The first secreted Tyrosine kinase

 描述（由申请人提供）：在现有文献中已广泛鉴定了体内分泌蛋白中广泛且结构保守的酪氨酸磷酸化模式，但负责这些磷酸化的激酶尚不清楚，因此无法对这些修饰进行功能分析。我们的实验室最近发现了第一个已知的分泌蛋白酪氨酸激酶（VLK）。VLK首先在早期外胚层中表达，并且对于正常器官发生是必需的。我们发现，VLK磷酸化广泛的分泌蛋白，在分泌，细胞外基质（ECM）的形成和ECM重塑的调节中发挥作用。在许多情况下，由VLK磷酸化的位点对应于先前发现在体内发生的分泌蛋白中的酪氨酸磷酸化位点。我们还发现，在刺激的血小板分泌过程中，VLK与内源性ATP共同释放，以驱动细胞外的从头酪氨酸磷酸化。这些发现为ECM和其他分泌蛋白的动态调控研究开辟了广阔的新领域。在这个提议中，我们计划研究分泌途径中VLK的调节，鉴定内源性表达VLK的细胞中VLK磷酸化的分泌靶点，并开始确定VLK磷酸化如何改变特定分泌或分泌途径驻留蛋白的功能。我们将详细研究VLK分泌和磷酸化的调节所需的组件，无论是在血小板中响应生理刺激，并在永生化细胞分泌VLK组成或响应刺激。我们将检查这些细胞类型中的每一种中的磷酸化分泌组，以及这些磷酸化对VLK的依赖性。然后，我们将建立在这种鉴定的生理VLK基板在分化的细胞类型，以建立VLK如何修改特定的基板功能。 这些研究将为分泌途径和细胞外环境中蛋白质活性的动态调节确定一种新的机制。

 DESCRIPTION (provided by applicant): Broad and structurally conserved patterns of tyrosine phosphorylation in secreted proteins in vivo have been widely identified in the existing literature but the kinase(s) responsible for these phosphorylations have been obscure, precluding functional analyses of these modifications. Our laboratory has recently identified the first known secreted protein tyrosine kinase (VLK). VLK is first expressed in the early epiblast, and is essential for normal organogenesis. We find that VLK phosphorylates a wide range of secreted proteins with established roles in the regulation of secretion, extracellular matrix (ECM) formation, and ECM remodeling. In many cases, sites phosphorylated by VLK correspond to sites of tyrosine phosphorylation in secreted proteins previously found to occur in vivo. We have also found that, during stimulated platelet secretion, VLK is co-released with endogenous ATP to drive de novo tyrosine phosphorylation outside the cell. These finding open up a broad new area in the study of dynamic regulation of ECM and other secreted proteins. In this proposal, we plan to investigate the regulation of VLK within the secretory pathway, identify secreted targets for VLK phosphorylation in cells that express VLK endogenously, and begin to establish how VLK phosphorylation modifies the function of specific secreted or secretory pathway resident proteins. We will examine in detail the components necessary for regulation of VLK secretion and phosphorylation, both in platelets in response to physiological stimuli, and in immortalized cells that secrete VLK either constitutively or in response to stimuli. We will examine the phosphorylated secretome in each of these cell types, and the dependence of these phosphorylations on VLK. We will then build on this identification of physiological VLK substrates in differentiated cell types to establish how VLK modifies specific substrate functions. These studies will define a new mechanism for the dynamic regulation of the activity of proteins in the secretory pathway and in the extracellular environment.