Characterization of Leupaxin in Osteoclast Sealing Zone

破骨细胞封闭区 Leupaxin 的表征

• 批准号: 6617483
• 负责人: ANANDA GUPTA
• 金额: $ 24.74万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-04-01 至 2007-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6617483
• 关键词: SDS polyacrylamide gel electrophoresis; autoradiography; biological signal transduction; cell adhesion molecules; cell migration; cell morphology; enzyme induction /repression; enzyme substrate; focal adhesion kinase; immunoprecipitation; laboratory mouse; nucleic acid sequence; osteoclasts; osteogenesis; phase contrast microscopy; phosphorylation; physiologic bone resorption; polymerase chain reaction; protein binding; protein protein interaction; protein structure function; protein tyrosine kinase; protein tyrosine phosphatase; tissue /cell culture; western blottings

DESCRIPTION (provided by applicant): The principal resorptive cells of bone, the osteoclasts, attach to bone through integrin-dependent adhesion structures known as podosomes. Prior to initiation of bone resorption, these podosomes form putative precursors of the sealing zone, and are similar to focal adhesions. Several protein-tyrosine kinases and phosphorylated proteins exist within the podosomal signaling complex. Integrin activation results in an increased tyrosine-phosphorylation of the nonreceptor protein tyrosine kinase (Pyk2) and induces its association with other signaling molecules. The characterization of the wide array of signaling molecules contained within the osteoclast (OC) podosomal complex is largely incomplete. The aim of this grant application is to characterize the role of Leupaxin (LPXN), a protein that we have identified as a novel component of the OC podosomal signaling complex. LPXN is a cytoplasmic tyrosine-phosphorylated protein that shares homology with the focal adhesion protein, paxillin. We have found that LPXN is associated with both the protein-tyrosine kinases, Pyk2 and FAK, and with the protein-tyrosine phosphatase, (PTP)-PEST, at the podosomal complex, suggesting regulation by phosphorylation / dephosphorylation mechanisms. LPXN was also found to associate with the ARF-GTPase paxillin kinase linker, p95PKL, the p21GTPase-activated kinase, PAK, and the structural focal adhesion actin-binding protein, actopaxin. Both tumor necrosis factor-alpha, and an extracellular matrix protein, such as osteopontin, were found to stimulate tyrosine-phosphorylation of LPXN. Overexpression of LPXN evoked numerous cytoplasmic projections at the leading edge of the cell, resembling a motile phenotype. Finally, in vitro inhibition of LPXN expression in the OC led to a decrease in resorptive capacity. We hypothesize that LPXN functions as an adaptor protein, and is a critical nucleating component of the OC podosomal signaling complex. This study will determine the role of LPXN in the regulation of OC activity, regulation of LPXN by protein tyrosine kinases, and functional significance of interaction between LPXN and PTP-PEST. We propose that LPXN, as a podosomal scaffold protein, plays a role in OC motility and resorption by regulating adhesion and turnover of the podosomal complex. The identification of LPXN as a new component of the OC adhesion zone adds a new complexity to their regulation, and may provide a pharmacological target in bone resorption.

描述(申请人提供)：骨的主要吸收细胞，破骨细胞，通过被称为足小体的整合素依赖的黏附结构附着在骨上。在骨吸收开始之前，这些足小体形成封闭区的假定前体，类似于局灶性粘连。几种蛋白酪氨酸激酶和磷酸化的蛋白质存在于后体信号复合体中。整合素激活导致非受体蛋白酪氨酸激酶(PYK2)的酪氨酸磷酸化增加，并诱导其与其他信号分子的联系。破骨细胞(OC)足体复合体中包含的广泛的信号分子的特征在很大程度上是不完整的。这项资助申请的目的是描述亮蛋白(LPXN)的作用，LPXN是一种我们已经确定的OC足体信号复合体的新成分。LPXN是一种胞质酪氨酸磷酸化蛋白，与焦点黏附蛋白paxlin有相同的同源性。我们发现，LPXN既与蛋白酪氨酸激酶PYK2和FAK有关，也与后体复合体上的蛋白酪氨酸磷酸酶(PTP)-PEST有关，这表明LPXN通过磷酸化/去磷酸化机制进行调节。LPXN还与ARF-GTP酶、p95PKL、p21GTP酶激活的激酶PAK和结构粘着斑肌动蛋白结合蛋白actopaxin相关联。肿瘤坏死因子-α和一种细胞外基质蛋白，如骨桥蛋白，都被发现能刺激LPXN的酪氨酸磷酸化。LPXN的过度表达在细胞的前缘引起了大量的细胞质投射，类似于一种运动表型。最后，在体外抑制LPXN在OC中的表达导致了吸收能力的下降。我们假设LPXN作为一个接头蛋白发挥作用，是OC足体信号复合体的关键成核成分。本研究将确定LPXN在OC活性调节中的作用，蛋白酪氨酸激酶对LPXN的调节，以及LPXN与PTP-PEST相互作用的功能意义。我们认为，LPXN作为一种足体支架蛋白，通过调节足体复合体的黏附和周转，在OC的运动和吸收中发挥作用。LPXN是OC粘附区的一种新成分，为其调控增加了新的复杂性，并可能为骨吸收提供一个药理靶点。