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T Cell Activation-dependent Association between the p85 Subunit of the Phosphatidylinositol 3-Kinase and Grb2/Phospholipase C-γ1-binding Phosphotyrosyl Protein pp36/38 (∗)

  • Toru Fukazawa
    Affiliations
    (1) Lymphocyte Biology Section, Department of Rheumatology and Immunology, Boston, Massachusetts 02115
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  • Kris A. Reedquist
    Footnotes
    Affiliations
    (1) Lymphocyte Biology Section, Department of Rheumatology and Immunology, Boston, Massachusetts 02115
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  • Govindaswamy Panchamoorthy
    Footnotes
    Affiliations
    (1) Lymphocyte Biology Section, Department of Rheumatology and Immunology, Boston, Massachusetts 02115
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  • Stephen Soltoff
    Affiliations
    (3) Division of Signal Transduction, Beth Israel Hospital and the Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115
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  • Thomas Trub
    Affiliations
    (2) Research Division, Joslin Diabetes Center, Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts 02115
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  • Brian Druker
    Affiliations
    (4) Division of Hematology and Medical Oncology, Oregon Health Sciences University, Portland, Oregon 97201
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  • Lewis Cantley
    Affiliations
    (3) Division of Signal Transduction, Beth Israel Hospital and the Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115
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  • Steven E. Shoelson
    Affiliations
    (2) Research Division, Joslin Diabetes Center, Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts 02115
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  • 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 Bldg., Rm. 514, 250 Longwood Ave., Boston, MA 02115. Tel.: 617-432-1557; Fax: 617-432-2799
    Affiliations
    (1) Lymphocyte Biology Section, Department of Rheumatology and Immunology, Boston, Massachusetts 02115
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  • Author Footnotes
    ∗ This work was supported by National Institutes of Health Grants R29-AI28508 and AR36308 and Geyer Foundation grants (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.
    § 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 25, 1995DOI:https://doi.org/10.1074/jbc.270.34.20177
      Tyrosine phosphorylation of cellular proteins is an early and an essential step in T cell receptor-mediated lymphocyte activation. Tyrosine phosphorylation of transmembrane receptor chains (such as ζ and CD3 chains) and membrane-associated proteins provides docking sites for SH2 domains of adaptor proteins and signaling enzymes, resulting in their recruitment in the vicinity of activated receptors. pp36/38 is a prominent substrate of early tyrosine phosphorylation upon stimulation through the T cell receptor. The tyrosine-phosphorylated form of pp36/38 is membrane-associated and directly interacts with phospholipase C-γ1 and Grb2, providing one mechanism to recruit downstream effectors to the cell membrane. Here, we demonstrate that in Jurkat T cells, pp36/38 associates with the p85 subunit of phosphatidylinositol 3-kinase (PI-3-K p85) in an activation-dependent manner. Association of pp36/38 with PI-3-K p85 was confirmed by transfection of a hemagglutinin-tagged p85α cDNA into Jurkat cells followed by anti-hemagglutinin immunoprecipitation. In vitro binding experiments with glutathione S-transferase fusion proteins of PI-3-K p85 demonstrated that the SH2 domains, but not the SH3 domain, mediated binding to pp36/38. This binding was selectively abrogated by phosphopeptides that bind to p85 SH2 domains with high affinity. Filter binding assays demonstrated that association between pp36/38 and PI-3-K p85 SH2 domains was due to direct binding. These results strongly suggest the role of pp36/38 in recruiting PI-3-K to the cell membrane and further support the idea that pp36/38 is a multifunctional docking protein for SH2 domain-containing signaling proteins in T cells.

      INTRODUCTION

      Engagement of the T cell receptor (TCR)
      The abbreviations used are: TCR
      T cell receptor
      PI-3-K
      phosphatidylinositol 3-kinase
      PI-3-K p85
      p85 subunit of PI-3-K
      HA
      hemagglutinin
      GST
      glutathione S-transferase
      PAGE
      polyacrylamide gel electrophoresis
      PVDF
      polyvinylidene difluoride
      ECL
      enhanced chemiluminescence.
      by the 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.
      ). Studies with receptor tyrosine kinases, such as the epidermal growth factor receptor, have elucidated the role of tyrosine phosphorylation in recruiting downstream signaling proteins. Specific phosphotyrosyl peptide motifs serve as docking sites for the SH2 (Src homology 2) domains of signaling proteins, allowing them to form stable complexes with the activated receptors (
      • Pawson T.
      • Gish G.D.
      ,
      • Schlessinger J.
      ,
      • Downward J.
      ). However, unlike growth factor receptors(
      • Schlessinger J.
      ), the TCR/CD3 components lack intrinsic tyrosine kinase domains and 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.
      ). Two Src family kinases, the TCR/CD3-associated p59fyn (Fyn) (
      • Timson Gauen L.K.
      • Kong A.N.
      • Samelson L.E.
      • Shaw A.S.
      ) and the CD4/8-associated p56lck (Lck)(
      • 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.
      ), and a distinct cytoplasmic tyrosine kinase, ZAP-70(
      • Chan A.C.
      • Iwashima M.
      • Turck C.W.
      • Weiss A.
      ,
      • Iwashima M.
      • Irving B.A.
      • van Oers N.S.
      • Chan A.C.
      • Weiss A.
      ), have been demonstrated to play critical and apparently nonoverlapping roles in T cell activation. It is now widely accepted that these cytoplasmic tyrosine kinases phosphorylate the cytoplasmic tails of CD3 and ζ chains and other membrane-associated proteins, which serve as docking sites for SH2 domain-containing proteins(
      • Weiss A.
      • Littman D.R.
      ,
      • Perlmutter R.M.
      • Levin S.D.
      • Appleby M.W.
      • Anderson S.J.
      • Alberola-Ila J.
      ). For example, tyrosine phosphorylation of CD3 and ζ chains by the Src family kinases allows binding to SH2 domains of ZAP-70 (
      • Iwashima M.
      • Irving B.A.
      • van Oers N.S.
      • Chan A.C.
      • Weiss A.
      ) and adaptor protein Shc(
      • Ravichandran K.S.
      • Lee K.K.
      • Songyang Z.
      • Cantley L.C.
      • Burn P.
      • Burakoff S.J.
      ,
      • Osman N.
      • Lucas S.C
      • Turner H.
      • Cantrell D.
      ). The latter protein is itself phosphorylated, providing a binding site for Grb2. This is thought to translocate Grb2-bound guanine nucleotide exchanger m-Sos to the cell membrane, thus leading to Ras activation(
      • Ravichandran K.S.
      • Lee K.K.
      • Songyang Z.
      • Cantley L.C.
      • Burn P.
      • Burakoff S.J.
      ), although ζ-Shc-Grb2-Sos complexes have been difficult to demonstrate(
      • Osman N.
      • Lucas S.C
      • Turner H.
      • Cantrell D.
      ). Grb2 also associates with other proteins through its SH3 domains, such as the p120cbl(
      • Fukazawa T.
      • Reedquist K.A.
      • Trub T.
      • Soltoff S.
      • Panchamoorthy G.
      • Druker B.
      • Cantley L.
      • Shoelson S.E.
      • Band H.
      ,
      • Donovan J.A.
      • Wange R.L.
      • Langdon W.Y.
      • Samelson L.E.
      ,
      • Meisner H.
      • Conway B.R.
      • Hartley D.
      • Czech M.P.
      ) and p76-SLP (
      • Jackman J.K.
      • Motto D.G.
      • Sun Q.
      • Tanemoto M.
      • Turck C.W.
      • Pelz G.A.
      • Koretzky G.A.
      • Findell P.R.
      ) proteins, which are also likely to be recruited to the cell membrane in this fashion.
      While tyrosine-phosphorylated CD3/ζ chains provide an important set of motifs to recruit signaling proteins, these are unlikely to directly interact with all signaling proteins that are recruited to the TCR, given the preference of various SH2 domains for specific peptide sequences at positions +1 to +3 following phosphotyrosine(
      • 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.
      ). Thus, it is important to identify additional SH2 domain targets and to characterize their specific preferences for different SH2 domains. Recent studies have revealed a likely role for a polypeptide (pp36/38) in recruiting downstream effectors to the cell membrane in activated T cells. pp36/38 represents a dominant substrate of TCR-associated tyrosine kinases in primary T cells as well as in the Jurkat human T cell line(
      • Weber J.R.
      • Bell G.M.
      • Han M.Y.
      • Pawson T.
      • Imboden J.B.
      ,
      • Gilliland L.K.
      • Schieven G.L.
      • Norris N.A.
      • Kanner S.B.
      • Aruffo A.
      • Ledbetter J.A.
      ,
      • Sieh M.
      • Batzer A.
      • Schlessinger J.
      • Weiss A.
      ,
      • Buday L.
      • Egan S.E.
      • Viciana P.R.
      • Cantrell D.A.
      • Downward J.
      ). Importantly, pp36/38 was shown to coimmunoprecipitate with phospholipase C-γ1 and Grb2, and SH2 domains of these proteins directly complexed with pp36/38 in vitro(
      • Weber J.R.
      • Bell G.M.
      • Han M.Y.
      • Pawson T.
      • Imboden J.B.
      ,
      • Gilliland L.K.
      • Schieven G.L.
      • Norris N.A.
      • Kanner S.B.
      • Aruffo A.
      • Ledbetter J.A.
      ,
      • Sieh M.
      • Batzer A.
      • Schlessinger J.
      • Weiss A.
      ,
      • Buday L.
      • Egan S.E.
      • Viciana P.R.
      • Cantrell D.A.
      • Downward J.
      ). Since tyrosine-phosphorylated pp36/38 was found to be membrane-associated(
      • Sieh M.
      • Batzer A.
      • Schlessinger J.
      • Weiss A.
      ,
      • Buday L.
      • Egan S.E.
      • Viciana P.R.
      • Cantrell D.A.
      • Downward J.
      ), these results strongly suggested a possible role of this protein in recruiting critical signaling proteins to the membrane.
      The phosphatidylinositol 3-kinase (PI-3-K) enzyme, which phosphorylates the D-3 position of the inositol ring to generate a unique class of lipid second messengers, has been implicated in signaling through a number of growth factor, cytokine, and other receptors(
      • Fry M.J.
      • Waterfield M.D.
      ,
      • Kapeller R.
      • Cantley L.C.
      ). In T lymphocytes, stimulation through the TCR leads to an increase in the PI-3-K activity and activation-dependent incorporation of this enzyme into phosphotyrosyl protein complexes(
      • Thompson P.A.
      • Gutkind J.S.
      • Robbins K.C.
      • Ledbetter J.A.
      • Bolen J.B.
      ,
      • Ward S.G.
      • Reif K.
      • Ley S.
      • Fry M.J.
      • Waterfield M.D.
      • Cantrell D.A.
      ,
      • Prasad K.V.
      • Janssen O.
      • Kapeller R.
      • Raab M.
      • Cantley L.C.
      • Rudd C.E.
      ,
      • Reif K.
      • Gout I.
      • Waterfield M.D.
      • Cantrell D.A.
      ,
      • Carrera A.C.
      • Rodriguez-Borlado L.
      • Martinez-Alonso C.
      • Merida I.
      ,
      • Exley M.
      • Varticovski L.
      • Peter M.
      • Sancho J.
      • Terhorst C.
      ). However, p110 or p85 subunits of PI-3-K are not tyrosine-phosphorylated upon TCR triggering(
      • Ward S.G.
      • Reif K.
      • Ley S.
      • Fry M.J.
      • Waterfield M.D.
      • Cantrell D.A.
      ,
      • Reif K.
      • Gout I.
      • Waterfield M.D.
      • Cantrell D.A.
      ), indicating that incorporation of PI-3-K into phosphotyrosyl complexes occurs through interactions with other proteins. The regulatory p85 subunit of PI-3-K (PI-3-K p85) possesses an SH3 domain and two SH2 domains (
      • Skolnik E.Y.
      • Margolis B.
      • Mohammadi M.
      • Lowenstein E.
      • Fischer R.
      • Drepps A.
      • Ullrich A.
      • Schlessinger J.
      ) and has been implicated in mediating protein-protein interactions that regulate the activity and localization of PI-3-K(
      • Fry M.J.
      • Waterfield M.D.
      ,
      • Kapeller R.
      • Cantley L.C.
      ). Here, we demonstrate that pp36/38 associates with the p85 subunit of PI-3-K. In vitro binding experiments demonstrate that this binding is direct and involves the PI-3-K p85 SH2 domains. These results provide further evidence for the role of pp36/38 as a multifunctional SH2 domain docking adaptor in TCR signaling.

      MATERIALS AND METHODS

      Peptides

      The following phosphotyrosyl peptides were synthesized and high pressure liquid chromatography-purified as described(
      • Domchek S.M.
      • Auger K.R.
      • Chatterjee S.
      • Burke Jr., T.R.
      • Shoelson S.E.
      ) : the pYEEI motif peptide corresponding to Tyr-324 of the hamster medium-sized tumor antigen (EPQpYEEIPIYL; boldface letters indicate the SH2-binding motif) and its unphosphorylated version (YEEI); the pYVNV motif peptide corresponding to Tyr-317 of the Shc protein (PSpYVNVQNL) and its mutant version, pYVAV (PSpYVAVQNL); and the pYMNM motif peptide corresponding to CD28 Tyr-191 (HSDpYMNMTPR)(
      • 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.
      ).

      Antibodies

      The monoclonal antibodies used in this work were as follows: 4G10 (anti-pY; IgG2a)(
      • Druker B.
      • Mamon T.
      • Roberts T.
      ), 2Ad2 (anti-CD3∊; IgM) (a gift from Dr. Ellis Reinherz, Dana-Farber Cancer Institute, Boston), SPV-T3b (anti-CD3∊; IgG2a)(
      • Spits H.
      • Keizer G.
      • Borst J.
      • Terhorst C.
      • Hekman A.
      • de Vries J.E.
      ), anti-phospholipase C-γ1 (a mixture of monoclonal antibodies; Upstate Biotechnology, Inc.), and 12CA5 (anti-influenza hemagglutinin (HA) epitope tag; IgG2b)(
      • Wilson I.A.
      • Niman H.L.
      • Houghten R.A.
      • Cherenson A.R.
      • Connolly M.L.
      • Lerner R.A.
      ). The polyclonal rabbit antibodies used were as follows: normal rabbit serum from young adult unimmunized rabbits (negative control), anti-PI-3-K p85 (α and β) (against the C-terminal 19-kDa fragment; Transduction Laboratories, Lexington, KY), and anti-Grb2 (against amino acids 195-217 of human Grb2) and anti-p120cbl (against a 15-amino acid synthetic peptide corresponding to the C terminus of Cbl) (both from Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Goat anti-GST antibody was from Pharmacia Biotech Inc.

      GST Fusion Proteins

      Murine Grb2 (
      • Suen K.L.
      • Bustelo X.R.
      • Pawson T.
      • Barbacid M.
      ) (provided by Mike Moran, University of Toronto) or its SH2 domain and the following fragments of PI-3-K p85α (
      • Skolnik E.Y.
      • Margolis B.
      • Mohammadi M.
      • Lowenstein E.
      • Fischer R.
      • Drepps A.
      • Ullrich A.
      • Schlessinger J.
      ) (provided by Joseph Schlessinger, New York University Medical Center) were expressed in the pGEX-3X vector (Pharmacia Biotech Inc.): p85 SH2(N) (amino acids 321-440), p85 SH3/SH2(N) (amino acids 1-440), and p85 SH2(N+C) (amino acids 321-725). p85 SH3 (amino acids 1-80) in pGEX-2T has been described (
      • Kapeller R.
      • Prasad K.V.S.
      • Janssen O.
      • Hou W.
      • Schaffhausen B.S.
      • Rudd C.E.
      • Cantley L.C.
      ).
      Fusion proteins were affinity-purified on glutathione-Sepharose beads (Pharmacia Biotech Inc.) using the Triton X-100-soluble fraction of isopropyl-1-thio-β-D-galactopyranoside-induced Escherichia coli (DH5α strain) as described(
      • Fukazawa T.
      • Reedquist K.A.
      • Trub T.
      • Soltoff S.
      • Panchamoorthy G.
      • Druker B.
      • Cantley L.
      • Shoelson S.E.
      • Band H.
      ,
      • Panchamoorthy G.
      • Fukazawa T.
      • Stolz L.
      • Payne G.
      • Reedquist K.
      • Shoelson S.
      • Zhou S.
      • Cantley L.
      • Walsh C.
      • Band H.
      ,
      • Reedquist K.A.
      • Fukazawa T.
      • Druker B.
      • Panchamoorthy G.
      • Shoelson S.E.
      • Band H.
      ). Proteins were quantitated by the Bradford assay (Bio-Rad) against a bovine serum albumin standard and analyzed on Coomassie gels to confirm quantitation and to assess purity (usually >95%).

      Transient Expression of PI-3-K p85 in SV40 Large T Antigen-expressing Jurkat Derivative JMC-T

      pCG.p85-HA (
      • Klippel A.
      • Escobedo J.A.
      • Hu Q.
      • Williams L.T.
      ) (a gift from Dr. L. Williams, University of California, San Francisco) encodes murine PI-3-K p85α tagged on its C terminus with the influenza HA epitope (for monoclonal antibody 12CA5) (
      • Wilson I.A.
      • Niman H.L.
      • Houghten R.A.
      • Cherenson A.R.
      • Connolly M.L.
      • Lerner R.A.
      ) under control of the SV40 promoter. The JMC-T cell line was derived by electroporation of Jurkat JMC with pCMVneo.SVT (SV40 large T DNA sequences cloned in the pCMVneo vector downstream of the cytomegalovirus promoter(
      • Baker S.J.
      • Markowitz S.
      • Fearon E.R.
      • Wilson J.K.V.
      • Vogelstein B.
      ); a gift from V. Band, New England Medical Center, Boston). High SV40 T antigen expression was verified by immunoprecipitation (data not shown). 4 × 107 cells in 0.5 ml of phosphate-buffered saline (137 mM NaCl, 15.7 mM NaH2PO4, 1.47 mM KH2PO4, 2.68 mM KCl, pH 7.4) were mixed with 50 μg of pCG.p85-HA plasmid DNA and subjected to electroporation as described (
      • Bukowski J.F.
      • Morita C.T.
      • Tanaka Y.
      • Bloom B.R.
      • Brenner M.B.
      • Band H.
      ). Cells were used after 72 h.

      Activation of Jurkat Cells

      Cells were washed and resuspended in RPMI 1640 medium with 20 mM HEPES at 1-2 × 108/ml and then incubated either without (control) or with anti-CD3 monoclonal antibody (SPV-T3b or 2Ad2; 1:200 ascites) for 2 min or as specified. Cells were lysed at 5 × 107/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 each leupeptin and pepstatin, 1 mM vanadate, and 10 mM sodium fluoride).

      Binding of Cellular Polypeptides in Lysates to GST Fusion Proteins

      10 μg of purified GST fusion proteins noncovalently coupled to glutathione-Sepharose beads were rocked with lysate from 5 × 107 cells for 1 h at 4°C and washed six times in lysis buffer. Bound proteins were solubilized in Laemmli sample buffer with 2-mercaptoethanol, resolved by SDS-polyacrylamide gel electrophoresis (PAGE) under reducing conditions, and subjected to immunoblotting.

      Immunoprecipitations and Gel Electrophoresis of Proteins

      Antibodies were added to lysates (5 × 107 cell equivalents) precleared with Staphylococcus aureus Cowan I strain (Pansorbin, Calbiochem). After 1-2 h of rocking at 4°C, 20 μl of protein A-Sepharose 4B beads (Pharmacia Biotech Inc.) were added, and incubation was continued for 45-60 min. Beads were washed six times in lysis buffer, and bound proteins were solubilized in Laemmli sample buffer and subjected to SDS-PAGE and immunoblotting.

      Immunoblotting

      Polypeptides were transferred to PVDF membranes (Immobilon-P, Millipore Corp., Bedford, MA), which were blocked at room temperature with 2% gelatin (Bio-Rad) in TBS-T (10 mM Tris, pH 8, 150 mM NaCl2, 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 protein A-horseradish peroxidase (1:20,000; Cappel-Organon Technika, Durham, NC) for 1 h at room temperature, washed six times, and subjected to enhanced chemiluminescence (ECL) detection according to the supplier's recommendations (Renaissance™ chemiluminescence reagent and Reflection™ autoradiographic film, DuPont NEN). 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.

      Direct Binding of GST Fusion Proteins to Phosphotyrosyl Proteins Immobilized on PVDF Membranes

      Anti-pY immunoprecipitates from 108 Jurkat T cells were resolved by SDS-PAGE and transferred to PVDF membranes. After blocking with 2% gelatin in TBS-T, membranes were rocked with 2.5 μg/ml soluble GST fusion proteins in lysis buffer containing 1 mg/ml gelatin for 1 h at 4°C. Membranes were washed six times and serially incubated with goat anti-GST antiserum (1:200; Pharmacia Biotech Inc.) and protein A-horseradish peroxidase in TBS-T with 0.1% Triton X-100 for 1 h each. Washed filters were subjected to ECL detection.

      Measurement of the PI-3-K 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 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-K activity, PI (Avanti Polar Lipids) plus [γ-32P]ATP (10 μCi/sample) was added to the immunoprecipitates for 10 min at room temperature. The PI was 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 [γ-32P]ATP was added in a mixture that provided a final concentration of 50 μM ATP, 5 mM MgCl2, 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 TLC plates (Silica Gel 60, MCB reagents, Merck) and developed in chloroform/methanol/water/ammonium hydroxide (60:47:11.3:2), and lipid species were visualized by autoradiography.

      RESULTS

      Coimmunoprecipitation of pp36/38 with the p85 Subunit of PI-3-K in Anti-CD3-stimulated Jurkat T Cells

      Prior studies have shown that the PI-3-K activity as well as the PI-3-K p85 protein are recruited into phosphotyrosyl protein complexes upon TCR triggering(
      • Thompson P.A.
      • Gutkind J.S.
      • Robbins K.C.
      • Ledbetter J.A.
      • Bolen J.B.
      ,
      • Ward S.G.
      • Reif K.
      • Ley S.
      • Fry M.J.
      • Waterfield M.D.
      • Cantrell D.A.
      ,
      • Prasad K.V.
      • Janssen O.
      • Kapeller R.
      • Raab M.
      • Cantley L.C.
      • Rudd C.E.
      ,
      • Reif K.
      • Gout I.
      • Waterfield M.D.
      • Cantrell D.A.
      ,
      • Carrera A.C.
      • Rodriguez-Borlado L.
      • Martinez-Alonso C.
      • Merida I.
      ,
      • Exley M.
      • Varticovski L.
      • Peter M.
      • Sancho J.
      • Terhorst C.
      ); anti-p85 immunoblotting of anti-pY immunoprecipitates from anti-CD3-stimulated Jurkat cells confirmed this observation (Fig. 1A, lanes11 and 12). Furthermore, anti-pY immunoprecipitates were also associated with a substantial level of phosphoinositide kinase activity (Fig. 1B). However, the PI-3-K subunits themselves are not tyrosine-phosphorylated upon TCR triggering (
      • Ward S.G.
      • Reif K.
      • Ley S.
      • Fry M.J.
      • Waterfield M.D.
      • Cantrell D.A.
      ,
      • Reif K.
      • Gout I.
      • Waterfield M.D.
      • Cantrell D.A.
      ) (data not shown), suggesting a role for interactions of the p85 SH2 domains with other proteins in mediating PI-3-K recruitment into phosphotyrosyl protein complexes. As we (
      • Fukazawa T.
      • Reedquist K.A.
      • Trub T.
      • Soltoff S.
      • Panchamoorthy G.
      • Druker B.
      • Cantley L.
      • Shoelson S.E.
      • Band H.
      ) and others (
      • Meisner H.
      • Conway B.R.
      • Hartley D.
      • Czech M.P.
      ) have recently demonstrated, one of these proteins is p120cbl; this is seen as a 120-kDa tyrosine-phosphorylated band in anti-p85 immunoprecipitates from activated cells (Fig. 1A, lane6) that comigrates with directly immunoprecipitated p120cbl(lanes 9 and 10) and by coimmunoprecipitation of the p85 protein in anti-Cbl immunoprecipitates (anti-p85 blot; lanes 9 and 10). Additional tyrosine-phosphorylated polypeptides migrating at 100, 75 (seen upon longer exposures), and 36-38 kDa were also observed in anti-p85 immunoprecipitates; the 36-38-kDa polypeptide consistently showed the highest phosphotyrosine content. Similar results were observed using a different anti-PI-3-K p85 antibody (a gift from Dr. B. Schaffhausen, Tufts University, Boston) (data not shown). The p85-associated 36-38-kDa phosphoprotein migrated identically to pp36/38 associated with Grb2 (lanes 3 and 4) and phospholipase C-γ1 (lanes7 and 8)(
      • Weber J.R.
      • Bell G.M.
      • Han M.Y.
      • Pawson T.
      • Imboden J.B.
      ,
      • Gilliland L.K.
      • Schieven G.L.
      • Norris N.A.
      • Kanner S.B.
      • Aruffo A.
      • Ledbetter J.A.
      ,
      • Sieh M.
      • Batzer A.
      • Schlessinger J.
      • Weiss A.
      ,
      • Buday L.
      • Egan S.E.
      • Viciana P.R.
      • Cantrell D.A.
      • Downward J.
      ). In keeping with the association of Grb2 and PI-3-K p85 with a shared set of phosphotyrosyl proteins (p120cbl, p100, p75, and pp36/38), coimmunoprecipitation of p85 with Grb2 (lanes 3 and 4) and of Grb2 with p85 (lanes 5 and 6) was also observed. Thus, in addition to phospholipase C-γ1 and Grb2(
      • Weber J.R.
      • Bell G.M.
      • Han M.Y.
      • Pawson T.
      • Imboden J.B.
      ,
      • Gilliland L.K.
      • Schieven G.L.
      • Norris N.A.
      • Kanner S.B.
      • Aruffo A.
      • Ledbetter J.A.
      ,
      • Sieh M.
      • Batzer A.
      • Schlessinger J.
      • Weiss A.
      ,
      • Buday L.
      • Egan S.E.
      • Viciana P.R.
      • Cantrell D.A.
      • Downward J.
      ), pp36/38 appears to associate with the p85 subunit of PI-3-K.
      Figure thumbnail gr1
      Figure 1:In vivo association of pp36/38 with PI-3-K p85 in anti-CD3-stimulated Jurkat T cells. A, immunoprecipitates from lysates of 5 × 107 unstimulated (-) or anti-CD3 (SPV-T3b)-stimulated (+) Jurkat cells with the indicated antibodies (I.P.; shown on top) or whole cell lysates (106 cells) were resolved by SDS-9% PAGE, transferred to PVDF membranes, and subjected to immunoblotting with the antibodies indicated on the right, followed by protein A-horseradish peroxidase and ECL detection. Immunoprecipitated species are indicated on left. PLCγ1, phospholipase C-γ1; Ig, immunoglobulin heavy chain; NRS, normal rabbit serum. The threelowerpanels represent a reprobing of different parts of the filter shown in the toppanel. B, association of the PI-3-K activity with phosphotyrosyl proteins. Immunoprecipitations, carried out as described for A, were subjected to lipid kinase assay as described under “Materials and Methods.” The reaction products were subjected to TLC and visualized by autoradiography. PIP, phosphorylated inositides.

      Association of pp36/38 with Transfected Epitope-tagged PI-3-K p85

      To rule out the possibility that coimmunoprecipitation of pp36/38 with PI-3-K p85 may reflect cross-reactivity of the anti-p85 antibody, we examined the interaction of endogenous pp36/38 with transfected PI-3-K p85α tagged near its C terminus with the anti-HA antibody epitope. HA-tagged PI-3-K p85α was transiently transfected into a SV40 large T antigen-expressing Jurkat cell line (JMC-T; see “Materials and Methods”). After 72 h, lysates of unstimulated or anti-CD3-stimulated cells were subjected to immunoprecipitations and immunoblotting (Fig. 2). Anti-PI-3-K p85 immunoblotting of the whole cell lysates (lanes11 and 12versus23 and 24) and of anti-PI-3-K p85 (lanes5 and 6 versus17 and 18) or anti-HA tag (lanes 7 and 8 versus19 and 20) immunoprecipitates demonstrated the overexpression of tagged PI-3-K p85 specifically in the p85-HA transfectant. Anti-pY immunoblotting showed that the anti-HA antibody coimmunoprecipitated pp36/38 (and p120cbl) specifically in p85-HA-transfected cells (lanes 7 and 8 versus19 and 20); tyrosine phosphorylation of these associated polypeptides increased upon activation, similar to that observed in anti-PI-3-K p85 immunoprecipitates from p85-HA-transfected (lanes 17 and 18) and mock-transfected (lanes 5 and 6) cells. Again, p85-associated pp36/38 comigrated with Grb2-associated pp36/38 (lanes 9 and 10 and lanes21 and 22). These transfection analyses confirmed the association of pp36/38 with PI-3-K p85 in Jurkat T cells.
      Figure thumbnail gr2
      Figure 2:In vivo association of pp36/38 with transfected HA-tagged PI-3-K p85α. JMC-T cells were either mock-transfected or transfected with pCG.85-HA plasmid DNA (p85-HA transfectant) and analyzed after 72 h. Immunoprecipitates from 5 × 107 unstimulated(-) or anti-CD3 (SPV-T3b)-stimulated (+) cells were subjected to immunoblotting with the indicated antibodies, followed by protein A-horseradish peroxidase and ECL detection. I.P., immunoprecipitate; NRS, normal rabbit serum; HA, influenza hemagglutinin epitope recognized by monoclonal antibody 12CA5. Immunoprecipitated species are shown on the left. The p85 and Grb2 blots represent serial reprobing of the filter used for the anti-pY blot.

      Kinetics of Tyrosine Phosphorylation of PI-3-K p85-associated pp36/38

      pp36/38 is one of the relatively early substrates of tyrosine phosphorylation upon TCR triggering(
      • Weber J.R.
      • Bell G.M.
      • Han M.Y.
      • Pawson T.
      • Imboden J.B.
      ,
      • Sieh M.
      • Batzer A.
      • Schlessinger J.
      • Weiss A.
      ,
      • Buday L.
      • Egan S.E.
      • Viciana P.R.
      • Cantrell D.A.
      • Downward J.
      ); increased phosphorylation is detectable in whole cell lysates within 10 s, is maximal by ∼2 min, and declines thereafter (Fig. 3, toppanel). To determine the kinetics of tyrosine phosphorylation of PI-3-K p85-associated pp36/38, we carried out anti-pY immunoblotting of anti-p85 immunoprecipitates from Jurkat cells stimulated for various time periods. Phosphorylation of PI-3-K p85-associated pp36/38 (bottompanel) was comparable to that observed in whole cell lysates (toppanel) and on the Grb2-associated pp36/38 (middlepanel). Anti-p85 and anti-Grb2 immunoblotting confirmed that equal amounts of these proteins were immunoprecipitated at each time point.
      Figure thumbnail gr3
      Figure 3:Time course of tyrosine phosphorylation of PI-3-K p85- and Grb2-associated pp36/38 upon T cell activation. Whole cell lysates (106 cells; toppanel), anti-Grb2 immunoprecipitates (5 × 107 cells; second and thirdpanels), or anti-PI-3-K p85 immunoprecipitates (fourth and fifthpanels) from unstimulated(-) or anti-CD3 (2Ad2)-stimulated (+) Jurkat cells were resolved by SDS-PAGE and subjected to immunoblotting with the antibodies shown on the right, followed by protein A-horseradish peroxidase and ECL detection. Time of anti-CD3 stimulation is shown in seconds (s) or minutes (m). Anti-Grb2 and anti-p85 immunoblots represent reprobing of the respective filters. I.P., immunoprecipitate.

      pp36/38 Selectively Binds to SH2 Domains of PI-3-K p85

      To assess the role of PI-3-K p85 SH2 and SH3 domains in pp36/38 binding, we incubated the PI-3-K p85-derived GST fusion proteins with lysates of anti-CD3-stimulated Jurkat T cells and detected the bound proteins by anti-pY immunoblotting (Fig. 4A). As positive controls, GST fusion proteins of Grb2 or its SH2 domain were also included. pp36/38 binding was observed with PI-3-K p85 fusion proteins that included either the two SH2 domains (SH2(N+C); lane5) or SH2(N) together with the SH3 domain (lane6). In contrast, no pp36/38 binding was observed with the p85 SH3 fusion protein (lane4), although this protein was active, as shown by its binding to p120cbl (data not shown). These results suggest that pp36/38 interacts with the SH2 domains (but not the SH3 domain) of PI-3-K p85.
      Figure thumbnail gr4
      Figure 4:Binding of pp36/38 to GST fusion proteins of PI-3-K p85 and selective inhibition of binding by p85 SH2-specific phosphotyrosyl peptides. A, SH2 domains (but not the SH3 domain) of PI-3-K p85 are capable of binding to pp36/38. Binding reactions were carried out by incubating lysate from 2 × 107 anti-CD3 (SPV-T3b)-stimulated Jurkat cells (in a 2-ml volume) with 10 μg of the indicated GST fusion proteins noncovalently immobilized on glutathione-Sepharose beads (5 μl of packed beads) for 1 h. Whole cell lysate (106 cells) or binding reactions were subjected to anti-pY immunoblotting, followed by protein A-horseradish peroxidase and ECL detection. Only the pp36/38 portion of the blot is shown. B, binding of pp36/38 to GST-p85 SH2(N+C) is selectively abrogated by PI-3-K p85 SH2-specific phosphotyrosyl peptides. Competing peptides were separately added 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 described for A. Peptides were EPQpYEEIPIYL (pYEEI), EPQYEEIPIYL (YEEI), PSpYVNVQNL (pYVNV), PSpYVAVQNL (pYVAV), HSDpYMNMTPR (pYMNM), and HSDYMNMTPR (YMNM). -, no peptide. pYMNM is p85 SH2-specific; pYVNV is Grb2 SH2-specific; and pYEEI is Src SH2-specific.

      Specific Peptide Inhibition of p36/38 Binding to PI-3-K p85 Fusion Proteins

      To further delineate the mechanism of pp36/38-PI-3-K p85 interaction, we determined the effects of specific phosphotyrosyl peptides on pp36/38 binding to the GST-p85 SH2(N+C) fusion protein (Fig. 4B). Binding of pp36/38 to p85 SH2(N+C) was significantly inhibited at 0.5 μM and nearly completely abrogated at a 4 μM concentration of the p85 SH2-specific pYMNM peptide (lanes12 and 13); in contrast, 100 μM unphosphorylated YMNM peptide (lane16) and pYEEI (Src SH2-specific) (lane17), YEEI (lane18), and pYVAV (lane20) peptides failed to inhibit binding, and only partial inhibition was seen with 100 μM pYVNV peptide (Grb2 SH2-specific; lane19). Binding of pp36/38 to Grb2 was nearly completely inhibited by 4 μM pYVNV (lane3), but was only slightly decreased by 100 μM pYMNM peptide (lane9) and was unaffected by other peptides. These results demonstrate the crucial requirement of phosphotyrosyl peptide recognition in binding between p85 SH2 domains and pp36/38.

      PI-3-K p85 SH2 Domain Binding to pp36/38 Is Direct

      Since the above coimmunoprecipitation and fusion protein binding experiments were carried out under nondenaturing detergent conditions, it remained possible that the pp36/38-PI-3-K p85 interaction might be indirectly mediated through another protein, such as Grb2. To assess direct binding, anti-pY immunoprecipitates of unstimulated or anti-CD3-stimulated Jurkat cells were resolved by SDS-PAGE; transferred to PVDF membrane; and subjected to blotting with GST or with the GST-Grb2 or GST-p85 SH2(N+C) fusion protein. Binding of fusion proteins was detected with an anti-GST antibody. Compared to background binding with GST, GST-Grb2 showed specific binding to pp36/38 and additional polypeptides in anti-pY immunoprecipitates from activated cells (Fig. 5, lane6). Notably, a prominent binding of the p85 SH2(N+C) fusion protein to pp36/38 was observed (lane10) in addition to its binding to several other phosphoproteins. Equal loading of phosphotyrosyl proteins in each blot was demonstrated by anti-pY reimmunoblotting of the membranes. These results demonstrate that pp36/38 directly interacts with the SH2 domains of PI-3-K p85.
      Figure thumbnail gr5
      Figure 5:Direct binding of PI-3-K p85 SH2(N+C) fusion protein to membrane-immobilized pp36/38. Anti-pY immunoprecipitates from 108 unstimulated(-) or anti-CD3 (SPV-T3b)-stimulated (+) Jurkat T cells were resolved by SDS-PAGE, transferred to PVDF membrane, and incubated with 2.5 μg/ml GST (leftpanel, lanes 1 and 2), GST-Grb2 (middlepanel, lanes 5 and 6), or GST-p85 SH2(N+C) (right panel, lanes 9 and 10) in lysis buffer containing 1 mg/ml gelatin. After 1 h of incubation at 4°C, filters were probed with anti-GST antibody, followed by protein A-horseradish peroxidase and ECL detection. Each blot was reprobed with anti-pY antibody (lanes 3 and 4, 7 and 8, and 11 and 12) and shows similar phosphotyrosyl protein signals. pp36/38 and p120cbl are indicated.

      DISCUSSION

      Stimulation through the TCR leads to an increase in the PI-3-K activity and activation-dependent incorporation of this enzyme into phosphotyrosyl protein complexes(
      • Thompson P.A.
      • Gutkind J.S.
      • Robbins K.C.
      • Ledbetter J.A.
      • Bolen J.B.
      ,
      • Ward S.G.
      • Reif K.
      • Ley S.
      • Fry M.J.
      • Waterfield M.D.
      • Cantrell D.A.
      ,
      • Prasad K.V.
      • Janssen O.
      • Kapeller R.
      • Raab M.
      • Cantley L.C.
      • Rudd C.E.
      ,
      • Reif K.
      • Gout I.
      • Waterfield M.D.
      • Cantrell D.A.
      ,
      • Carrera A.C.
      • Rodriguez-Borlado L.
      • Martinez-Alonso C.
      • Merida I.
      ,
      • Exley M.
      • Varticovski L.
      • Peter M.
      • Sancho J.
      • Terhorst C.
      ). Since p110 or p85 subunits of PI-3-K are not tyrosine-phosphorylated upon TCR triggering (
      • Ward S.G.
      • Reif K.
      • Ley S.
      • Fry M.J.
      • Waterfield M.D.
      • Cantrell D.A.
      ,
      • Reif K.
      • Gout I.
      • Waterfield M.D.
      • Cantrell D.A.
      ) (data not shown), the incorporation of this enzyme into phosphotyrosyl complexes must occur through its interactions with other proteins. It has been shown that SH3 domains of the Src family tyrosine kinases can interact in vitro with PI-3-K p85 by recognizing its proline-rich peptide motifs(
      • Prasad K.V.
      • Janssen O.
      • Kapeller R.
      • Raab M.
      • Cantley L.C.
      • Rudd C.E.
      ,
      • Kapeller R.
      • Prasad K.V.S.
      • Janssen O.
      • Hou W.
      • Schaffhausen B.S.
      • Rudd C.E.
      • Cantley L.C.
      ,
      • Pleiman C.M.
      • Clark M.R.
      • Gauen L.K.
      • Winitz S.
      • Coggeshall K.M.
      • Johnson G.L.
      • Shaw A.S.
      • Cambier J.C.
      ). However, we observed a significantly lower level of PI-3-K activity associated with immunoprecipitates of Fyn tyrosine kinase compared with that observed in anti-pY immunoprecipitates (
      • Fukazawa T.
      • Reedquist K.A.
      • Trub T.
      • Soltoff S.
      • Panchamoorthy G.
      • Druker B.
      • Cantley L.
      • Shoelson S.E.
      • Band H.
      ) (data not shown). An alternate mechanism to recruit PI-3-K into signaling complexes is through binding of the SH3 and SH2 domains of its p85 subunit to signaling proteins. Recent studies have identified one such protein. We (
      • Fukazawa T.
      • Reedquist K.A.
      • Trub T.
      • Soltoff S.
      • Panchamoorthy G.
      • Druker B.
      • Cantley L.
      • Shoelson S.E.
      • Band H.
      ) and others (
      • Meisner H.
      • Conway B.R.
      • Hartley D.
      • Czech M.P.
      ) have demonstrated a primarily SH2 domain-mediated association of PI-3-K p85 with p120cbl, a rapidly tyrosine-phosphorylated protein in T cells, and a substantial level of PI-3-K activity coimmunoprecipitated with p120cbl. In the present report, we have identified and characterized the interaction of PI-3-K p85 with pp36/38, a major substrate of TCR-dependent tyrosine phosphorylation that has been shown to also associate with Grb2 and phospholipase C-γ1 in T cells(
      • Weber J.R.
      • Bell G.M.
      • Han M.Y.
      • Pawson T.
      • Imboden J.B.
      ,
      • Gilliland L.K.
      • Schieven G.L.
      • Norris N.A.
      • Kanner S.B.
      • Aruffo A.
      • Ledbetter J.A.
      ,
      • Sieh M.
      • Batzer A.
      • Schlessinger J.
      • Weiss A.
      ,
      • Buday L.
      • Egan S.E.
      • Viciana P.R.
      • Cantrell D.A.
      • Downward J.
      ).
      Using both parental and transfected Jurkat T cells, we have demonstrated in vivo association between pp36/38 and PI-3-K p85. pp36/38 was the most prominent phosphotyrosyl protein associated with PI-3-K p85. In vitro binding experiments using GST fusion proteins demonstrated that pp36/38 (at least the fraction that is detectable with the anti-pY antibody) interacts specifically with the SH2 domains (but not the SH3 domain) of PI-3-K p85. Peptide competition experiments further demonstrated the requirement for phosphotyrosyl peptide motif recognition. Finally, filter binding assays clearly demonstrated that pp36/38 directly binds to PI-3-K p85. Consistent with this result, PI-3-K p85-associated pp36/38 was still observed (although reduced) after complete immunodepletion of Grb2 and its associated pp36/38 (data not shown).
      It is likely that pp36/38, together with other p85-associated proteins like p120cbl(
      • Fukazawa T.
      • Reedquist K.A.
      • Trub T.
      • Soltoff S.
      • Panchamoorthy G.
      • Druker B.
      • Cantley L.
      • Shoelson S.E.
      • Band H.
      ,
      • Meisner H.
      • Conway B.R.
      • Hartley D.
      • Czech M.P.
      ), plays an important role in recruiting PI-3-K into TCR-induced signaling complexes. We suggest that the interaction of PI-3-K p85 with TCR-induced phosphotyrosyl proteins is likely to complement other previously described mechanisms, such as Fyn SH3 binding to proline-rich regions of PI-3-K p85(
      • Prasad K.V.
      • Janssen O.
      • Kapeller R.
      • Raab M.
      • Cantley L.C.
      • Rudd C.E.
      ,
      • Kapeller R.
      • Prasad K.V.S.
      • Janssen O.
      • Hou W.
      • Schaffhausen B.S.
      • Rudd C.E.
      • Cantley L.C.
      ,
      • Pleiman C.M.
      • Clark M.R.
      • Gauen L.K.
      • Winitz S.
      • Coggeshall K.M.
      • Johnson G.L.
      • Shaw A.S.
      • Cambier J.C.
      ), to recruit this enzyme into TCR signaling. Binding of tyrosine-phosphorylated proteins or peptides to PI-3-K p85 has been demonstrated to activate the associated 110-kDa catalytic subunit of PI-3-K(
      • Myers Jr., M.G.
      • Backer J.M.
      • Sun X.J.
      • Shoelson S.
      • Hu P.
      • Schlessinger J.
      • Yoakim M.
      • Schaffhausen B.
      • White M.F.
      ,
      • Carpenter C.L.
      • Auger K.R.
      • Chaudhuri M.
      • Yoakim M.
      • Schaffhausen B.
      • Shoelson S.
      • Cantley L.C.
      ). Therefore, it is quite possible that binding of pp36/38 to PI-3-K p85 may also activate the PI-3-K enzyme.
      The pp36/38-PI-3-K interaction was primarily observed in activated cells, reflecting the fact that at present we can detect pp36/38 only as a phosphotyrosyl protein. Consistent with a TCR activation-dependent association between pp36/38 and PI-3-K p85, in vitro binding and peptide competition experiments demonstrated that pp36/38-PI-3-K p85 interactions were exclusively SH2-mediated. However, we cannot rule out an activation-independent complex of unphosphorylated pp36/38 with PI-3-K. Such analyses await identification of the pp36/38 protein.
      It is of significant interest that pp36/38 interacts directly with the SH2 domains of PI-3-K p85 (this study) as well as those of phospholipase C-γ1 and Grb2 ((
      • Weber J.R.
      • Bell G.M.
      • Han M.Y.
      • Pawson T.
      • Imboden J.B.
      ,
      • Gilliland L.K.
      • Schieven G.L.
      • Norris N.A.
      • Kanner S.B.
      • Aruffo A.
      • Ledbetter J.A.
      ,
      • Sieh M.
      • Batzer A.
      • Schlessinger J.
      • Weiss A.
      ,
      • Buday L.
      • Egan S.E.
      • Viciana P.R.
      • Cantrell D.A.
      • Downward J.
      ) and this study). Given the specificity of these different SH2 domains for distinct phosphotyrosyl 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.
      ), it is likely that various SH2 domain-containing signaling proteins can concurrently interact with pp36/38. Coimmunoprecipitation between Grb2 and PI-3-K p85 (Fig. 1A) and between Grb2 and phospholipase C-γ1 (Fig. 1A) (
      • Sieh M.
      • Batzer A.
      • Schlessinger J.
      • Weiss A.
      ) is consistent with this proposal. In additional experiments, we have observed that pp36/38 coimmunoprecipitates with Lck when Jurkat T cells are stimulated and that the Lck SH2 (but not SH3) domain can bind to pp36/38 in vitro (data not shown). Thus, pp36/38 and other proteins with multiple SH2-binding sites (e.g. p120cbl) (
      • Fukazawa T.
      • Reedquist K.A.
      • Trub T.
      • Soltoff S.
      • Panchamoorthy G.
      • Druker B.
      • Cantley L.
      • Shoelson S.E.
      • Band H.
      ) are likely to substitute for multiple SH2 docking sites that are created on growth factor receptors (such as the epidermal growth factor receptor) by autophosphorylation. In this regard, it is noteworthy that pp36/38 in its tyrosine-phosphorylated form has been demonstrated to be cell membrane-associated(
      • Sieh M.
      • Batzer A.
      • Schlessinger J.
      • Weiss A.
      ,
      • Buday L.
      • Egan S.E.
      • Viciana P.R.
      • Cantrell D.A.
      • Downward J.
      ).

      Acknowledgments

      We thank Drs. Paul Anderson, Vimla Band, Brian Schaffhausen, Mike Moran, Ellis Reinherz, Lewis Williams, and Joseph Schlessinger for critical reagents and Rob Littlefield for artwork.

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