MOLECULAR MECHANISM OF C-SRC REGULATION IN OSTEOCLASTS

描述(改编自申请人的摘要)：创建 原癌基因c-Src的零突变证实了 C-Src在破骨细胞中的表达对破骨细胞的作用和产生 褶皱的边界和再吸收的空隙。这种分子及其上游 调节器和下游底物(S)，代表着关键和中心 破骨细胞性骨吸收的共同途径与方式无关 破骨细胞受到刺激。C-Src的主要监管者最近 被鉴定为c-CSK，一种胞质酪氨酸激酶，磷酸化 C-Src在Tyr 527，并使c-Src无效。然而，c-Src仍然是 在Tyr 527处被磷酸化，即使在CSK缺乏的细胞中也是如此，这表明 存在其他与CSK相关的激酶。这些与CSK相关的激酶是 以组织或细胞特有的方式分布，这意味着 CSK相关激酶可能在组织特异性功能中发挥作用 C-源本应用程序的目标是了解源 蛋白表达和酪氨酸激酶活性在 破骨细胞性骨吸收，并鉴定Src特异性底物(S)。 具体的目标是：1)通过以下方法确定精确的分子机制 哪些c-Src在破骨细胞中受调控，通过(A)克隆和表达 CSK相关的激酶，(B)研究CSK相关的激酶对 C-Src活性，(C)检测脑脊液中CSK相关蛋白的表达 破骨细胞，在体外和体内，和测定之间的关系 C-Src与CSK、CSK相关基因的表达及亚细胞定位 吸收骨的细胞中的激酶和c-Src底物，(D)确定 通过检测CSK相关蛋白在破骨细胞中的作用 反义寡核苷酸的表达，以及全长和 突变的显性-阴性的CSK相关激酶，以及(E)确定 促骨因子对亚细胞定位和酪氨酸的影响 C-Src、CSK、CSK相关的激酶和c-Src底物的磷酸化，以及 将这些变化与对骨吸收的影响相关联；以及2) 确定破骨细胞中c-Src的特异性靶底物(S)，以及 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.