The first secreted Tyrosine kinase

 描述(申请人提供)：在体内分泌性蛋白质中广泛的和结构保守的酪氨酸磷酸化模式已经在现有的文献中被广泛识别，但负责这些磷酸化的激酶(S)一直不清楚，排除了对这些修饰的功能分析。我们实验室最近发现了第一个已知的分泌型蛋白酪氨酸激酶(VLK)。VLK首先在上胚层早期表达，是正常器官发生所必需的。我们发现VLK可磷酸化多种分泌蛋白，并在分泌、细胞外基质(ECM)形成和ECM重塑中发挥重要作用。在许多情况下，VLK的磷酸化位点对应于以前在体内发现的分泌蛋白中的酪氨酸磷酸化位点。我们还发现，在受刺激的血小板分泌过程中，VLK与内源性ATP共同释放，推动细胞外的从头酪氨酸磷酸化。这些发现为研究细胞外基质和其他分泌性蛋白的动态调控开辟了一个广阔的新领域。在这个计划中，我们计划研究VLK在分泌途径中的调节，在内源性表达VLK的细胞中确定VLK磷酸化的分泌靶点，并开始建立VLK磷酸化如何改变特定的分泌或分泌途径驻留蛋白的功能。我们将详细研究调节VLK分泌和磷酸化所需的成分，包括血小板对生理刺激的反应，以及结构性或对刺激反应分泌VLK的永生化细胞。我们将研究每种细胞类型中的磷酸化分泌体，以及这些磷酸化对VLK的依赖性。然后，我们将在鉴定分化细胞类型中的生理VLK底物的基础上，确定VLK如何修改特定的底物功能。 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定义一种新的机制。

 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.