Cichowski K, Brugge J S, Brass L F
Department of Medicine, University of Pennsylvania, Philadelphia, 19104, USA.
J Biol Chem. 1996 Mar 29;271(13):7544-50. doi: 10.1074/jbc.271.13.7544.
The vav proto-oncogene product, p95vav or Vav, is primarily expressed in hematopoietic cells and has been shown to be a substrate for tyrosine kinases. Although its function is unknown, Vav shares a region of homology with DBL, an exchange factor for the Rho family of GTP-binding proteins. The presence of this domain and the observation that cells transformed with Vav display prominent stress fibers and focal adhesions similar to those that are observed in RhoA transformed cells suggests that Vav may play a role in regulating the actin cytoskeleton. We have, therefore, examined Vav phosphorylation in platelets, which undergo dramatic cytoskeletal reorganization in response to agonists. Two potent platelet agonists, thrombin (via its G protein-coupled receptor) and collagen (via its interaction with the alpha2beta1 integrin), caused Vav to become phosphorylated on tyrosine. Weaker platelet agonists, including ADP, epinephrine and the thromboxane A2 analog, U46619, did not. The phosphorylation of Vav in response to thrombin was maximal within 15 s and was unaffected by aspirin, inhibitors of aggregation, or the presence of the ADP scavenger, apyrase. Vav phosphorylation was also observed when platelets became adherent to immobilized collagen (via integrin alpha2beta1), fibronectin (via integrin alpha5beta1), and fibrinogen (via integrin alphaIIbbeta3). These results show that Vav phosphorylation by tyrosine kinases 1) occurs during platelet activation by potent agonists, 2) also occurs when platelets adhere to biologically relevant matrix proteins, 3) requires neither platelet aggregation nor the release of secondary agonists such as ADP and TxA2, and 4) can be initiated by at least some members of two additional classes of receptors, G protein-coupled receptors and integrins, providing further evidence that both of these can couple to tyrosine kinases.
原癌基因vav的产物p95vav或Vav主要在造血细胞中表达，并且已被证明是酪氨酸激酶的底物。尽管其功能尚不清楚，但Vav与DBL存在同源区域，DBL是GTP结合蛋白Rho家族的交换因子。该结构域的存在以及用Vav转化的细胞显示出与RhoA转化细胞中观察到的类似的明显应力纤维和粘着斑，这表明Vav可能在调节肌动蛋白细胞骨架中起作用。因此，我们研究了血小板中的Vav磷酸化情况，血小板在激动剂作用下会发生显著的细胞骨架重组。两种强效血小板激动剂，凝血酶（通过其G蛋白偶联受体）和胶原蛋白（通过其与α2β1整合素的相互作用），可使Vav在酪氨酸上发生磷酸化。较弱的血小板激动剂，包括ADP、肾上腺素和血栓素A2类似物U46619，则不会。凝血酶作用下Vav的磷酸化在15秒内达到最大值，不受阿司匹林、聚集抑制剂或ADP清除剂腺苷三磷酸双磷酸酶的影响。当血小板通过整合素α2β1粘附于固定化胶原蛋白、通过整合素α5β1粘附于纤连蛋白以及通过整合素αIIbβ3粘附于纤维蛋白原时，也观察到了Vav磷酸化。这些结果表明，酪氨酸激酶介导的Vav磷酸化：1）在强效激动剂激活血小板的过程中发生；2）在血小板粘附于生物学相关的基质蛋白时也会发生；3）既不需要血小板聚集，也不需要释放ADP和TxA2等二级激动剂；4）可由另外两类受体中的至少一些成员启动，即G蛋白偶联受体和整合素，这进一步证明了这两者都可与酪氨酸激酶偶联。
