MOLECULAR MECHANISM OF C-SRC REGULATION IN OSTEOCLASTS

描述（改编自申请人的摘要）： c-Src原癌基因的无效突变确定了表达 c-Src在破骨细胞中的表达对于破骨细胞的作用和 边缘皱褶和吸收陷窝。 这种分子及其上游 调节剂和下游底物，代表了关键和核心的 骨细胞骨吸收的共同途径， 破骨细胞受到刺激。 最近，c-Src的主要调节剂是 被鉴定为c-Csk，一种胞质酪氨酸激酶， c-Src位于Tyr 527处，并使c-Src失活。 然而，C-Src仍然是 在Tyr 527处磷酸化，即使在Csk缺陷细胞中，也表明 其他CSK相关激酶的存在。 这些Csk相关激酶是 以组织或细胞特异性方式分布，这意味着 csk相关激酶可能在csk介导的组织特异性功能中发挥作用 c-Src。 本申请的目标是理解Src 蛋白质表达和酪氨酸激酶活性在 骨细胞骨吸收，并鉴定Src特异性底物。 具体目的是：1）确定精确的分子机制， 其中c-Src在破骨细胞中受到调节，通过（a）克隆和表达c-Src， （B）检查Csk相关激酶对Csk相关激酶的作用， c-Src活性，（c）检测Csk相关激酶在C-Src中的表达。 破骨细胞，在体外和体内，并确定之间的关系 c-Src与Csk、Csk相关蛋白的表达及亚细胞定位 激酶和c-Src底物，（d）测定骨吸收细胞中的 破骨细胞中Csk相关激酶的功能， 反义寡脱氧核苷酸的表达，以及完整和 突变的显性阴性Csk相关激酶，和（e）确定 成骨因子对亚细胞定位和酪氨酸的影响 c-Src、Csk、Csk相关激酶和c-Src底物的磷酸化，和 将这些变化与对骨吸收的影响相关联;和2） 鉴定破骨细胞中c-Src特异性的靶底物，和 解开破骨细胞中使用的下游信号通路。

DESCRIPTION (Adapted from the Applicant's Abstract): The creation of the null mutation for the c-Src proto-oncogene established that the expression of c-Src in osteoclasts is essential for osteoclast action and generation of ruffled borders and resorption lacunae. This molecule, and its upstream regulators and downstream substrate(s), represent the critical and central common pathway in osteoclastic bone resorption independent of how osteoclasts are stimulated. The major regulator of c-Src has recently been identified as c-Csk, a cytoplasmic tyrosine kinase which phosphorylates c-Src at Tyr 527 and renders c-Src inactive. However, c-Src is still phosphorylated at Tyr 527, even in Csk-deficient cells, suggesting the presence of other Csk-related kinases. These Csk-related kinases are distributed in a tissue- or cell-specific manner, implying that the Csk-related kinases may play a role in tissue-specific functions mediated by c-Src. The goals of the present application are to understand how Src protein expression and tyrosine kinase activity is regulated during osteoclastic bone resorption, and to identify Src-specific substrate(s). The Specific Aims are: 1) to determine the precise molecular mechanisms by which c-Src is regulated in osteoclasts, by (a) cloning and expressing the Csk-related kinases, (b) examining the effects of Csk-related kinases on c-Src activity, (c) examining the expression of the Csk-related kinases in osteoclasts, in vitro and in vivo, and determining the relationship between expression and subcellular localization of c-Src with Csk, Csk-related kinases and c-Src substrates in cells resorbing bone, (d) determining the function of the Csk-related kinases in osteoclasts by examining the effects of antisense oligodeoxynucleotides, and over-expression of intact and mutated dominant-negative Csk-related kinases, and (e) determining the effects of osteotropic factors on subcellular localization and tyrosine phosphorylation of c-Src, Csk, Csk-related kinases and c-Src substrates, and correlating these changes with effects on bone resorption; and 2) to identify the target substrate(s) specific for c-Src in osteoclasts, and unravel the downstream signaling pathways utilized in osteoclasts.