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 recently 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 of tyrosine phosphorylation of phospholipase Cgamma2. 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-mobilizing action of a collagen-related peptide (CRP) is kinase-dependent, inhibited by the microinjection of the tandem SH2 domains of Syk and abolished in Syk-deficient mice. Furthermore, the CRP response is abolished by the Src family kinase inhibitor PP1 and inhibited in Fyn-deficient mice. In contrast, the calcium response to the G-protein-linked receptor agonist thrombin is not significantly altered under these conditions. These results provide direct evidence of the functional importance of Fyn and Syk in collagen receptor signaling and support the megakaryocyte as a model for the study of proteins involved in this pathway.
Melford
S K
SK
Department of Pharmacology, University of Oxford, Mansfield Road, Oxford OX1 3QT, United Kingdom.
Turner
M
M
Briddon
S J
SJ
Tybulewicz
V L
VL
Watson
S P
SP
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002352:Carrier Proteins; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004792:Enzyme Precursors / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins; D008297:Male; D008533:Megakaryocytes / Q000187:drug effects* / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008845:Microinjections; D010770:Phosphotransferases / Q000037:antagonists & inhibitors; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051076:Proto-Oncogene Proteins c-fyn; D015398:Signal Transduction; D019311:Staurosporine / Q000494:pharmacology; D000072377:Syk Kinase
D002352:Carrier Proteins; D004791:Enzyme Inhibitors; D004792:Enzyme Precursors; D047908:Intracellular Signaling Peptides and Proteins; D011506:Proteins; D011518:Proto-Oncogene Proteins; C007105:c1q-binding proteins; D010770:Phosphotransferases; D011505:Protein-Tyrosine Kinases; C494960:Fyn protein, mouse; D051076:Proto-Oncogene Proteins c-fyn; D000072377:Syk Kinase; C000606897:Syk protein, mouse; D019311:Staurosporine; D002118:Calcium