Characterization of Leupaxin in Osteoclast Sealing Zone

破骨细胞封闭区 Leupaxin 的表征

• 批准号: 6727433
• 负责人: ANANDA GUPTA
• 金额: $ 25.21万
• 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/6727433
• 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（由申请人提供）：骨的主要吸收细胞，破骨细胞，通过整合素依赖性粘附结构（称为足体）附着在骨上。在开始骨吸收之前，这些足体形成了密封区的假定前体，并且类似于局灶性粘连。在足体信号传导复合物中存在几种蛋白 - 酪氨酸激酶和磷酸化的蛋白质。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关联。在破骨细胞（OC）中构成的大量信号分子的表征在很大程度上是不完整的。该赠款应用的目的是表征Leupaxin（LPXN）的作用，该蛋白质已确定为OC Podosomal信号复合物的新成分。 LPXN是一种细胞质酪氨酸磷酸化蛋白，与局灶性粘附蛋白Paxillin共享同源性。我们发现，LPXN与蛋白质酪氨酸激酶，Pyk2和FAK以及蛋白质 - 酪氨酸磷酸酶（PTP） - 预留蛋白 - 蛋白 - 磷酸化酶（PTP） - 蛋白质磷酸化酶（PTP）相关，这表明通过磷酸化 /去磷酸化机制来调节。还发现LPXN与ARF-GTPase Paxillin激酶接头，P95PKL，P21GTPase激活激活激酶，PAK和结构粘着肌动蛋白结合蛋白Actopaxin，pak。发现肿瘤坏死因子-Alpha和细胞外基质蛋白（例如骨桥蛋白）都刺激LPXN的酪氨酸磷酸化。 LPXN的过表达引起了细胞前缘的大量细胞质投影，类似于运动表型。最后，在OC中对LPXN表达的体外抑制导致吸收能力降低。我们假设LPXN充当衔接蛋白，并且是OC足体信号复合物的关键成核成分。这项研究将确定LPXN在OC活性，蛋白酪氨酸激酶对LPXN的调节中的作用，以及LPXN和PTP-PEST之间相互作用的功能意义。我们提出，LPXN作为大型体积支架蛋白，通过调节足体复合物的粘附和周转，在OC运动和吸收中起作用。将LPXN鉴定为OC粘附区的新组成部分，为其调节增添了新的复杂性，并可能在骨吸收中提供了药理目标。