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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：
10631700
负责人：
Jonathan A Cooper
金额：
$ 21.52万
依托单位：
FRED HUTCHINSON CANCER 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 2023-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10631700
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 (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) 蛋白可能是通过去磷酸化或 pY 特异性泛素化后的蛋白酶体降解而失活泛素 E3 连接酶。我们重点关注多亚基 Cullin 5-RING 连接酶 (CRL5)，它与 pY 相互作用蛋白质通过细胞因子信号传导抑制子 (SOCS) 接头蛋白。我们过去的研究表明各种 CRL5-SOCS 复合物在正常上皮细胞中对于抑制致癌 PTK、Src 至关重要。什么时候我们抑制 Cullin 5 或特定 SOCS 基因的表达，Src 被激活，细胞迁移、增殖和转变受到刺激。增加 Src 活动是必要的，但对于迁移和迁移来说还不够 Cullin 5 缺陷细胞的增殖，表明 CRL5 具有额外的底物。我们过去的研究有确定了两种对细胞迁移至关重要的底物。在这里，我们建议扩展这些研究，以了解 CRL5-SOCS 如何调节上皮细胞迁移。我们的科学前提是鉴定迁移中关键 CRL5-SOCS 底物和功能的研究对于理解正常和转化的至关重要细胞生物学。在上一个资助期间，我们使用创新的实时成像、光遗传学和突变分析来揭示CRL5-SOCS6调节信号支架Cas（Crk- 相关基质），位于迁移上皮细胞前缘的粘着斑位点内。我们还用过定量蛋白质组学来识别其他 CRL5 底物并测试它们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性 3）作为第二种 CRL5-SOCS6 底物，调节细胞迁移。最后，我们发现 SOCS2 与 SOCS6 一样，定位于粘着斑位点并且调节细胞骨架动力学。我们提出了三个广泛的目标来扩展这些观察并理解分子机制。第一的，我们将了解 BCAR3 磷酸化在 BCAR3 稳定性和粘着斑动力学中的重要性。其次，BCAR3 与 Cas 结合并协同：每种蛋白质都可以稳定另一种蛋白质的降解。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 调节粘着斑动力学的机制。这些目标共同将扩展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。