SYK AND FYN ARE REQUIRED BY MOUSE MEGAKARYOCYTES FOR THE RISE IN INTRACELLULAR CALCIUM-INDUCED BY A COLLAGEN-RELATED PEPTIDE

Stimulation of platelets by collagen leads to activation of a tyrosine kinase cascade resulting in secretion and aggregation. We have recent ly shown that this pathway involves rapid tyrosine phosphorylation of an Fc receptor gamma chain, which contains an immunoreceptor tyrosine- based activation motif(ITAM), enabling interaction with the tandem SH2 domains of the tyrosine kinase Syk. Activation of Syk lies upstream o f tyrosine phosphorylation of phospholipase C gamma 2. In the present study we sought to test directly the role of the ITAM/Syk interaction and the role of the Src-related kinases in collagen receptor signaling using mouse megakaryocytes. We demonstrate that the calcium-mobilizin g action of a collagen-related peptide (CRP) is kinase-dependent, in h ibited by the microinjection of the tandem SH2 domains of Syk and abol ished in Syk-deficient mice. Furthermore, the CRP response is abolishe d by the Src family kinase inhibitor PPI and inhibited in Fyn-deficien t mice. In contrast, the calcium response to the G-protein-linked rece ptor agonist thrombin is not significantly altered under these conditi ons. These results provide direct evidence of the functional importanc e of Fyn and Syk in collagen receptor signaling and support the megaka ryocyte as a model for the study of proteins involved in this pathway.