Melford S K, Turner M, Briddon S J, Tybulewicz V L, Watson S P
Department of Pharmacology, University of Oxford, Mansfield Road, Oxford OX1 3QT, United Kingdom.
J Biol Chem. 1997 Oct 31;272(44):27539-42. doi: 10.1074/jbc.272.44.27539.
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.
胶原蛋白对血小板的刺激会导致酪氨酸激酶级联反应的激活，从而引发分泌和聚集。我们最近发现，该信号通路涉及Fc受体γ链的快速酪氨酸磷酸化，该链含有基于免疫受体酪氨酸的激活基序（ITAM），能够与酪氨酸激酶Syk的串联SH2结构域相互作用。Syk的激活位于磷脂酶Cγ2酪氨酸磷酸化的上游。在本研究中，我们试图使用小鼠巨核细胞直接测试ITAM/Syk相互作用的作用以及Src相关激酶在胶原蛋白受体信号传导中的作用。我们证明，胶原蛋白相关肽（CRP）的钙动员作用是激酶依赖性的，可被显微注射Syk的串联SH2结构域所抑制，并在Syk缺陷小鼠中消失。此外，CRP反应被Src家族激酶抑制剂PP1消除，并在Fyn缺陷小鼠中受到抑制。相比之下，在这些条件下，对G蛋白偶联受体激动剂凝血酶的钙反应没有明显改变。这些结果直接证明了Fyn和Syk在胶原蛋白受体信号传导中的功能重要性，并支持将巨核细胞作为研究参与该信号通路的蛋白质的模型。
