EFFECT OF PHOSPHORYLATION ON INTERACTION OF P130CAS WITH AND-34

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 这个项目试图确定为什么一种特定的蛋白质，和-34， NSP蛋白家族成员与p130Cas相关，p130Cas是一种焦点黏附适配蛋白，通常被认为是一种整合黏附相关信号的Src底物。And-34/BCAR3/NSP2(BCAR3)过表达，而不是NSP1或NSP3过表达，可诱导人乳腺癌细胞产生抗雌激素耐药。BCAR3在上皮MCF-7细胞中的过表达增加了一种磷酸化的p130Cas物种的水平，该物种在SDS PAGE上迁移较慢，而NSP-1和NSP3分别诱导适度的或没有磷酸化。相反，可诱导的shRNA下调间充质细胞中BCAR3的表达会导致这种p130Cas的丢失 磷酸化。NSP3的S丝氨酸/脯氨酸富含结构域被替换为和- 34/BCAR3具有诱导p130Cas磷酸化的能力。磷酸氨基酸分析表明，BCAR3可诱导p130Cas丝氨酸磷酸化。质谱仪鉴定了p130Cas丝氨酸139、437和639的磷酸化。P130Cas丝氨酸磷酸化在MDA-231细胞与纤维连接蛋白黏附数小时后发生，并依赖于BCAR3的表达。BCAR3基因敲除也改变了p130Cas的定位，将MDA-231的生长转化为上皮样生长，其特征是显著的粘附性和缺乏片层脂膜 殖民地边界的投影。这些研究表明，BCAR3调节p130Cas丝氨酸的磷酸化，这种磷酸化是黏附依赖的，不同于以前很好描述的Fak和Src激酶介导的p130Cas酪氨酸磷酸化，并且与侵袭性表型相关。这些结果已被接受发表在《细胞信号》杂志上。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. This project seeks to establish why over-expression of a specific protein, AND-34, NSP protein family members associate with p130Cas, a focal adhesion adapter protein best known as a Src substrate that integrates adhesion-related signaling. Over-expression of AND-34/BCAR3/NSP2 (BCAR3), but not NSP1 or NSP3, induces anti-estrogen resistance in human breast cancer cell lines. BCAR3 over-expression in epithelial MCF-7 cells augments levels of a phosphorylated p130Cas species that migrates more slowly on SDS PAGE while NSP-1 and NSP3 induce modest or no phosphorylation, respectively. Conversely, reduction in BCAR3 expression in mesenchymal MDA-231 cells by inducible shRNA results in loss of such p130Cas phosphorylation. Replacement of NSP3's serine/proline-rich domain with that of AND- 34/BCAR3 instills the ability to induce p130Cas phosphorylation. Phospho-amino acid analysis demonstrates that BCAR3 induces p130Cas serine phosphorylation. Mass spectrometry identified phosphorylation at p130Cas serines 139, 437 and 639. p130Cas serine phosphorylation occurs physiologically hours after adhesion of MDA-231 cells to fibronectin and is dependent upon BCAR3 expression. BCAR3 knockdown also alters p130Cas localization and converts MDA-231 growth to an epithelioid pattern characterized by striking cohesiveness and lack of lamellipodial projections at colony borders. These studies suggest that BCAR3 regulates p130Cas serine phosphorylation that is adhesion-dependent, temporally distinct from previously well-characterized rapid Fak and Src kinase-mediated p130Cas tyrosine phosphorylation and that correlates with invasive phenotype. These results have been accepted for publication in Cellular Signalling.