Syk interacts with tyrosine-phosphorylated proteins in human platelets activated by collagen and cross-linking of the Fcγ-IIA receptor

F. Yanaga, A. Poole, J. Asselin, R. Blake, G. L. Schieven, E. A. Clark, C. L. Law, S. P. Watson

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136 Citations (Scopus)


Activation of human platelets by cross-linking of the platelet low-affinity IgG receptor, the Fcγ receptor IIA (Fcγ-RIIA), or by collagen is associated with rapid phosphorylation on tyrosine of the non-receptor tyrosine kinase syk. Phosphorylation is still observed, albeit sometimes reduced, in the presence of a combination of a protein kinase C inhibitor, Ro 31-8220, and the intracellular calcium chelator, BAPTA-AM, demonstrating independence from phosphoinositide-specific phospholipase C (PLC) activity. In contrast, the combination of Ro 31-8220 and BAPTA-AM completely inhibits phosphorylation of syk in thrombin-stimulated platelets. Phosphorylation of syk increases its autophosphorylation activity measured in a kinase assay performed on syk immunoprecipitates. Fcγ-RIIA also undergoes phosphorylation in syk immunoprecipitates from platelets activated by cross-linking of Fcγ-RIIA but not by collagen, suggesting that it associates with the kinase. Consistent with this, tyrosine-phosphorylated Fcγ-RIIA is precipitated by a glutathione S-transferase (GST) fusion protein containing the tandem src homology (SH2) domains of syk from Fcγ-RIIA-but not collagen-activated cells. Two uncharacterized tyrosine-phosphorylated proteins of 40 and 65 kDa are uniquely precipitated by a GST fusion protein containing the tandem syk-SH2 domains in collagen-stimulated platelets. A peptide based on the antigen recognition activation motif(ARAM) of Fcγ-RIIA, and phosphorylated on the two tyrosine residues found within this region, selectively binds syk from lysates of resting platelets; this interaction is not seen with a non-phosphorylated peptide. Kinase assays on Fcγ-RIIA immunoprecipitates reveal the constitutive association of an unidentified kinase activity in resting cells which phosphorylates a 67 kDa protein. Syk is not detected in Fcγ-RIIA immunoprecipitates from resting cells but associates with the receptor following activation and, together with Fcγ-RIIA, is phosphorylated in the kinase assay in vitro. These results demonstrate that syk is activated by Fcγ-RIIA cross-linking and collagen, independent of PLC, suggesting that it may have an important role in the early events associated with platelet activation. The association of syk with Fcγ-RIIA appears to be mediated through the tandem SH2 domains in syk and the ARAM motif of Fcγ-RIIA. A similar interaction may underlie the response to collagen, suggesting that its signalling receptor contains an ARAM motif.

Original languageEnglish
Pages (from-to)471-478
Number of pages8
JournalBiochemical Journal
Issue number2
Publication statusPublished - 1995

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology


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