A mechanism for tyrosine phosphorylation of extracellular matrix proteins

摘要 细胞外基质中的大量蛋白质是磷酸化的；但几乎所有已知的激酶 在细胞内运作，不能催化分泌蛋白质的磷酸化。最近发现的 两个分泌的激酶，Fam20C和VLK，代表着在理解蛋白质如何 外部细胞可以在多个位置被磷酸化。然而，许多胞外蛋白是 在明显不是这两个激酶靶点的位置发生磷酸化。因此，额外的分泌型激酶 必须存在，这引发了如何最好地识别它们并确定它们可能的功能的问题。在这 应用研究人员描述了生物信息学策略如何导致发现隐秘的变种 跨膜、配体激活的成纤维细胞生长因子受体酪氨酸激酶(FGFRs) 由成纤维细胞生长因子结合域组成的变体直接连接到激活域。 根据初步数据，分泌的FGFR2激酶变异体在多个组织中表达 在小鼠身上，研究人员已经构建了几种在培养细胞中表达该激酶的结构，他们 已经产生了可以被诱导在不同细胞和组织中表达这种激酶的小鼠。在Aim 1中，体外 体内研究旨在确定分泌的FGFR2激酶在磷酸化过程中的特异性 皮肤、肺和颅面(头骨)组织中细胞外基质蛋白的含量。在AIM中，设计了2个实验 为了深入了解分泌的FGFR2激酶在小鼠肺发育中的作用，以及在三种情况下- 肺组织的立体器官培养，使细胞和分子机制的研究可能 受分泌的FGFR2激酶调节。如果成功，这些研究很可能会开辟新的天地，引领 为未来的研究揭开了理解细胞如何相互作用和调节的全新篇章 细胞外基质分子的性质。

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.