Syk and Fyn Are Required by Mouse Megakaryocytes for the Rise in Intracellular Calcium Induced by a Collagen-related Peptide*
- Steven K. Melford‡,
- Martin Turner§,
- Stephen J. Briddon,
- Victor L. J. Tybulewicz§ and
- Stephen P. Watson¶
- From the Department of Pharmacology, University of Oxford, Mansfield Road, Oxford OX1 3QT and the §National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, United Kingdom
Abstract
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 γ 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 Cγ2. 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.
Footnotes
-
↵* This work was supported by the Wellcome Trust, the British Heart Foundation, and the Medical Research Council.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.
-
↵‡ Recipient of a British Heart Foundation studentship.
-
↵¶ Royal Society Research Fellow. To whom correspondence should be addressed. Tel.: 44-1865-271592; Fax: 44-1865-271853; E-mail:steve.watson{at}pharmacology.ox.ac.uk.
-
↵1 The abbreviations used are: PLC, phospholipase C; SH2, Src homology 2; CRP, collagen-related peptide; FcR-γ chain, Fc receptor γ chain; IP3R, inositol 1,4,5-trisphosphate receptor.
-
↵2 B. Gross, S. K. Melford, and S. P. Watson, manuscript in preparation.
-
- Received June 30, 1997.
- Revision received August 14, 1997.
