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Fc Receptor-mediated Platelet Activation Is Dependent on Phosphatidylinositol 3-Kinase Activation and Involves p120cbl*

  • Abdelhafid Saci
    Footnotes
    Affiliations
    From INSERM U428, Faculté de Pharmacie, UniversitéParis-V, 75270 Paris, France
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  • Sabine Pain
    Footnotes
    Affiliations
    From INSERM U428, Faculté de Pharmacie, UniversitéParis-V, 75270 Paris, France
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  • Francine Rendu
    Affiliations
    From INSERM U428, Faculté de Pharmacie, UniversitéParis-V, 75270 Paris, France
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  • Christilla Bachelot-Loza
    Correspondence
    To whom correspondence should be addressed: INSERM U428, Faculté de Pharmacie, 4 ave. de l'Observatoire, F-75006, Paris, France. Tel.: 33-1-53-73-96-19; Fax: 33-1-44-07-17-72;
    Affiliations
    From INSERM U428, Faculté de Pharmacie, UniversitéParis-V, 75270 Paris, France
    Search for articles by this author
  • Author Footnotes
    * 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.
    ‡ Supported by Diagnostica-Stago (Asnière, France).
    § Supported by Ministère de la Recherche et de la Technologie, France.
Open AccessPublished:January 22, 1999DOI:https://doi.org/10.1074/jbc.274.4.1898
      The platelet receptor for the Fc domain of IgGs (FcγRIIa) triggers intracellular signaling through protein tyrosine phosphorylations leading to platelet aggregation. In this study, we focused on the adaptor protein p120cbl (Cbl), which became tyrosine-phosphorylated after platelet activation induced by antibodies. Cbl phosphorylation was dependent on Fc receptor engagement. An association of Cbl with the p85 subunit of phosphatidylinositol 3-kinase (PI 3-K) occurred in parallel with Cbl tyrosine phosphorylation. We showed by in vitro experiments that Cbl/p85 association was mediated by the Src homology 3 domain of p85/PI 3-K and the proline-rich region of Cbl. Inhibition of PI 3-K activity by wortmannin led to the blockade of both platelet aggregation and serotonin release mediated by FcγRIIa engagement, whereas it only partly inhibited those induced by thrombin. Thus, PI 3-K may play a crucial role in the initiation of platelet responses after FcγRIIa engagement. Our results suggest that Cbl is involved in platelet signal transduction by the recruitment of PI 3-K to the FcγRIIa pathway, possibly by increasing PI 3-K activity.
      PI 3-K
      phosphatidylinositol 3-kinase
      FcγRIIa
      platelet receptor for the Fc domain of IgGs
      mAb
      monoclonal antibody
      RAM
      rabbit polyclonal F(ab)′2 anti-mAb
      GST
      glutathioneS-transferase
      IP
      immunoprecipitation
      PAGE
      polyacrylamide gel electrophoresis
      SH
      Src homology
      PY
      phosphotyrosine.
      The Cbl protein is the product of the c-cblprotooncogene, the cellular homologue of the v-cbl oncogene present in the Cas-NS-1 retrovirus, which induces pre-B cell lymphomas and myeloid leukemias (
      • Langdon W.Y.
      • Hartley J.W.
      • Klinken S.P.
      • Ruscetti S.K.
      • Morse III, H.C.
      ,
      • Blake T.J.
      • Shapiro M.
      • Morse H.C.
      • Langdon W.Y.
      ). Cbl is found in a wide range of hemopoietic cell lineages and some nonhemopoietic tissues such as lung, brain, and testis (
      • Langdon W.Y.
      • Hyland C.D.
      • Grumont R.J.
      • Morse III, H.C.
      ). A deletion in the c-cbl sequence (62% of the C-terminal domain) involving functional domains, such as the leucine zipper motif and proline-rich region, converts this protooncogene into the transforming one (
      • Savage P.D.
      • Shapiro M.
      • Langdon W.Y.
      • Geurts van Kessel A.D.
      • Seuanez H.N.
      • Akao Y.
      • Croce C.
      • Morse III, H.C.
      • Kersey J.H.
      ). Unlike the product of v-cbl localized in both the cytoplasm and the nucleus, the c-cbl product (p120cbl) is exclusively cytoplasmic (
      • Blake T.J.
      • Heath K.G.
      • Langdon W.Y.
      ). A tumorigenic form of Cbl was detected in the 70Z/3 pre-B cell lymphoma, in which Cbl tyrosine phosphorylation is increased as a result of a deletion of 17 amino acids in the Cbl sequence (
      • Andoniou C.E.
      • Thien C.B.F.
      • Langdon W.Y.
      ). Cbl is also heavily tyrosine-phosphorylated in v-src-transformed hemopoietic cells (
      • Andoniou C.E.
      • Thien C.B.F.
      • Langdon W.Y.
      ).
      Cbl becomes tyrosine-phosphorylated after cell stimulation through a wide range of receptors including B and T cell receptors (
      • Donovan J.A.
      • Wange R.L.
      • Langdon W.Y.
      • Samelson L.E.
      ,
      • Kim T.J.
      • Kim Y.-T.
      • Pillai S.
      ,
      • Reedquist K.A.
      • Fukazawa T.
      • Panchamoorthy G.
      • Langdon W.Y.
      • Shoelson S.E.
      • Druker B.J.
      • Band H.
      ,
      • Panchamoorthy G.
      • Fukazawa T.
      • Myake S.
      • Soltoff S.
      • Reedquist K.
      • Druker B.
      • Shoelson S.
      • Cantley L.C.
      • Band H.
      ,
      • Buday L.
      • Khwaja A.
      • Sipeki S.
      • Faragó A.
      • Downward J.
      ), various growth factor receptors (
      • Odai H.
      • Sasaki K.
      • Iwamatsu A.
      • Hanazono Y.
      • Tanaka T.
      • Mitani K.
      • Yazaki Y.
      • Hirai H.
      ,
      • Sattler M.
      • Salgia R.
      • Durstin M.A.
      • Prasad K.V.
      • Griffin J.D.
      ,
      • Barber D.L.
      • Mason J.M.
      • Fukazawa T.
      • Reedquist K.A.
      • Druker B.J.
      • Band H.
      • Andrea A.
      ,
      • Meisner H.
      • Czech M.P.
      ,
      • Galisteo M.L.
      • Dikic I.
      • Batzer A.G.
      • Langdon W.Y.
      • Schlessinger J.
      ,
      • Bowtell D.D.
      • Langdon W.Y.
      ,
      • Soltoff S.P.
      • Cantley L.C.
      ,
      • Wisniewski D.
      • Strife A.
      • Clarkson B.
      ,
      • Naccache P.H.
      • Gilbert C.
      • Barabé F.
      • Al-Shami A.
      • Mahana W.
      • Bourgoin S.G.
      ), integrins (
      • Ojaniemi M.
      • Martin S.S.
      • Dolfi F.
      • Olefsky J.M.
      • Vuori K.
      ,
      • Manié S.N.
      • Sattler M.
      • Astier A.
      • Phifer J.S.
      • Canty T.
      • Morimoto C.
      • Druker B.J.
      • Salgia R.
      • Griffin J.D.
      • Freedman A.S.
      ,
      • Sattler M.
      • Salgia R.
      • Shrikhande G.
      • Verma S.
      • Uemura N.
      • Law S.F.
      • Golemis E.A.
      • Griffin J.D.
      ), and receptors for the Fc domain of IgG (
      • Naccache P.H.
      • Gilbert C.
      • Barabé F.
      • Al-Shami A.
      • Mahana W.
      • Bourgoin S.G.
      ,
      • Tanaka S.
      • Neff L.
      • Baron R.
      • Levy J.B.
      ,
      • Marcilla A.
      • Rivero-Lezcano O.M.
      • Agarwal A.
      • Robbins K.C.
      ). The primary structure of Cbl shows no homology with any catalytic domain but contains a number of tyrosine residues that can be phosphorylated and a proline-rich region (
      • Blake T.J.
      • Shapiro M.
      • Morse H.C.
      • Langdon W.Y.
      ). Cbl has been shown to bind to a number of signaling proteins, such as tyrosine kinases Src, Lyn, Fyn, Lck, Blk, Syk, the lipid kinase phosphatidylinositol 3-kinase (PI 3-K),1 phospholipase Cγ, and the adaptor proteins Grb2 and Vav (
      • Kim T.J.
      • Kim Y.-T.
      • Pillai S.
      ,
      • Reedquist K.A.
      • Fukazawa T.
      • Panchamoorthy G.
      • Langdon W.Y.
      • Shoelson S.E.
      • Druker B.J.
      • Band H.
      ,
      • Panchamoorthy G.
      • Fukazawa T.
      • Myake S.
      • Soltoff S.
      • Reedquist K.
      • Druker B.
      • Shoelson S.
      • Cantley L.C.
      • Band H.
      ,
      • Ojaniemi M.
      • Martin S.S.
      • Dolfi F.
      • Olefsky J.M.
      • Vuori K.
      ,
      • Hunter S.
      • Koch B.L.
      • Anderson S.M.
      ,
      • Kitanaka A.
      • Ito C.
      • Nishigaki H.
      • Campana D.
      ,
      • Hartley D.
      • Corvera S.
      ,
      • Tanaka S.
      • Amling M.
      • Neff L.
      • Peyman A.
      • Uhlmann E.
      • Levy J.B.
      • Baron R.
      ,
      • Marengère L.E.M.
      • Mirtsos C.
      • Kozieradzki I.
      • Veillette A.
      • Mak T.W.
      • Penninger J.M.
      ,
      • Ribon V.
      • Saltiel A.R.
      ,
      • Sattler M.
      • Salgia R.
      • Okuda K.
      • Uemura N.
      • Durstin M.A.
      • Pisick E.
      • Xu G.
      • Li J.J.
      • Prasad K.V.
      • Griffin J.D.
      ). Cbl phosphorylation on serine residues has also been reported in phorbol ester-activated T cells, allowing its interaction with 14.3.3 protein (
      • Liu Y.C.
      • Liu Y.
      • Elly C.
      • Yoshida H.
      • Lipkowitz S.
      • Altman A.
      ). Finally, Cbl contains a phosphotyrosine binding domain in the N-terminal region that directly binds to phosphorylated ZAP-70 in activated T cells (
      • Lupher Jr., M.L.
      • Reedquist K.A.
      • Miyake S.
      • Langdon W.Y.
      • Band H.
      ).
      A regulator activity has recently been described for Cbl when overexpressed in mast cells, in which it regulates p72sykactivity (
      • Ota Y.
      • Samelson L.E.
      ). Cbl is also proposed to regulate the T cell receptor-mediated Ras pathway activation via its association with Grb2 in T cells (
      • Rellahan B.L.
      • Graham L.J.
      • Stoica B.
      • DeBell K.E.
      • Bonvini E.
      ). In interleukin 4-treated B cells, Cbl is tyrosine-phosphorylated and associated with p85/PI 3-K, and overexpression of Cbl enhances PI 3-K activity, mitogenic activity, and cell survival (
      • Ueno H.
      • Sasaki K.
      • Honda H.
      • Nakamoto T.
      • Yamagata T.
      • Miyagawa K.
      • Mitani K.
      • Yazaki Y.
      • Hirai H.
      ). Taken together, the data suggest a role for Cbl in multiple signaling pathways of different cell types.
      In human platelets, Cbl has been identified and shown to be constitutively associated with the Grb2 adaptor protein and tyrosine-phosphorylated after thrombopoietin activation (
      • Hunter S.
      • Koch B.L.
      • Anderson S.M.
      ). Thus, Cbl seems to be implicated in signal transduction after thrombopoietin binding to c-mpl. However, the role of Cbl or its possible involvement in platelet signal transduction mediated by other receptors has not yet been documented. Platelet activation is mediated by a wide variety of agonists, including thrombin, thromboxane A2, ADP, and adhesion molecules such as von Willebrand factor and collagen. Some antibodies directed against antigens on the platelet membrane (e.g. the tetraspanin CD9, glycoprotein IV, and the integrin αIIb3) are also able to induce platelet activation. In most cases, the activation induced by IgGs is dependent on the binding of their Fc domain on the specific receptor, FcγRIIa (
      • Rubinstein E.
      • Boucheix C.
      • Worthington R.E.
      • Carroll R.C.
      ).
      In the present study, we have investigated the involvement of Cbl in platelet signaling after FcγRIIa engagement. Two models of platelet activation involving the Fc receptor were used: the cross-linking of FcγRIIa and bridging of the CD9 antigen with FcγRIIa by an activating monoclonal antibody (mAb) (anti-CD9, Syb). Our results demonstrate that after FcγRIIa engagement, Cbl was heavily tyrosine-phosphorylated. In parallel with Cbl phosphorylation, we found that Cbl was associated with p85/PI 3-K. Moreover, the use of wortmannin, an inhibitor of PI 3-K, abolished platelet aggregation and release induced by antibodies, underlining the crucial role of PI 3-K during immunological activation. The results suggest an important role for Cbl in FcγRIIa-mediated platelet activation, possibly through the regulation of PI 3-K activity.

      DISCUSSION

      The adaptor protein Cbl has been identified in a variety of cells, including platelets, but its involvement in platelet signaling remains uncharacterized. The present work was devoted to studying the involvement of Cbl in signal transduction after platelet activation induced by FcγRIIa cross-linking or Syb antibody (anti-CD9), which activates platelets via FcγRIIa. We demonstrated strong and rapid tyrosine phosphorylation of Cbl in platelets activated through FcγRIIa. In addition, we showed that after platelet activation, p85/PI 3-K association with Cbl correlates with the intensity of Cbl tyrosine phosphorylation. Furthermore, we showed that the PI 3-K inhibitor wortmannin abolished antibody-mediated platelet responses, indicating a crucial role for PI 3-K in antibody-induced platelet activation.
      Cbl was not significantly tyrosine-phosphorylated in resting platelets, but it became phosphorylated during platelet activation depending on the agonist used. After activation by FcγRIIa cross-linking, Cbl was strongly and rapidly tyrosine-phosphorylated. To a lesser extent, Syb induced a similar Cbl phosphorylation to that obtained after FcγRIIa cross-linking. The former difference in the Cbl phosphorylation was probably attributable to distinct modes of platelet activation induced by Syb and FcγRIIa cross-linking, as suggested by others (
      • Qi R.
      • Ozaki Y.
      • Kuroda K.
      • Asazuma N.
      • Yatomi Y.
      • Satoh K.
      • Nomura S.
      • Kume S.
      ). That specific binding of Syb antibody to its antigen (CD9), in the presence of IV.3 (anti-FcγR), did not induce Cbl phosphorylation indicates that Cbl tyrosine phosphorylation occurred after FcγRIIa engagement. These results suggest that, unlike thrombin, which induced a faint and slow Cbl tyrosine phosphorylation, Fc receptor engagement strongly involves Cbl at the first steps of platelet signaling. Another protein involved in the first steps of FcγRIIa-mediated signal transduction is the tyrosine kinase Syk (
      • Yanaga F.
      • Poole A.
      • Asselin J.
      • Blake R.
      • Schieven G.L.
      • Clark E.A.
      • Law C.
      • Watson S.P.
      ). The latter could be a potential candidate to phosphorylate Cbl in platelets, because it was previously demonstrated to participate in Cbl phosphorylation in activated T cells (
      • Feshchenko E.A.
      • Langdon W.Y.
      • Tsygankov A.Y.
      ). We could not, however, detect any Cbl association with Syk after platelet activation. Cbl tyrosine phosphorylation was transient, suggesting an action of tyrosine phosphatase(s) on phosphorylated Cbl. This is supported by the fact that in the presence of the protein tyrosine phosphatase inhibitor phenylarsine oxide, Cbl phosphorylation remained stable for up to 10 min of platelet activation (data not shown).
      Because Cbl association with p85/PI 3-K has been suggested to increase PI 3-K activity in a number of cell systems (
      • Sattler M.
      • Salgia R.
      • Durstin M.A.
      • Prasad K.V.
      • Griffin J.D.
      ,
      • Hunter S.
      • Koch B.L.
      • Anderson S.M.
      ,
      • Ueno H.
      • Sasaki K.
      • Honda H.
      • Nakamoto T.
      • Yamagata T.
      • Miyagawa K.
      • Mitani K.
      • Yazaki Y.
      • Hirai H.
      ), we examined the association of Cbl with PI 3-K in platelets. We found that Cbl/PI 3-K association was negligible in resting and thrombin-activated platelets. In contrast, Cbl was strongly associated with PI 3-K after FcγRIIa-mediated platelet activation. In in vitroexperiments, using GST fusion proteins, we did not find any association between the N-SH2 or C-SH2 domains of p85 and Cbl in resting or Syb-activated platelets. In contrast, full p85 and the p85 SH3 domain precipitated Cbl from resting and Syb-activated platelet lysates. It is thus most likely that Cbl and p85 associate via the SH3 domain of p85 and the proline-rich region of Cbl.
      We could not exclude, however, that in vivo Cbl/PI 3-K association may require tyrosine phosphorylation of Cbl. Indeed, our experiments favor a relationship between these two events. In Nb2 cells, constitutive Cbl/PI 3-K association is mediated by the Cbl proline-rich region and the p85 SH3 domain, whereas increased Cbl/p85 association was proposed to occur through both the p85 SH2 and SH3 domains after cell activation and Cbl tyrosine phosphorylation (
      • Hunter S.
      • Koch B.L.
      • Anderson S.M.
      ). In fact, both SH2 and SH3 domains of p85 interact with Cbl in other cells (
      • Soltoff S.P.
      • Cantley L.C.
      ,
      • Ojaniemi M.
      • Martin S.S.
      • Dolfi F.
      • Olefsky J.M.
      • Vuori K.
      ,
      • Sattler M.
      • Salgia R.
      • Okuda K.
      • Uemura N.
      • Durstin M.A.
      • Pisick E.
      • Xu G.
      • Li J.J.
      • Prasad K.V.
      • Griffin J.D.
      ,
      • Jain S.K.
      • Langdon W.Y.
      • Varticovski L.V.
      ). Interestingly, Soltoff and Cantley (
      • Soltoff S.P.
      • Cantley L.C.
      ) suggested that engagement of the p85 SH2 domain exposes the SH3 domain, which can then further interact with Cbl and increase the affinity of p85 for Cbl. The authors proposed that Cbl could act as an adaptor protein that recruits PI 3-K in the epidermal growth factor-mediated activation of PC12 cells (
      • Soltoff S.P.
      • Cantley L.C.
      ). Moreover, Cbl has a Tyr-X-X-Met motif, which could associate with a p85 SH2 domain if phosphorylated on tyrosine (
      • Cantley L.C.
      • Auger K.R.
      • Carpenter C.
      • Duckworth B.
      • Graziani A.
      • Kapeller R.
      • Soltoff S.
      ). Alternatively, the tyrosine phosphorylation of Cbl could be necessary for its relocalization near PI 3-K. In that respect, Tanaka et al. (
      • Tanaka S.
      • Neff L.
      • Baron R.
      • Levy J.B.
      ) showed that in epidermal growth factor-activated macrophages and fibroblasts, Cbl tyrosine phosphorylation may be accompanied by its subcellular translocation. A last hypothesis could be that in resting platelets, the Cbl proline-rich region may not be in a conformation that allows its association with the p85 SH3 domain. After platelet activation and Cbl phosphorylation, a conformational change in Cbl would render possible the association between the two proteins. By analogy, in stimulated fibroblasts, a phosphotyrosine-dependent conformational change of Cbl was proposed to transiently expose the Cbl N-terminal region, permitting interaction with platelet-derived growth factor receptor α (
      • Bonita D.P.
      • Miyake S.
      • Lupher Jr., M.L.
      • Langdon W.Y.
      • Band H.
      ).
      To determine whether Cbl tyrosine phosphorylation and association with PI 3-K occurred before PI 3-K activation, we used wortmannin to inhibit PI 3-K activity. We found that wortmannin had no effect on Cbl phosphorylation or on Cbl/PI 3-K association induced by different agonists, which indicates that the two events occurred upstream of the lipid kinase activation (data not shown). However, a noncovalent association between PI 3-K and FcγRIIa has been previously shown (
      • Chacko G.W.
      • Brandt J.T.
      • Coggeshall K.M.
      • Anderson C.L.
      ). Therefore, tyrosine-phosphorylated Cbl would play a role in FcγRIIa-mediated platelet signaling by linking PI 3-K with the Fc receptor pathway, possibly by enhancing PI 3-K activity.
      That the level of Cbl/p85 association was stronger after Fc receptor engagement than after thrombin addition suggests a differential role for PI 3-K in the signaling induced by the two types of platelet activation. In the presence of wortmannin, platelet aggregation mediated through FcγRIIa was abolished. In contrast, and as previously demonstrated with thrombin receptor activating peptide (
      • Kovacsovics T.J.
      • Bachelot C.
      • Toker A.
      • Vlahos C.J.
      • Duckworth B.
      • Cantley L.C.
      • Hartwig J.H.
      ,
      • Zhang J.
      • Zhang J.
      • Shattil S.J.
      • Cunningham M.C.
      • Rittenhouse S.E.
      ), platelet aggregation induced by thrombin was only partly inhibited and became reversible. These data indicate that PI 3-K participates in the control of platelet aggregation, especially that which occurs after Fc receptor engagement. The crucial role of PI 3-K in FcγRIIa-mediated platelet activation was also confirmed by the demonstration that wortmannin strongly inhibited antibody-induced serotonin release from dense granules but only weakly inhibited serotonin release induced by thrombin. These results demonstrated that in platelets, PI 3-K activation was required to initiate platelet responses after FcγRIIa engagement. Thus, if Cbl increases PI 3-K activity as previously proposed, Cbl would also play a crucial role in platelet activation mediated through FcγRIIa .
      In conclusion, Cbl was strongly tyrosine-phosphorylated during FcγRIIa-mediated platelet activation, and levels of Cbl tyrosine phosphorylation paralleled levels of Cbl/PI 3-K association. Because PI 3-K activity appeared crucial in platelet responses dependent on FcγRIIa engagement, we suggest that Cbl participates in signal transduction mediated through the Fc receptor by enhancing PI 3-K activity. Thus, Cbl could be one of the key adaptor and regulator proteins in this system.

      Acknowledgments

      We thank Dr. M. Bryckaert and G. Chang for critical reading of the manuscript and S. Aitsiali for editorial help.

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