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Abstract：
Bruton\'s tyrosine kinase (Btk) and spleen tyrosine kinase (Syk) are major signaling proteins in human platelets that are implicated in atherothrombosis and thrombo-inflammation, but the mechanisms controlling their activities are not well understood. Previously, we showed that Syk becomes phosphorylated at S297 in glycoprotein VI (GPVI)-stimulated human platelets, which limits Syk activation. Here, we tested the hypothesis that protein kinases C (PKC) and A (PKA) and protein phosphatase 2A (PP2A) jointly regulate GPVI-induced Btk activation in platelets. The GPVI agonist convulxin caused rapid, transient Btk phosphorylation at S180 (pS180↑), Y223 and Y551, while direct PKC activation strongly increased Btk pS180 and pY551. This increase in Btk pY551 was also Src family kinase (SFK)-dependent, but surprisingly Syk-independent, pointing to an alternative mechanism of Btk phosphorylation and activation. PKC inhibition abolished convulxin-stimulated Btk pS180 and Syk pS297, but markedly increased the tyrosine phosphorylation of Syk, Btk and effector phospholipase Cγ2 (PLCγ2). PKA activation increased convulxin-induced Btk activation at Y551 but strongly suppressed Btk pS180 and Syk pS297. PP2A inhibition by okadaic acid only increased Syk pS297. Both platelet aggregation and PLCγ2 phosphorylation with convulxin stimulation were Btk-dependent, as shown by the selective Btk inhibitor acalabrutinib. Together, these results revealed in GPVI-stimulated platelets a transient Syk, Btk and PLCγ2 phosphorylation at multiple sites, which are differentially regulated by PKC, PKA or PP2A. Our work thereby demonstrated the GPVI-Syk-Btk signalosome as a tightly controlled protein kinase network, in agreement with its role in atherothrombosis.
摘要：
布鲁顿酪氨酸激酶（Btk）和脾酪氨酸激酶（Syk）是人血小板中主要的信号蛋白，与动脉粥样硬化和血栓炎症有关。但是控制其活动的机制还没有得到很好的理解。以前,我们表明Syk在糖蛋白VI（GPVI）刺激的人血小板中的S297处磷酸化，这限制了Syk的激活。这里,我们检验了蛋白激酶C（PKC）和A（PKA）以及蛋白磷酸酶2A（PP2A）共同调节GPVI诱导的血小板Btk激活的假设。GPVI激动剂惊厥素引起的迅速，S180时的瞬时Btk磷酸化（pS180^），Y223和Y551，而直接PKC活化则强烈增加了BtkpS180和pY551。BtkpY551的这种增加也是Src家族激酶（SFK）依赖性的，但令人惊讶的是独立于Syk，指向Btk磷酸化和激活的替代机制。PKC抑制消除了惊厥蛋白刺激的BtkpS180和SykpS297，但显着增加了Syk的酪氨酸磷酸化，Btk和效应磷脂酶Cγ2(PLCγ2)。PKA激活在Y551增加了惊厥素诱导的Btk激活，但强烈抑制了BtkpS180和SykpS297。冈田酸对PP2A的抑制仅增加SykpS297。惊厥素刺激下的血小板聚集和PLCγ2磷酸化都是Btk依赖性的，如选择性Btk抑制剂阿卡拉布替尼所示。一起,这些结果表明，在GPVI刺激的血小板中存在短暂的Syk，Btk和PLCγ2在多个位点磷酸化，它们受到PKC的不同调节，PKA或PP2A。因此，我们的工作证明了GPVI-Syk-Btk信号体是一个严格控制的蛋白激酶网络，与它在动脉粥样硬化中的作用一致。
