Abstract：
Receptor-induced tyrosine phosphorylation of spleen tyrosine kinase (Syk) has been studied extensively in hematopoietic cells. Metabolic mapping and high-resolution mass spectrometry, however, indicate that one of the most frequently detected phosphorylation sites encompassed S297 (S291 in mice) located within the linker B region of Syk. It has been reported that Protein kinase C (PKC) phosphorylates Syk S297, thus influencing Syk activity. However, conflicting studies suggest that this phosphorylation enhances as well as reduces Syk activity. To clarify the function of this site, we generated Syk S291A knock-in mice. We used platelets as a model system as they possess Glycoprotein VI (GPVI), a receptor containing an immunoreceptor tyrosine-based activation motif (ITAM) which transduces signals through Syk. Our analysis of the homozygous mice indicated that the knock-in platelets express only one isoform of Syk, while the wild-type expresses two isoforms at 69 and 66 kDa. When the GPVI receptor was activated with collagen-related peptide (CRP), we observed an increase in functional responses and phosphorylations in Syk S291A platelets. This potentiation did not occur with AYPGKF or 2-MeSADP, although they also activate PKC isoforms. Although there was potentiation of platelet functional responses, there was no difference in tail bleeding times. However, the time to occlusion in the FeCl3 injury model was enhanced. These data indicate that the effects of Syk S291 phosphorylation represent a significant outcome on platelet activation and signaling in vitro but also reveals its multifaceted nature demonstrated by the differential effects on physiological responses in vivo.
What is the context Spleen tyrosine kinase (Syk) is present a number of cells and important in controlling the functions of various cells and organs.Syk is known to exist in two isoforms Syk L (long form or Syk A) and Syk S (short form or Syk B).It is known that phosphorylation events regulate Syk activation and activity.In several inflammatory disease conditions, Syk mutants are known to play a role.Phosphorylation of the Syk residue Serine 291 is known to occur, but its function in the regulation of Syk activation or activity is not known.What is new In this study, we generated a mutant mouse Syk S291A, which cannot be phosphorylated on serine residue. We evaluated the function of platelets isolated from these mice and compared them to platelets isolated from wild type littermates.We observed that the mutation in Syk L unexpectedly caused Syk S to disappear from a number of tissues.Platelet functions are enhanced in mutant mouse platelets compared to those from wild-type mice.What is the impact These studies enhance our understanding of the impact of Serine 291 phosphorylation on the function of Syk in platelets.
What is the context Spleen tyrosine kinase (Syk) is present a number of cells and important in controlling the functions of various cells and organs.Syk is known to exist in two isoforms Syk L (long form or Syk A) and Syk S (short form or Syk B).It is known that phosphorylation events regulate Syk activation and activity.In several inflammatory disease conditions, Syk mutants are known to play a role.Phosphorylation of the Syk residue Serine 291 is known to occur, but its function in the regulation of Syk activation or activity is not known.What is new In this study, we generated a mutant mouse Syk S291A, which cannot be phosphorylated on serine residue. We evaluated the function of platelets isolated from these mice and compared them to platelets isolated from wild type littermates.We observed that the mutation in Syk L unexpectedly caused Syk S to disappear from a number of tissues.Platelet functions are enhanced in mutant mouse platelets compared to those from wild-type mice.What is the impact These studies enhance our understanding of the impact of Serine 291 phosphorylation on the function of Syk in platelets.
摘要：
已经在造血细胞中广泛研究了受体诱导的脾酪氨酸激酶（Syk）的酪氨酸磷酸化。代谢图谱和高分辨率质谱，然而,表明最频繁检测的磷酸化位点之一包括位于Syk的接头B区域内的S297(小鼠中的S291)。据报道,蛋白激酶C(PKC)使SykS297磷酸化,从而影响Syk活性。然而,相互矛盾的研究表明，这种磷酸化增强和减少Syk活性。为了明确本网站的功能，我们产生了SykS291A敲入小鼠。我们使用血小板作为模型系统，因为它们具有糖蛋白VI（GPVI），一种含有免疫受体酪氨酸基活化基序(ITAM)的受体,其通过Syk转导信号。我们对纯合小鼠的分析表明，敲入的血小板仅表达一种Syk同工型，而野生型表达69和66kDa的两种同工型。当GPVI受体被胶原相关肽（CRP）激活时，我们观察到SykS291A血小板的功能反应和磷酸化增加。AYPGKF或2-MeSADP不会发生这种增强，尽管它们也激活PKC亚型。尽管血小板功能反应增强，尾部出血次数无差异。然而,FeCl3损伤模型的闭塞时间延长。这些数据表明SykS291磷酸化的作用代表了体外血小板活化和信号传导的显著结果,但也揭示了其通过对体内生理反应的差异作用所证明的多方面性质。
什么是背景脾酪氨酸激酶（Syk）存在许多细胞，在控制各种细胞和器官的功能方面很重要。已知Syk以两种同种型存在SykL(长形式或SykA)和SykS(短形式或SykB)。已知磷酸化事件调节Syk激活和活性。在几种炎症性疾病中，已知Syk突变体发挥作用。已知会发生Syk残基丝氨酸291的磷酸化，但其在Syk激活或活性调节中的功能尚不清楚。这项研究的新内容，我们产生了一个突变小鼠SykS291A,不能在丝氨酸残基上磷酸化。我们评估了从这些小鼠中分离的血小板的功能，并将其与从野生型同窝中分离的血小板进行了比较。我们观察到SykL中的突变意外地导致SykS从许多组织中消失。与野生型小鼠相比,突变小鼠血小板中的血小板功能增强。这些研究增强了我们对丝氨酸291磷酸化对血小板中Syk功能的影响的理解。
什么是背景脾酪氨酸激酶（Syk）存在许多细胞，在控制各种细胞和器官的功能方面很重要。已知Syk以两种同种型存在SykL(长形式或SykA)和SykS(短形式或SykB)。已知磷酸化事件调节Syk激活和活性。在几种炎症性疾病中，已知Syk突变体发挥作用。已知会发生Syk残基丝氨酸291的磷酸化，但其在Syk激活或活性调节中的功能尚不清楚。这项研究的新内容，我们产生了一个突变小鼠SykS291A,不能在丝氨酸残基上磷酸化。我们评估了从这些小鼠中分离的血小板的功能，并将其与从野生型同窝中分离的血小板进行了比较。我们观察到SykL中的突变意外地导致SykS从许多组织中消失。与野生型小鼠相比,突变小鼠血小板中的血小板功能增强。这些研究增强了我们对丝氨酸291磷酸化对血小板中Syk功能的影响的理解。
