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，通过整合素依赖性粘附结构（称为podosomes）附着于骨。在骨吸收开始之前，这些podosome形成了封闭区的假定前体，并且与局灶性粘连相似。几种蛋白酪氨酸激酶和磷酸化蛋白存在于足体信号复合物中。整合素活化导致非受体蛋白酪氨酸激酶（Pyk 2）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）足体复合物中包含的大量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亮帕素（LPXN）的作用，我们已经确定这种蛋白质是OC podosomal信号复合物的一种新组分。LPXN是一种细胞质酪氨酸磷酸化蛋白，与黏着斑蛋白桩蛋白具有同源性。我们已经发现LPXN与蛋白酪氨酸激酶Pyk 2和FAK以及蛋白酪氨酸磷酸酶（PTP）-PEST在足体复合物处相关，表明通过磷酸化/去磷酸化机制进行调节。还发现LPXN与ARF-GTP酶桩蛋白激酶接头p95 PKL、p21 GTP酶激活激酶PAK和结构粘着斑肌动蛋白结合蛋白actopaxin相关。发现肿瘤坏死因子-α和细胞外基质蛋白（如骨桥蛋白）都能刺激LPXN的酪氨酸磷酸化。LPXN的过表达诱发了细胞前缘的许多细胞质突起，类似于能动表型。最后，在体外抑制OC中LPXN表达导致再吸收能力降低。我们假设LPXN作为衔接蛋白发挥作用，并且是OC podosomal信号复合物的关键成核组分。本研究将探讨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粘附和周转，在OC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节增加了新的复杂性，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.