Advertisement

Monocyte Colony-stimulating Factor Stimulates Binding of Phosphatidylinositol 3-Kinase to Grb2·Sos Complexes in Human Monocytes *

  • Ahamed Saleem
    Affiliations
    Division of Cancer Pharmacology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115
    Search for articles by this author
  • Surender Kharbanda
    Correspondence
    To whom correspondence should be addressed.
    Affiliations
    Division of Cancer Pharmacology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115
    Search for articles by this author
  • Zhi-Min Yuan
    Affiliations
    Division of Cancer Pharmacology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115
    Search for articles by this author
  • Donald Kufe
    Affiliations
    Division of Cancer Pharmacology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115
    Search for articles by this author
  • Author Footnotes
    * This investigation was supported by United States Public Health Service Grant CA42802 awarded by the National Cancer Institute, DHHS. 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.
Open AccessPublished:May 05, 1995DOI:https://doi.org/10.1074/jbc.270.18.10380
      Monocyte colony-stimulating factor (M-CSF) is required for the proliferation of mononuclear phagocytes. The activated M-CSF receptor associates with phosphatidylinositol 3-kinase (PI 3-kinase). In the present studies, we demonstrate that M-CSF also induces direct interaction of PI 3-kinase (p85α subunit) with the SH2/SH3 adaptor protein Grb2. Tyrosine-phosphorylated PI 3-kinase interacts with the SH2 domain of Grb2. A pYRNE (pY408) site in PI 3-kinase is potentially involved in this interaction. The results also demonstrate that the PI 3-kinase·Grb2 complex associates with the guanine nucleotide exchange protein Sos. Since Sos binds to the SH3 domains of Grb2 and thereby associates with Ras at the cell membrane, formation of the PI 3-kinase·Grb2·Sos complex provides a potential mechanism for growth factor-induced interactions of PI 3-kinase and Ras.

      INTRODUCTION

      The M-CSF1(
      The abbreviations used are: M-CSF
      monocyte colony-stimulating factor
      DTT
      dithiothreitol
      PAGE
      polyacrylamide gel electrophoresis
      PBS
      phosphate-buffered saline.
      ) receptor is a transmembrane protein tyrosine kinase encoded by the c- fms gene (
      • Sherr C.J.
      • Rettenmier C.W.
      • Sacca R.
      • Roussel M.F.
      • Look A.T.
      • Stanley E.R.
      ,
      • Sherr C.
      ). M-CSF stimulates receptor dimerization and autophosphorylation at Tyr697, Tyr706, and Tyr721 in the kinase domain (
      • Van der Geer P.
      • Hunter T.
      ,
      • Reedijk M.
      • Liu X.
      • Geer P.V.D.
      • Letwin K.
      • Waterfield M.D.
      • Hunter T.
      • Pawson T.
      ). Studies have demonstrated that Tyr721 serves as a binding site for PI 3-kinase (
      • Reedijk M.
      • Liu X.
      • Geer P.V.D.
      • Letwin K.
      • Waterfield M.D.
      • Hunter T.
      • Pawson T.
      ), whereas the adaptor protein Grb2 associates with phosphotyrosine 697 (
      • Van der Geer P.
      • Hunter T.
      ). PI 3-kinase is a heterodimer consisting of an 85-kDa SH2 domain-containing subunit which links the catalytic 110-kDa subunit to tyrosine-phosphorylated proteins. PI 3-kinase is responsible for growth factor-induced phosphorylation of phosphoinositides at the 3’ position (
      • Auger K.R.
      • Serunian L.A.
      • Soltoff S.P.
      • Libby P.
      • Cantley L.C.
      ). One such phosphorylated product, phosphatidylinositol 4,5-triphosphate, has been implicated in the regulation of PKC ζ (
      • Nakanishi H.
      • Brewer K.A.
      • Exton J.H.
      ). Stimulation of PI 3-kinase activity has been associated with binding of the 85-kDa subunit to phosphotyrosine (
      • Backer J.M.
      • Myers Jr., M.G.
      • Shoelson S.E.
      • Chin D.J.
      • Sun X.J.
      • Miralpeiz M.
      • Hu P.
      • Margolis B.
      • Skolnik E.Y.
      • Schlessinger J.
      • White M.F.
      ,
      • Myers Jr., M.G.
      • Backer J.M.
      • Sun Z.J.
      • Shoelson S.
      • Hu P.
      • Schlessinger J.
      • Yoakim M.
      • Schaffhausen B.
      • White M.F.
      ). While tyrosine phosphorylation of PI 3-kinase may also be associated with increases in activity, recent studies have demonstrated that PI 3-kinase is directly activated by Ras (
      • Rodriguez-Viciana P.
      • Warne P.H.
      • Dhand R.
      • Vanhaesebroeck B.
      • Gout I.
      • Fry M.J.
      • Waterfield M.D.
      • Downward J.
      ). The mechanisms, however, responsible for growth factor-induced PI 3-kinase activity and the basis for the interaction with Ras are unclear.
      In the present studies, we demonstrate that M-CSF induces direct association of the p85α subunit of PI 3-kinase with the SH2 domain of Grb2. Our finding that M-CSF also induces the formation of a PI 3-kinase·Grb2·Sos complex supports a potential role for PI 3-kinase in Ras signaling pathways in monocytes.

      EXPERIMENTAL PROCEDURES

      Monocyte Isolation and Culture

      Human monocytes were isolated from the peripheral blood of healthy volunteers by Ficoll-Paque separation, followed by adherence for 1 h and removal of the nonadherent cells (
      • Nakamura T.
      • Lin L.-L.
      • Kharbanda S.
      • Knopf J.
      • Kufe D.
      ,
      • Sariban E.
      • Mitchell T.
      • Kufe D.
      ). The monocytes were treated with 1000 units/ml human recombinant M-CSF (specific activity, 1.90 × 106 units/ml; Genetics Institute, Cambridge, MA).

      Antibodies and Fusion Proteins

      Anti-p85α and anti-Sos antibodies were purchased from Santa Cruz Biotechnology (San Diego, CA). Anti-Grb2 and anti-M-CSF receptor antibodies were obtained from Transduction Laboratory (Lexington, KY) and Upstate Biotechnology, Inc. (Lake Placid, NY), respectively. GST-Grb2 (full length), GST-Grb2 SH2, GST-Grb2 N-SH3, and GST-Grb2 C-SH3 fusion proteins were purchased from Santa Cruz Biotechnology.

      Immunoprecipitations and Immunoblotting

      Cell lysates were prepared by resuspending cells for 30 min on ice in lysis buffer (50 mM Tris, pH 7.6, 1% Brij-96, 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium vanadate, 1 mM DTT, 10 mM sodium fluoride, and 10 μg/ml each of leupeptin and aprotinin). Equal amounts of proteins (250-300 μg) were immunoprecipitated by incubation with anti-M-CSF-receptor, anti-p85α, anti-Grb2, or anti-Sos for 2 h at 4°C and then with protein A-Sepharose (Pharmacia Biotech Inc.) for an additional 30 min. The resulting precipitates were washed four times with lysis buffer and resolved by SDS-PAGE under reducing conditions. Proteins were then transferred to nitrocellulose by semi-dry transfer (Bio-Rad), blocked by incubation in 5% dry milk in PBST (0.5% Tween 20 in PBS), and then probed with appropriate antibodies. The blots were developed by ECL (Amersham Corp.).

      Fusion Protein Binding Assays

      The fusion proteins GST, GST-Grb2, GST-Grb2 SH2, GST-Grb2 N-SH3, or GST-Grb2 C-SH3 were purified by affinity chromatography using glutathione-Sepharose beads and equilibrated in lysis buffer. Cell lysates were incubated with 2 μg of immobilized GST, GST-Grb2, GST-Grb2 SH2, GST-Grb2 N-SH3, or GST-Grb2 C-SH3 fusion proteins for 2 h at 4°C. The resulting protein complexes were washed three times with lysis buffer containing 0.1% detergent and boiled for 5 min in SDS sample buffer. The complexes were then separated by 7.5% SDS-PAGE and subjected to silver staining or immunoblot analysis with anti-p85α.

      Peptide Synthesis and Competition Assays

      Peptide was synthesized using Fmoc (N-(9-fluorenyl)methoxycarbonyl)-Tyr(PO3Me2)-OH for incorporation of phosphotyrosine and subsequently purified by ether precipitation and preparative reverse-phase high-pressure liquid chromatography (HPLC). Amino acid analysis was used to confirm the sequence of the p85α-derived phosphopeptide: LINHpYRNESLAQ. GST-Grb2 SH2 protein was incubated in the presence or absence of 50 μM tyrosine-phosphorylated synthetic peptide for 1 h at 4°C. The fusion proteins were incubated with lysates from M-CSF-treated monocytes and the adsorbates then analyzed by immunoblotting with anti-p85α.

      Second Immunoprecipitation Assays

      M-CSF-treated cell lysates were immunoprecipitated with anti-p85α for 2 h at 4°C. Immune complexes were released by boiling in 50 mM Tris-HCl, pH 8.0, 0.5% SDS, and 1 mM DTT and then subjected to 1) a second immunoprecipitation with anti-p85α or 2) precipitations with GST or GST-Grb2. The resulting protein precipitates were analyzed by immunoblotting with anti-p85α.

      PI 3-Kinase Assays

      Assays for PI 3-kinase were performed as described (
      • Sung C.K.
      • Sanchez-Margalet V.
      • Goldfien I.D.
      ). Briefly, PI 3-kinase activity was measured directly in immune complexes containing 2 μg/ml phosphatidylinositol (Avanti Polar Lipids, Alabaster, AL), 20 mM HEPES, pH 7.2, 0.5 mM EGTA, 0.5 mM sodium phosphate, 10 mM MgCl2, and [γ-32P]ATP. The reaction was stopped by addition of 4 N HCl and chloroform/methanol (1:1). The organic layer was separated and spotted on a Silica Gel-60 plate (Sigma) and analyzed by thin layer chromatography.

      RESULTS AND DISCUSSION

      Studies in Rat-2 fibroblasts which express the mouse c- fms gene have demonstrated that M-CSF stimulation is associated with binding of PI 3-kinase to the activated M-CSF receptor (
      • Reedijk M.
      • Liu X.
      • Geer P.V.D.
      • Letwin K.
      • Waterfield M.D.
      • Hunter T.
      • Pawson T.
      ). In order to confirm these findings in a physiological system, we stimulated human peripheral blood monocytes with M-CSF and assayed anti-M-CSF receptor immunoprecipitates for the p85α subunit of PI 3-kinase. While there was no detectable anti-p85α reactivity in the immunoprecipitates from unstimulated monocytes, M-CSF treatment rapidly induced binding of p85α to M-CSF receptors (Fig. 1 A). Reprobing the same filter with anti-Tyr(P) demonstrated a M-CSF-dependent increase in reactivity with an 85-kDa protein (Fig. 1 B). These results supported binding of PI 3-kinase to the activated M-CSF receptor and tyrosine phosphorylation of the p85α subunit.
      Figure thumbnail gr1
      Figure 1:Association of PI 3-kinase with M-CSF receptors. Human peripheral blood monocytes were stimulated with M-CSF (1000 units/ml) for 5 min at 37°C. Lysates from control and M-CSF-treated monocytes were immunoprecipitated with anti-M-CSF receptor antibody. The proteins were resolved by 7.5% SDS-PAGE and immunoblotted with anti-p85α (A) or anti-Tyr(P) (B). One of the three independent experiments is shown.
      Other studies in Rat-2 fibroblasts have shown that mutation of Tyr721 in the M-CSF receptor blocks binding of PI 3-kinase and inhibits M-CSF-dependent growth (
      • Reedijk M.
      • Liu X.
      • Geer P.V.D.
      • Letwin K.
      • Waterfield M.D.
      • Hunter T.
      • Pawson T.
      ,
      • Van der Geer P.
      • Hunter T.
      ). While these results support the involvement of PI 3-kinase in growth factor-induced proliferation, recent work has also demonstrated that PI 3-kinase coimmunoprecipitates with Ras (
      • Sjolander A.
      • Yamamoto
      • Huber B.E.
      • Lapetina E.G.
      ,
      • Sjolander A.
      • Lapetina E.G.
      ) and that PI 3-kinase is regulated by Ras (
      • Rodriguez-Viciana P.
      • Warne P.H.
      • Dhand R.
      • Vanhaesebroeck B.
      • Gout I.
      • Fry M.J.
      • Waterfield M.D.
      • Downward J.
      ). These findings suggest that localization of PI 3-kinase at the cell membrane, perhaps through SH2 or SH3 interactions, is necessary for direct activation by Ras. In addressing this issue, we asked whether the SH2-SH3 adaptor protein Grb2, which binds to the guanine nucleotide exchange protein Sos (son of sevenless) and thereby to the Ras activation pathway (
      • Clark S.G.
      • Stern M.J.
      • Horowitz H.R.
      ,
      • Simon M.A.
      • Dodson G.S.
      • Rubin G.M.
      ,
      • Lowenstein E.J.
      • Daly R.J.
      • Batzer A.G.
      • Li W.
      • Margolis B.
      • Lammers R.
      • Ullrich A.
      • Schlessinger J.
      ), interacts with PI 3-kinase. Lysates from control and M-CSF-treated monocytes were subjected to immunoprecipitation with anti-Grb2. Immunoblotting of the precipitates with anti-p85α revealed increased reactivity following M-CSF stimulation (Fig. 2 A). In the reciprocal experiment, analysis of anti-p85α immunoprecipitates with anti-Grb2 confirmed an M-CSF-dependent association between these two proteins (Fig. 2 B).
      Figure thumbnail gr2
      Figure 2:Association of PI 3-kinase with Grb2. A, monocytes were stimulated with M-CSF for 5 min. Lysates were subjected to immunoprecipitation with anti-Grb2 antibody. The immunoprecipitated proteins were resolved by 7.5% SDS-PAGE and analyzed by immunoblotting with anti-p85α antibody. B, anti-p85α immunoprecipitates from control and M-CSF-treated cell lysates were analyzed by 10% SDS-PAGE and immunoblotting with anti-Grb2 antibody. One representative experiment out of four is shown.
      In order to determine whether the interaction between Grb2 and p85α is direct, we prepared anti-p85α immunoprecipitates from M-CSF-treated monocytes. The precipitates were subjected to SDS-PAGE, transferred to nitrocellulose, and then incubated with GST-Grb2 (full length). After washing the filters thoroughly, we assayed Grb2 binding by immunoblotting with anti-Grb2. The finding that anti-Grb2 reactivity is detectable at a molecular mass of 85 kDa supports a direct interaction of Grb2 with the p85α subunit of PI 3-kinase (Fig. 3 A). In order to confirm these findings, we prepared anti-p85α immunoprecipitates from M-CSF-treated monocytes and then released the proteins by boiling the immune complexes in 0.5% SDS and 1 mM DTT. After diluting SDS to 0.1% by lysis buffer, secondary protein precipitations were performed using GST or GST-Grb2. Analysis of the second precipitates by immunoblotting with anti-p85α confirmed direct interaction of Grb2 with p85α (Fig. 3 B). Secondary anti-p85α immunoprecipitates were used as a positive control in this experiment (Fig. 3 B).
      Figure thumbnail gr3
      Figure 3:Direct binding of PI 3-kinase and Grb2. A, lysates from M-CSF-stimulated monocytes were subjected to immunoprecipitation with anti-p85α. The proteins were separated by 7.5% SDS-PAGE and transferred to nitrocellulose. The filters were then incubated with 50 μg/ml GST-Grb2 (full length) or GST at 4°C for 2 h. After washing thoroughly with PBST, immunoblotting was performed by anti-Grb2 antibody. B, lysates from M-CSF-treated monocytes were immunoprecipitated by anti-p85α. Proteins were released from the precipitates by boiling the immune complexes in 0.5% SDS, 1 mM DTT buffer. Secondary protein precipitations were performed by incubating either with GST, GST Grb2, or anti-p85α and the resulting protein complexes were analyzed by immunoblotting with p85α. Two independent experiments showed similar results. C, lysates from control (lanes 2, 4, 6, and 8) and M-CSF-treated (lanes 1, 3, 5, 7, and 9) monocytes were incubated with GST, GST-Grb2 (full length), GST-Grb2 N-SH3, GST-Grb2 SH2, and GST-Grb2 C-SH3 proteins immobilized on glutathione-Sepharose. The bound proteins were resolved by 7.5% SDS-PAGE and immunoblotted with anti-p85α antibody. D, GST-Grb2 SH2 fusion protein was preincubated (1 h, 4°C) in the absence (-) or presence (+) of 50 μM tyrosine-phosphorylated synthetic peptide (LINHpYRNESLAQ). The fusion protein was then incubated with lysate from M-CSF-treated monocytes and the adsorbate analyzed by immunoblotting with anti-p85α.
      This interaction was further analyzed using GST fusion proteins prepared from full-length Grb2, the SH3 (carboxyl and amino-terminal) and SH2 domains of Grb2. Adsorbates obtained with GST-Grb2 (full length) revealed increased binding of p85α when using lysates from M-CSF-stimulated, as compared with control, monocytes (Fig. 3 C). A low level of p85α binding to the Grb2 SH3 domains was obtained when using lysates from both control and M-CSF-treated cells (Fig. 3 C). In contrast, adsorbates obtained with GST-Grb2 SH2 demonstrated a M-CSF-dependent increase in binding of p85α (Fig. 3 C). The SH2 domain of Grb2 interacts with proteins that contain the pYXNX motif (
      • Lowenstein E.J.
      • Daly R.J.
      • Batzer A.G.
      • Li W.
      • Margolis B.
      • Lammers R.
      • Ullrich A.
      • Schlessinger J.
      ,
      • Rozakis-Adcock M.
      • McGlade J.
      • Mbamalu G.
      • Pelicci G.
      • Daly R.
      • Li W.
      • Batzer A.
      • Thomas S.
      • Brugge J.
      • Pelicci P.G.
      • Schlessinger J.
      • Pawson T.
      ,
      • Pelicci G.
      • Lanfrancone L.F.
      • Grignani L.
      • McGlade J.
      • Cavallo F.
      • Forni G.
      • Nicoletti I.
      • Grignani F.
      • Pawson T.
      • Pelicci P.G.
      ). To define the site in PI 3-kinase responsible for the association with the Grb2 SH2 domain, we identified a potential candidate sequence at Tyr408 which is followed by RNE. A chemically phosphorylated synthetic peptide corresponding to this site (amino acids 404-415) was used in competition assays. Preincubation of GST-Grb2 SH2 with the peptide inhibited binding of PI 3-kinase from lysates of M-CSF-treated monocytes (Fig. 3 D). These findings indicate that PI 3-kinase interacts directly with the SH2 domain of Grb2 and that the pYRNE (Y408) in p85α may be the binding site.
      The SH3 domains of Grb2 bind to Sos (
      • Rozakis-Adcock M.
      • McGlade J.
      • Mbamalu G.
      • Pelicci G.
      • Daly R.
      • Li W.
      • Batzer A.
      • Thomas S.
      • Brugge J.
      • Pelicci P.G.
      • Schlessinger J.
      • Pawson T.
      ,
      • Chardin P.
      • Camonis J.
      • Gale W.J.
      • Aelst L.V.
      • Schlessinger J.
      • Wingler M.H.
      • Bar-Sagi D.
      ,
      • Egan S.E.
      • Giddings B.W.
      • Brooks M.W.
      • Buday L.
      • Sizeland A.M.
      • Weinberg R.A.
      ,
      • Gale W.N.
      • Kaplan S.
      • Lowenstein E.J.
      • Schlessinger J.
      • Bar-Sagi D.
      ,
      • Li N.
      • Batzer A.
      • Daly R.
      • Yajnik V.
      • Skolnik E.
      • Chardin P.
      • Bar-Sagi D.
      • Margolis B.
      • Schlessinger J.
      ,
      • Skolnik E.Y.
      • Lee C.H.
      • Batzer A.
      • Vicentini L.M.
      • Zhou M.
      • Daly R.
      • Myers M.J.
      • Baker J.M.
      • Ullrich A.
      • White M.F.
      • Schlessinger J.
      ). This interaction of Sos with Grb2 translocates Sos to the plasma membrane where it increases the exchange of GDP for GTP on membrane-bound Ras (
      • Egan S.E.
      • Giddings B.W.
      • Brooks M.W.
      • Buday L.
      • Sizeland A.M.
      • Weinberg R.A.
      ,
      • Li N.
      • Batzer A.
      • Daly R.
      • Yajnik V.
      • Skolnik E.
      • Chardin P.
      • Bar-Sagi D.
      • Margolis B.
      • Schlessinger J.
      ). Since p85α binds to the SH2 domain of Grb2, and PI 3-kinase is khown to be a direct target of Ras (
      • Rodriguez-Viciana P.
      • Warne P.H.
      • Dhand R.
      • Vanhaesebroeck B.
      • Gout I.
      • Fry M.J.
      • Waterfield M.D.
      • Downward J.
      ), we asked whether PI 3-kinase associates with Sos. Immunoblot analysis of p85α immunoprecipitates with anti-Sos demonstrated an increased association of p85α and Sos in M-CSF-treated, as compared with control, monocytes (Fig. 4 A). Moreover, analysis of anti-Sos immunoprecipitates with anti-p85α demonstrated a M-CSF-dependent increase in the association of these proteins (Fig. 4 B). These findings and the demonstration that PI 3-kinase and Sos bind to the SH2 and SH3 domains, respectively, of Grb2 support the formation of a PI 3-kinase·Grb2·Sos complex. Since Ras regulates PI 3-kinase (
      • Rodriguez-Viciana P.
      • Warne P.H.
      • Dhand R.
      • Vanhaesebroeck B.
      • Gout I.
      • Fry M.J.
      • Waterfield M.D.
      • Downward J.
      ), we asked whether PI 3-kinase exhibits an increase in activity when associated with the Grb2·Sos complex. The results demonstrate that anti-Grb2 immunoprecipitates exhibit M-CSF-dependent increases (approximately 20-fold) in activity of PI 3-kinase (Fig. 4 C). Similar findings were obtained with the anti-Sos immunoprecipitates (Fig. 4 D). These results demonstrate that activation of PI 3-kinase in the anti-Grb2 or anti-Sos immunoprecipitates occurs in response to M-CSF (Fig. 4, C and D).
      Figure thumbnail gr4
      Figure 4:M-CSF-dependent activation of PI 3-kinase in anti-Grb2 and anti-Sos immunoprecipitates. A, lysates from control and M-CSF-treated monocytes were subjected to immunoprecipitation with anti-p85α. Lysates from M-CSF-treated monocytes were also subjected to immunoprecipitation with anti-Grb2. The immunoprecipitates were analyzed by immunoblotting with anti-Sos. B, anti-Sos immunoprecipitates from control and M-CSF-treated cell lysates were subjected to immunoblotting with anti-p85α. C, lysates from control and M-CSF-treated monocytes were immunoprecipitated with anti-Grb2. M-CSF-treated cell lysates were also subjected to immunoprecipitation with anti-p85α. The immune complexes were extensively washed and assayed for precipitable PI 3-kinase activity by the addition of PI and [γ-32P]ATP. The lipids were extracted with chloroform, separated by TLC, and analyzed by autoradiography. The origin and position of phosphatidylinositol phosphate (PIP) are indicated. D, lysates from control and M-CSF-treated monocytes were immunoprecipitated with anti-Sos antibody. M-CSF-treated cell lysates were also subjected to immunoprecipitation with anti-p85α and normal rabbit serum (NRS). The immune complexes were assayed for precipitable PI 3-kinase activity as described above. One of the three independent experiments is shown.
      PI 3-kinase directly associates with and is stimulated by activated receptor PTKs (
      • Van der Geer P.
      • Hunter T.
      ). The interaction of p85α with receptor phosphotyrosines may induce structural alterations that result in activation of the p110 catalytic subunit (
      • Backer J.M.
      • Myers Jr., M.G.
      • Shoelson S.E.
      • Chin D.J.
      • Sun X.J.
      • Miralpeiz M.
      • Hu P.
      • Margolis B.
      • Skolnik E.Y.
      • Schlessinger J.
      • White M.F.
      ,
      • Susa M.
      • Vulevic S.
      • Lane H.
      • Thomas G.
      ). Tyrosine phosphorylation of p85α may also be responsible for increases in activity of PI 3-kinase (
      • Ren C.L.
      • Morio T.
      • Fu S.M.
      • Geha R.S.
      ,
      • Karnitz L.M.
      • Sutor S.L.
      • Abraham S.L.
      ), whereas other work has implicated Ras as a regulator of the intrinsic phosphoinositide kinase activity (
      • Rodriguez-Viciana P.
      • Warne P.H.
      • Dhand R.
      • Vanhaesebroeck B.
      • Gout I.
      • Fry M.J.
      • Waterfield M.D.
      • Downward J.
      ). The present findings demonstrate that M-CSF induces tyrosine phosphorylation of p85α and that a pYXNX-like motif potentially contributes to binding of p85α to the SH2 domain of Grb2. While the tyrosine kinases involved in phosphorylation of PI 3-kinase could be of either the receptor or nonreceptor types, this event would be necessary for the formation of a complex with Grb2. The demonstration that PI 3-kinase binds to a pool of Grb2 associated with Sos also provides a potential mechanism for PI 3-kinase to complex with Ras through Sos/Ras interactions. Other studies have demonstrated that the insulin receptor substrate 1 interacts with both PI 3-kinase p85α and the SH2 domain of Grb2 (
      • Skolnik E.Y.
      • Lee C.H.
      • Batzer A.
      • Vicentini L.M.
      • Zhou M.
      • Daly R.
      • Myers M.J.
      • Baker J.M.
      • Ullrich A.
      • White M.F.
      • Schlessinger J.
      ,
      • Myers Jr., M.G.
      • Wang L.-M.
      • Sun X.J.
      • Zhang Y.
      • Yenush L.
      • Schlessinger J.
      • Pierce J.H.
      • White M.F.
      ). Insulin receptor substrate 1 or related proteins, such as 4PS (
      • Wang L.M.
      • Myers Jr., M.G.
      • Sun X.J.
      • Aaronson S.A.
      • White M.
      • Pierce J.H.
      ), may therefore also associate with a PI 3-kinase·Grb2 complex by binding to p85α. In any event, the present results provide the first evidence for binding of PI 3-kinase to an adaptor protein and support a mechanism for growth factor-induced regulation of PI 3-kinase by Ras. Since other studies have suggested that PI 3-kinase can also function upstream to Ras (
      • Yamauchi K.
      • Holt K.
      • Pessin J.E.
      ), the formation of a PI 3-kinase·Grb2·Sos complex could similarly contribute to the regulation of Ras.

      REFERENCES

        • Sherr C.J.
        • Rettenmier C.W.
        • Sacca R.
        • Roussel M.F.
        • Look A.T.
        • Stanley E.R.
        Cell. 1985; 41: 665-676
        • Sherr C.
        Trends Genet. 1991; 7: 398-402
        • Van der Geer P.
        • Hunter T.
        Mol. Cell. Biol. 1990; 10: 2991-3002
        • Reedijk M.
        • Liu X.
        • Geer P.V.D.
        • Letwin K.
        • Waterfield M.D.
        • Hunter T.
        • Pawson T.
        EMBO J. 1992; 11: 1365-1372
        • Van der Geer P.
        • Hunter T.
        EMBO J. 1993; 12: 5161-5172
        • Auger K.R.
        • Serunian L.A.
        • Soltoff S.P.
        • Libby P.
        • Cantley L.C.
        Cell. 1989; 57: 167-175
        • Nakanishi H.
        • Brewer K.A.
        • Exton J.H.
        J. Biol. Chem. 1993; 268: 13-16
        • Backer J.M.
        • Myers Jr., M.G.
        • Shoelson S.E.
        • Chin D.J.
        • Sun X.J.
        • Miralpeiz M.
        • Hu P.
        • Margolis B.
        • Skolnik E.Y.
        • Schlessinger J.
        • White M.F.
        EMBO J. 1992; 11: 3469-3479
        • Myers Jr., M.G.
        • Backer J.M.
        • Sun Z.J.
        • Shoelson S.
        • Hu P.
        • Schlessinger J.
        • Yoakim M.
        • Schaffhausen B.
        • White M.F.
        Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10350-10354
        • 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
        • Nakamura T.
        • Lin L.-L.
        • Kharbanda S.
        • Knopf J.
        • Kufe D.
        EMBO J. 1992; 11: 4917-4922
        • Sariban E.
        • Mitchell T.
        • Kufe D.
        Nature. 1985; 316: 64-66
        • Sung C.K.
        • Sanchez-Margalet V.
        • Goldfien I.D.
        J. Biol. Chem. 1994; 269: 12503-12507
        • Sjolander A.
        • Yamamoto
        • Huber B.E.
        • Lapetina E.G.
        Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7908-7912
        • Sjolander A.
        • Lapetina E.G.
        Biochem. Biophys. Res. Commun. 1992; 169: 1503-1508
        • Clark S.G.
        • Stern M.J.
        • Horowitz H.R.
        Nature. 1992; 356: 340-344
        • Simon M.A.
        • Dodson G.S.
        • Rubin G.M.
        Cell. 1993; 73: 169-177
        • Lowenstein E.J.
        • Daly R.J.
        • Batzer A.G.
        • Li W.
        • Margolis B.
        • Lammers R.
        • Ullrich A.
        • Schlessinger J.
        Cell. 1992; 70: 431-442
        • Rozakis-Adcock M.
        • McGlade J.
        • Mbamalu G.
        • Pelicci G.
        • Daly R.
        • Li W.
        • Batzer A.
        • Thomas S.
        • Brugge J.
        • Pelicci P.G.
        • Schlessinger J.
        • Pawson T.
        Nature. 1992; 360: 689-692
        • Pelicci G.
        • Lanfrancone L.F.
        • Grignani L.
        • McGlade J.
        • Cavallo F.
        • Forni G.
        • Nicoletti I.
        • Grignani F.
        • Pawson T.
        • Pelicci P.G.
        Cell. 1992; 70: 93-104
        • Chardin P.
        • Camonis J.
        • Gale W.J.
        • Aelst L.V.
        • Schlessinger J.
        • Wingler M.H.
        • Bar-Sagi D.
        Science. 1993; 260: 1338-1343
        • Egan S.E.
        • Giddings B.W.
        • Brooks M.W.
        • Buday L.
        • Sizeland A.M.
        • Weinberg R.A.
        Nature. 1993; 363: 45-51
        • Gale W.N.
        • Kaplan S.
        • Lowenstein E.J.
        • Schlessinger J.
        • Bar-Sagi D.
        Nature. 1993; 363: 88-92
        • Li N.
        • Batzer A.
        • Daly R.
        • Yajnik V.
        • Skolnik E.
        • Chardin P.
        • Bar-Sagi D.
        • Margolis B.
        • Schlessinger J.
        Nature. 1993; 363: 85-88
        • Skolnik E.Y.
        • Lee C.H.
        • Batzer A.
        • Vicentini L.M.
        • Zhou M.
        • Daly R.
        • Myers M.J.
        • Baker J.M.
        • Ullrich A.
        • White M.F.
        • Schlessinger J.
        EMBO J. 1993; 12: 1929-1936
        • Van der Geer P.
        • Hunter T.
        Mol. Cell. Biol. 1991; 11: 4698-4709
        • Susa M.
        • Vulevic S.
        • Lane H.
        • Thomas G.
        J. Biol. Chem. 1992; 267: 6905-6909
        • Ren C.L.
        • Morio T.
        • Fu S.M.
        • Geha R.S.
        J. Exp. Med. 1994; 179: 673-680
        • Karnitz L.M.
        • Sutor S.L.
        • Abraham S.L.
        J. Exp. Med. 1994; 179: 1799-1808
        • Myers Jr., M.G.
        • Wang L.-M.
        • Sun X.J.
        • Zhang Y.
        • Yenush L.
        • Schlessinger J.
        • Pierce J.H.
        • White M.F.
        Mol. Cell. Biol. 1994; 14: 3577-3587
        • Wang L.M.
        • Myers Jr., M.G.
        • Sun X.J.
        • Aaronson S.A.
        • White M.
        • Pierce J.H.
        Science. 1993; 261: 1591-1594
        • Yamauchi K.
        • Holt K.
        • Pessin J.E.
        J. Biol. Chem. 1993; 268: 14597-14600