Dorahy D J, Berndt M C, Burns G F
Cancer Research Unit, Faculty of Medicine, University of Newcastle, N.S.W., Australia.
Biochem J. 1995 Jul 15;309 ( Pt 2)(Pt 2):481-90. doi: 10.1042/bj3090481.
Platelet activation is accompanied by a cascade of kinase reactions in which numerous specific proteins are phosphorylated on tyrosine. These events are strictly dependent upon functional activation of an integrin receptor, generally alpha IIb beta 3 (also known as glycoprotein IIb-IIIa). It is not known how alpha IIb beta 3 regulates protein tyrosine kinase activation and, in particular, neither this nor any other integrin has been shown to associate with a protein tyrosine kinase. We employed chemical crosslinking of intact platelets with the bifunctional reagents dithiobis(succinimidyl propionate) (DSP) (lipid-soluble) and dithiobis(sulphosuccinimidyl propionate) (DTSSP) (lipid-insoluble) followed by in vitro kinase assays of immunoprecipitated proteins to identify kinase activity associated with alpha IIb beta 3 in intact platelets. It was found that DSP but not DTSSP crosslinked kinase activity to alpha IIb beta 3, suggesting an internal association. In these immunoprecipitates from DSP-crosslinked platelets, the in vitro kinase reaction revealed a complex of several phosphoproteins in association with alpha IIb beta 3. This association was not seen when the resting platelets were lysed before crosslinking, indicating the specificity of the reaction in crosslinking only molecules in preformed spatial association. The beta 3 subunit of alpha IIb beta 3 was identified as one of the phosphoproteins in the complex obtained after subjecting anti-beta 3 immunoprecipitates from lysates of DSP-treated platelets to an in vitro kinase reaction and SDS/PAGE analysis. Phosphorylation of this subunit is shown to be predominantly on tyrosine. Affinity purification of the crosslinked phosphoprotein complex with anti-beta 3 followed by elution and re-precipitation identified pp60c-src and pp54/58c-lyn as two protein tyrosine kinases associating with the integrin. These results suggest that, upon platelet activation, alpha IIb beta 3 may provide a transmembrane focus for proteins involved in signal transduction.
血小板活化伴随着一系列激酶反应，其中许多特定蛋白质在酪氨酸上发生磷酸化。这些事件严格依赖于整合素受体的功能活化，通常是αIIbβ3（也称为糖蛋白IIb-IIIa）。目前尚不清楚αIIbβ3如何调节蛋白酪氨酸激酶的活化，特别是，尚未证明该整合素或任何其他整合素与蛋白酪氨酸激酶相关联。我们使用双功能试剂二硫代双（琥珀酰亚胺丙酸酯）（DSP）（脂溶性）和二硫代双（磺基琥珀酰亚胺丙酸酯）（DTSSP）（脂不溶性）对完整血小板进行化学交联，然后对免疫沉淀的蛋白质进行体外激酶测定，以鉴定完整血小板中与αIIbβ3相关的激酶活性。发现DSP而非DTSSP将激酶活性交联至αIIbβ3，提示存在内部关联。在这些来自DSP交联血小板的免疫沉淀中，体外激酶反应显示与αIIbβ3相关的几种磷蛋白复合物。当静息血小板在交联前裂解时，未观察到这种关联，表明该反应仅交联预先形成空间关联的分子的特异性。αIIbβ3的β3亚基被鉴定为在对DSP处理血小板裂解物的抗β3免疫沉淀进行体外激酶反应和SDS/PAGE分析后获得的复合物中的磷蛋白之一。该亚基的磷酸化主要发生在酪氨酸上。用抗β3亲和纯化交联的磷蛋白复合物，然后洗脱并重新沉淀，鉴定出pp60c-src和pp54/58c-lyn为与整合素相关的两种蛋白酪氨酸激酶。这些结果表明，在血小板活化时，αIIbβ3可能为参与信号转导的蛋白质提供跨膜聚集点。
