Characterization of Leupaxin in Osteoclast Sealing Zone

描述（由申请人提供）：骨的主要吸收细胞，破骨细胞，通过被称为足质体的整合素依赖的粘附结构附着在骨上。在骨吸收开始之前，这些足质体形成推定的封闭区前体，类似于局灶性粘连。一些蛋白酪氨酸激酶和磷酸化蛋白存在于足质体信号复合体中。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关联。对破骨细胞（OC）足质体复合体内广泛的信号分子的表征在很大程度上是不完整的。这项资助申请的目的是表征Leupaxin （LPXN）的作用，LPXN是一种蛋白质，我们已经确定是OC podosomal signaling complex的一种新成分。LPXN是一种细胞质酪氨酸磷酸化蛋白，与局灶黏附蛋白paxillin具有同源性。我们发现LPXN与蛋白酪氨酸激酶Pyk2和FAK，以及蛋白酪氨酸磷酸酶(PTP)-PEST，在podosomal complex中都有关联，提示通过磷酸化/去磷酸化机制进行调控。LPXN还被发现与ARF-GTPase paxillin激酶连接物p95PKL、p21gtpase活化激酶PAK和结构局灶黏附肌动蛋白结合蛋白actopaxin相关。肿瘤坏死因子- α和细胞外基质蛋白，如骨桥蛋白，均可刺激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突起，类似于运动表型。最后，体外抑制OC中LPXN的表达导致吸收能力下降。我们假设LPXN作为一种接头蛋白起作用，并且是OC足囊体信号复合体的关键成核成分。本研究将确定LPXN在OC活性调控中的作用，蛋白酪氨酸激酶对LPXN的调控，以及LPXN与PTP-PEST相互作用的功能意义。我们认为LPXN作为一种足突支架蛋白，通过调节足突复合体的粘附和周转，在OC的运动和吸收中发挥作用。LPXN作为OC粘附区的新组分的鉴定为其调控增加了新的复杂性，并可能提供骨吸收的药理学靶点。

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.