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

• 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
• 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

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.

摘要 蛋白酪氨酸激酶（PTK）磷酸化参与细胞信号传导途径的蛋白质中的酪氨酸， 刺激细胞迁移、增殖和致癌转化。磷酸酪氨酸（pY）蛋白可以是 通过去磷酸化或通过蛋白酶体降解而失活 泛素E3连接酶。我们关注的是与pY相互作用的多亚基Cullin 5-RING连接酶（CRL 5）。 通过细胞因子信号转导抑制因子（SOCS）衔接蛋白来调节蛋白质。我们过去的研究表明， 各种CRL 5-SOCS复合物在正常上皮细胞中对于抑制致癌PTK Src是关键的。当 我们抑制Cullin 5或特异性SOCS基因的表达，Src被激活，细胞迁移、增殖和 转型受到刺激。增加Src活动是必要的，但不足以实现迁移， Cullin 5缺陷细胞的增殖，表明CRL 5具有额外的底物。我们过去的研究 确定了两个这样的基板是细胞迁移的关键。在这里，我们建议扩展这些研究， 了解CRL 5-SOCS如何调节上皮细胞迁移。我们的科学前提是 关键CRL 5-SOCS底物和功能的迁移是至关重要的理解正常和转化 细胞生物学 在上一个资助期间，我们使用了创新的活体成像、光遗传学和突变分析， 揭示CRL 5-SOCS 6调节信号支架Cas（Crk-1）的分子机制。 相关底物），在迁移上皮细胞前缘的粘着斑位点内。我们还使用 定量蛋白质组学，以确定其他CRL 5底物，并测试其在细胞迁移中的作用。我们特别 将BCAR 3（乳腺癌抗雌激素抗性3）鉴定为第二种CRL 5-SOCS 6底物， 调节细胞迁移。最后，我们发现SOCS 2和SOCS 6一样，定位于粘着斑， 调节细胞骨架动力学。 我们提出了三个广泛的目标，以扩大这些观察和理解的分子机制。第一、 我们将理解BCAR 3磷酸化在BCAR 3稳定性和粘着斑动力学中的重要性。 其次，BCAR 3与Cas结合并合作：每种蛋白质都稳定另一种蛋白质免于降解。我们将 了解BCAR 3-Cas相互稳定的机制和重要性。第三，我们将确定 SOCS 2调节粘着斑动力学的机制。总之，这些目标将扩大我们的 了解蛋白质酪氨酸磷酸化和泛素化如何协同调节细胞迁移。