Jackson P.
        • Baltimore D.
        • Cantley L.C.
        Mol. Cell. Biol. 1991; 11: 1107-1113
        • Zhang W.
        • Johnson J.D.
        • Rutter W.J.
        Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11317-11321
        • Tailor P.
        • Gilman J.
        • Williams S.
        • Couture C.
        • Mustelin T.
        J. Biol. Chem. 1997; 272: 5371-5376
        • Williams S.
        • Couture C.
        • Gilman J.
        • Jascur T.
        • Deckert M.
        • Altman A.
        • Mustelin T.
        Eur. J. Biochem. 1997; 245: 84-90
        • Couture C.
        • Baier G.
        • Altman A.
        • Mustelin T.
        Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5301-5305
        • Couture C.
        • Baier G.
        • Oetken C.
        • Williams S.
        • Telford D.
        • Marie-Cardine A.
        • Baier-Bitterlich G.
        • Fischer S.
        • Burn P.
        • Altman A.
        • Mustelin T.
        Mol. Cell. Biol. 1994; 14: 5249-5258
        • Couture C.
        • Deckert M.
        • Williams S.
        • Russo F.O.
        • Altman A.
        • Mustelin T.
        J. Biol. Chem. 1996; 271: 24294-24299
        • Couture C.
        • Songyang Z.
        • Jascur T.
        • Williams S.
        • Tailor P.
        • Cantley L.C.
        • Mustelin T.
        J. Biol. Chem. 1996; 271: 24880-24884
        • Tailor P.
        • Jascur T.
        • Williams S.
        • von Willebrand M.
        • Couture C.
        • Mustelin T.
        Eur. J. Biochem. 1996; 237: 736-742
        • von Willebrand M.
        • Baier G.
        • Couture C.
        • Burn P.
        • Mustelin T.
        Eur. J. Immunol. 1994; 24: 234-238
        • Luo K.
        • Hurley T.R.
        • Sefton B.M.
        Oncogene. 1990; 5: 921-923
        • Clark S.S.
        • McLaughlin J.
        • Timmonis M.
        • Pendergast A.M.
        • Ben-Neriah Y.
        • Dow L.
        • Rovera G.
        • Smith S.D.W.
        Science. 1988; 239: 775-777
        • Daley G.Q.
        • Van Etten R.A.
        • Baltimore D.
        Science. 1990; 247: 824-830
        • Elefanty A.G.
        • Hariharan I.K.
        • Cory S.
        EMBO J. 1990; 9: 1069-1078
        • Shtivelman E.
        • Lifshitz B.
        • Gale R.P.
        • Roe B.A.
        Nature. 1985; 315: 550-551
        • McGahon A.
        • Bissonnette R.
        • Schmitt M.
        • Cotter K.M.
        • Green D.R.
        • Cotter T.G.
        Blood. 1994; 83: 1179-1187
        • Amrein K.E.
        • Sefton B.M.
        Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 4247-4251
        • Marth J.D.
        • Cooper J.A.
        • King C.S.
        • Ziegler S.F.
        • Tinker D.A.
        • Overell R.W.
        • Krebs E.G.
        • Perlmutter R.M.
        Mol. Cell. Biol. 1988; 8: 540-550
        • Mustelin T.
        • Coggeshall K.M.
        • Altman A.
        Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 6302-6306
        • Hsi E.D.
        • Siegel J.N.
        • Minami Y.
        • Luong E.T.
        • Klausner R.D.
        • Samelson L.E.
        J. Biol. Chem. 1989; 264: 10836-10842
        • June C.
        • Fletcher M.C.
        • Ledbetter J.A.
        • Schieven G.L.
        • Siegel J.N.
        • Phillips A.F.
        • Samelson L.E.
        Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 7722-7727
        • Mustelin T.
        Src Family Tyrosine Kinases in Leukocytes. R. G. Landes Co., Austin/Georgetown, TX1994: 1-155
        • Mustelin T.
        Immunity. 1994; 1: 351-356
        • Mustelin T.
        • Coggeshall K.M.
        • Isakov N.
        • Altman A.
        Science. 1990; 247: 1584-1587
        • Chan A.C.
        • Iwashima M.
        • Turck C.W.
        • Weiss A.
        Cell. 1992; 71: 649-662
        • Stover D.R.
        • Furet P.
        • Lydon N.B.
        J. Biol. Chem. 1996; 21: 12481-12487
        • Waksman G.
        • Kominos D.
        • Robertson S.C.
        • Pant N.
        • Baltimore D.
        • Birge R.B.
        • Cowburn D.
        • Hanafusa H.
        • Mayer B.J.
        • Overduin M.
        • Resh M.D.
        • Rios C.B.
        • Silverman L.
        • Kuriyan J.
        Nature. 1992; 358: 646-653