Regulation of cell migration and signaling by phosphotyrosine and ubiquitin

磷酸酪氨酸和泛素对细胞迁移和信号传导的调节

基本信息

批准号：
10295778
负责人：
Jonathan A Cooper
金额：
$ 15.44万
依托单位：
FRED HUTCHINSON CANCER RESEARCH CENTER
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-08-01 至 2022-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10295778
关键词：
Adaptor Signaling Protein Adhesions Area BCAR1 gene BCAR3 gene Binding Breast Cancer Cell CISH gene Cancer Biology Cell Proliferation Cells Cellular biology Complex Cullin 5 Protein Epithelial Cells Focal Adhesions Funding Genes Goals Image Immunity Learning Ligase Link Malignant Neoplasms Methods Molecular Mutation Analysis Normal Cell Oncogenic Pathway interactions Phenotype Phosphorylation Phosphotyrosine Post-Translational Protein Processing Procedures Protein Dephosphorylation Protein Tyrosine Kinase Protein Tyrosine Phosphatase Proteins Proteomics Publishing Reagent Regulation Research Role Signal Pathway Signal Transduction Signaling Protein Site System Testing Tyrosine Tyrosine Phosphorylation Ubiquitin breast malignancies cancer cell cancer survival cell motility cell transformation innovation malignant breast neoplasm mammary migration multicatalytic endopeptidase complex novel strategies optogenetics prognostic indicator scaffold ubiquitin-protein ligase wound healing

项目摘要

ABSTRACT Protein tyrosine kinases (PTKs) phosphorylate tyrosine in proteins involved in cell signaling pathways that stimulate cell migration, proliferation and oncogenic transformation. Phosphotyrosine (pY) proteins may be inactivated by dephosphorylation or by proteasomal degradation following ubiquitylation by pY-specific ubiquitin E3 ligases. We are focusing on the multi-subunit Cullin 5-RING ligase (CRL5), which interacts with pY proteins through suppressors of cytokine signaling (SOCS) adaptor proteins. Our past studies have shown that various CRL5-SOCS complexes are critical in normal epithelial cells to repress an oncogenic PTK, Src. When we inhibit expression of Cullin 5 or specific SOCS genes, Src is activated and cell migration, proliferation and transformation are stimulated. Increased Src activity is necessary but not sufficient for the migration and proliferation of Cullin 5-deficient cells, suggesting that CRL5 has additional substrates. Our past studies have identified two such substrates that are critical for cell migration. Here we propose to extend these studies, to understand how CRL5-SOCS regulates epithelial cell migration. Our scientific premise is that the identification of key CRL5-SOCS substrates and functions in migration is critical for understanding normal and transformed cell biology. During the last funding period, we used innovative live imaging, optogenetics and mutational analysis to uncover the molecular mechanism by which CRL5-SOCS6 regulates the signaling scaffold, Cas (Crk- associated substrate), within focal adhesion sites at the leading edge of migrating epithelial cells. We also used quantitative proteomics to identify other CRL5 substrates and test their roles in cell migration. Specifically, we identified BCAR3 (breast cancer anti-estrogen resistance 3) as a second CRL5-SOCS6 substrate that regulates cell migration. Finally, we found that SOCS2, like SOCS6, localizes to focal adhesion sites and regulates cytoskeletal dynamics. We propose three broad aims to extend these observations and understand the molecular mechanisms. First, we will understand the importance of BCAR3 phosphorylation in BCAR3 stability and focal adhesion dynamics. Second, BCAR3 binds and cooperates with Cas: each protein stabilizes the other from degradation. We will understand the mechanism and importance of BCAR3-Cas mutual stabilization. Third, we will determine the mechanism by which SOCS2 regulates focal adhesion dynamics. Together, these aims will extend our understanding of how protein-tyrosine phosphorylation and ubiquitylation cooperate to regulate cell migration.

抽象的蛋白酪氨酸激酶（PTKS）在参与细胞信号通路的蛋白质中磷酸化酪氨酸刺激细胞迁移，增殖和致癌转化。磷酸酪氨酸（PY）蛋白可能是通过脱磷酸化或蛋白酶体降解在PY特异泛素E3连接酶。我们专注于与PY相互作用的多支亚基5环（CRL5）通过细胞因子信号传导（SOCS）衔接蛋白的抑制剂蛋白质。我们过去的研究表明在正常上皮细胞中，各种CRL5-SOCS络合物至关重要，以抑制致癌PTK，SRC。什么时候我们抑制Cullin 5或特定SOC基因的表达，SRC被激活，细胞迁移，增殖和刺激转化。 SRC活动增加是必要的，但不足以迁移和 Cullin 5缺陷细胞的增殖，表明CRL5具有其他底物。我们过去的研究有鉴定出对细胞迁移至关重要的两个这样的底物。在这里，我们建议将这些研究扩展到了解CRL5-SOC如何调节上皮细胞迁移。我们的科学前提是身份证明关键CRL5-SOC底物和迁移中的功能对于理解正常和转化至关重要细胞生物学。在最后一个资金期间，我们使用创新的实时成像，光遗传学和突变分析来发现CRL5-SOC6调节信号支架的分子机制，CAS（CRK-）相关的底物），在迁移上皮细胞前缘的焦点粘附位点内。我们也使用了定量蛋白质组学鉴定其他CRL5底物并测试其在细胞迁移中的作用。具体来说，我们确定BCAR3（乳腺癌抗雌激素耐药3）为第二个CRL5-SOCS6底物，调节细胞迁移。最后，我们发现SOCS2（例如SOCS6）本地化为焦点粘附部位，并且调节细胞骨架动力学。我们提出了三个广泛的目标，以扩展这些观察结果并了解分子机制。第一的，我们将了解BCAR3磷酸化在BCAR3稳定性和局灶性粘附动力学中的重要性。其次，BCAR3与CAS结合并合作：每种蛋白质稳定在降解中。我们将了解BCAR3-CAS相互稳定的机制和重要性。第三，我们将确定 SOCS2调节局灶性粘附动力学的机制。这些目标在一起将扩大我们的了解蛋白质酪氨酸磷酸化和泛素化如何配合以调节细胞迁移。