EFFECT OF PHOSPHORYLATION ON INTERACTION OF P130CAS WITH AND-34

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。列出的机构是 研究中心，而研究中心不一定是研究者所在的机构。 该项目旨在确定为什么在特定蛋白质AND-34的过表达后，NSP蛋白质家族成员与p130 Cas相关联，p130 Cas是一种粘着斑适配器蛋白，最为人所知的是整合粘附相关信号的Src底物。AND-34/BCAR 3/NSP 2（BCAR 3）而非NSP 1或NSP 3的过表达在人乳腺癌细胞系中诱导抗雌激素抗性。BCAR 3在上皮MCF-7细胞中的过表达增加了磷酸化的p130 Cas种类的水平，其在SDS PAGE上迁移更慢，而NSP-1和NSP 3分别诱导适度的磷酸化或不诱导磷酸化。相反，通过可诱导的shRNA减少间充质MDA-231细胞中的BCAR 3表达导致这种p130 Cas的丢失， 磷酸化NSP 3的富含丝氨酸/脯氨酸的结构域被AND-2000的结构域取代。 34/BCAR 3逐渐灌输诱导p130 Cas磷酸化的能力。磷酸-氨基酸分析表明BCAR 3诱导p130 Cas丝氨酸磷酸化。质谱鉴定了p130 Cas丝氨酸139、437和639处的磷酸化。p130 Cas丝氨酸磷酸化在MDA-231细胞粘附到纤连蛋白后数小时发生，并且依赖于BCAR 3表达。BCAR 3敲除还改变了p130 Cas定位，并将MDA-231生长转化为上皮样模式，其特征在于显著的内聚性和缺乏片状脂质 殖民地边界的投影这些研究表明，BCAR 3调节p130 Cas丝氨酸磷酸化是粘附依赖性的，时间上不同于以前充分表征的快速Fak和Src激酶介导的p130 Cas酪氨酸磷酸化，并与侵袭性表型相关。这些结果最近发表在Cellular Signalling（Makkinje A，Near RI，Infusini G，Vanden Borre P，Bloom A，Cai D，CostelloCE，Lerner A. AND-34/BCAR 3调节粘附依赖性p130 Cas丝氨酸磷酸化和乳腺癌细胞生长模式。A Makkinje等人，2009，21，1423-1435）。

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, upon 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 were recently published in Cellular Signalling (Makkinje A, Near RI, Infusini G, Vanden Borre P, Bloom A, Cai D, CostelloCE, Lerner A. AND-34/BCAR3 regulates adhesion-dependent p130Cas serine phosphorylation andbreast cancer cell growth pattern.Cell Signal. A Makkinje et al., 2009, 21, 1423-1435).