A mechanism for tyrosine phosphorylation of extracellular matrix proteins

总结 细胞外基质中的大量蛋白质被磷酸化;然而几乎所有已知的激酶 在细胞内起作用，不能催化分泌蛋白的磷酸化。最近发现的 两种分泌型激酶Fam20C和VLK代表了理解蛋白质如何 细胞外可以在多个位点磷酸化。然而，许多细胞外蛋白质是 在明显不是这两种激酶靶点的位点磷酸化。因此，额外的分泌激酶 这就提出了如何最好地识别它们并确定它们的功能的问题。在这 研究人员描述了生物信息学策略如何导致发现分泌的变异体， 跨膜、配体激活的成纤维细胞生长因子受体酪氨酸激酶（FGFR）; 变体由直接连接至激酶结构域的成纤维细胞生长因子结合结构域组成。 基于初步数据，显示分泌的Fgfr 2激酶变体在哺乳动物的多种组织中表达， 研究人员已经构建了几种用于在培养细胞中表达激酶的构建体， 已经产生了可以被诱导在不同细胞和组织中表达激酶的小鼠。目标1，体外 体内研究旨在确定分泌的FGFR 2激酶在磷酸化中的特异性 皮肤、肺和颅面（颅骨）组织中的细胞外基质蛋白。在Aim 2实验中， 以提供对分泌的FGFR2激酶在小鼠肺发育中的作用以及在三个- 肺组织的三维器官培养物，可以研究可能存在的细胞和分子机制 由分泌的FGFR2激酶调节。如果成功的话，这些研究很可能会开辟新的领域， 为未来的研究打开了一个全新的篇章，了解细胞如何相互作用和调节 细胞外基质分子的特性。

Summary A great number of proteins in the extracellular matrix are phosphorylated; yet practically all known kinases operate inside cells and are unable to catalyze phosphorylation of secreted proteins. The recent discovery of two secreted kinases, Fam20C and VLK, represents a major step forward in understanding how proteins outside cells can be phosphorylated at multiple sites. However, numerous extracellular proteins are phosphorylated at sites that are clearly not targets of these two kinases. Thus, additional secreted kinases must exist, raising questions about how to best identify them and determine what their functions may be. In this application the investigators describe how a bioinformatics strategy led to the discovery of secreted variants of transmembrane-spanning, ligand-activated, fibroblast growth factor receptor tyrosine kinases (FGFRs); with the variants consisting of the fibroblast growth factor-binding domain directly connected to the kinase domain. Based on preliminary data, showing that the secreted Fgfr2 kinase variant is expressed in multiple tissues in mice, the investigators have made several constructs for expression of the kinase in cultured cells and they have generated mice that can be induced to express the kinase in different cells and tissues. In Aim 1, in vitro and in vivo studies are directed at determining the specificity of the secreted FGFR2 kinase in phosphorylation of extracellular matrix proteins in skin, lung and craniofacial (skull) tissues. In Aim 2 experiments are designed to provide insights into the roles of the secreted FGFR2 kinase in developing lungs of mice and in three- dimensional organ cultures of lung tissues, allowing studies of cellular and molecular mechanisms that may be regulated by the secreted FGFR2 kinase. If successful, these studies are likely to break new ground and lead to future studies opening an entirely new chapter in the understanding of how cells interact with and regulate the properties of extracellular matrix molecules.