Connexin 43 Phosphorylation as a Regulatory Mechanism of Src Activity

Connexin 43 磷酸化作为 Src 活性的调节机制

• 批准号: 10580186
• 负责人: Anastasia Thevenin
• 金额: $ 40.91万
• 依托单位: MORAVIAN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-02-02 至 2026-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10580186
• 关键词: Amino Acids; Animals; Binding; Biological Sciences; C-terminal; Cell Communication; Cell Proliferation; Cell membrane; Cells; Cellular Structures; Chemicals; Co-Immunoprecipitations; Complex; Connexin 43; Connexins; Cytoplasm; Cytoskeleton; Development; Disease; Ectopic Expression; Endocytosis; Event; Family member; Gap Junctions; Goals; In Vitro; Individual; Knowledge; Life; MAPK8 gene; Malignant Neoplasms; Mammalian Cell; Mass Spectrum Analysis; Mediating; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Modification; Molecular; Oncogenic; Oranges; PTEN gene; Pathway interactions; Peptides; Phenotype; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphorylation Site; Phosphotransferases; Physiological; Process; Protein Biochemistry; Protein Kinase; Protein Tyrosine Kinase; Protein phosphatase; Proteins; Proto-Oncogenes; Regulation; Research; Role; SH3 Domains; Sampling; Scaffolding Protein; Series; Signal Pathway; Signal Transduction; Signaling Molecule; Site; Substrate Interaction; Tail; Testing; Therapeutic; Tumor Suppressor Proteins; Tyrosine-Kinase Oncogenes; Ubiquitination; Universities; Western Blotting; Work; cancer cell; design; experience; experimental study; gap junction channel; inhibitor; innovation; insight; intercellular communication; interest; mimetics; mutant; novel; p38 Mitogen Activated Protein Kinase; phosphoproteomics; prevent; protein-tyrosine kinase c-src; recruit; screening; tumor; tumor progression; undergraduate student

PROJECT SUMMARY Connexin 43 (Cx43) is a protein constituent of vertebrate gap junctions (GJs) - cellular structures key to direct cell-cell communication. Cx43-based GJs are known to be regulated through phosphorylation by several kinases on the cytoplasmic C-terminal tail (CT) of Cx43. Recent studies by the PI and others have indicated that Cx43-CT functions as a cell-signaling hub that interacts with and regulates a large number of kinases, phosphatases, scaffolding proteins, and cytoskeletal components. One of the kinases that interacts with and phosphorylates Cx43 is the proto-oncogenic tyrosine kinase, Src. Interestingly, ectopic expression of Cx43 in cancer cells with overactive Src leads to inhibition of Src activity and a decrease in cellular proliferation. While it is known that Src inhibition is due to its binding to Cx43-CT, along with the recruitment of two Src inhibitors (the phosphatase PTEN and C-terminal Src kinase, Csk), the precise molecular mechanisms that regulate these processes are not understood. We hypothesize that Cx43’s ability to recruit and inhibit Src is driven and regulated by a series of specific and sequential phosphorylation events taking place throughout Cx43-CT. Here, we propose three interconnected, but non-overlapping, specific aims, with the ultimate goals of (1) determining the specific sequence of phosphorylation events on Cx43 that regulate Cx43/Src interaction, and (2) developing Cx43-based peptides for use as Src inhibitors in cancer cells. In the first two aims, we will identify and interrogate particular phosphorylation sites as toggle switches that regulate the interactions between Cx43 and binding partners (e.g., Src, PTEN, Csk), with subsequent effects on signaling pathways and cell phenotypes. In the third aim, we will develop Cx43-based peptides and test their ability to modulate the Cx43/Src interaction and Src activity in living cells. To achieve these goals, we will combine expertise in protein biochemistry, kinase-substrate interactions (both in vitro and in cells), phosphoproteomics and mass spectrometry analyses of complex sample mixtures, and specific delivery of agents to cancer cells. These studies will substantially advance our molecular understanding of Src-mediated cell signaling, with a focus on the specific role of Cx43 as an interacting hub. It will also allow us to delineate multiple signaling pathways in both healthy and cancer cells. The Cx43-based peptides that we propose to develop will represent a novel way not only to tease out Cx43/Src regulatory mechanisms in live cells but also to target the aberrant activity of Src in cancers. We expect that the basic framework developed here for Cx43 can be extended to other connexins and to the examination of other signaling pathways. Finally, these studies will continue to provide invaluable research experience and professional development for many undergraduate Life Science majors at Moravian University.

项目摘要 连接蛋白43（Cx43）是脊椎动物间隙连接（GJs）的蛋白质成分， 细胞间通讯已知基于Cx43的GJ通过磷酸化由几种 Cx43的胞质C-末端尾（CT）上的激酶。PI和其他人最近的研究表明， Cx43-CT作为一个细胞信号中枢，与大量激酶相互作用并调节它们， 磷酸酶、支架蛋白和细胞骨架成分。一种激酶， 磷酸化Cx43是原癌酪氨酸激酶Src。有趣的是，Cx43的异位表达， 具有过度活性Src的癌细胞导致Src活性的抑制和细胞增殖的降低。而 已知Src抑制是由于其与Cx43-CT结合，沿着两种Src抑制剂的募集 (the磷酸酶PTEN和C-末端Src激酶，Csk），调节细胞凋亡的精确分子机制， 这些过程不被理解。我们假设Cx43招募和抑制Src的能力是由驱动的， 通过在Cx43-CT中发生的一系列特异性和顺序磷酸化事件来调节。 在这里，我们提出三个相互关联但不重叠的具体目标，最终目标是（1） 确定调节Cx43/Src相互作用的Cx43上磷酸化事件的特定序列，和 (2)开发基于Cx43的肽用作癌细胞中的Src抑制剂。在前两个目标中，我们将 识别和询问特定的磷酸化位点，作为调节相互作用的拨动开关 在Cx43和结合配偶体之间（例如，Src、PTEN、Csk），随后对信号传导途径产生影响， 细胞表型在第三个目标中，我们将开发基于Cx43的肽，并测试它们调节细胞凋亡的能力。 活细胞中Cx43/Src相互作用和Src活性。为了实现这些目标，我们将联合收割机在蛋白质方面的专业知识 生物化学，激酶-底物相互作用（体外和细胞内），磷酸化蛋白质组学和质谱 复杂样品混合物的光谱分析，以及向癌细胞特异性递送药剂。 这些研究将大大推进我们对Src介导的细胞信号传导的分子理解， 重点关注Cx43作为交互枢纽的特定作用。它还能让我们描绘出多种信号 健康细胞和癌细胞中的信号通路。我们建议开发的Cx43肽将代表 这是一种新的方法，不仅可以梳理出活细胞中的Cx43/Src调节机制，而且可以靶向异常的 Src在癌症中的活性。我们希望这里为Cx43开发的基本框架可以扩展到 其他连接蛋白和其他信号通路的检查。最后，这些研究将继续 为许多生命科学本科生提供宝贵的研究经验和专业发展 在摩拉维亚大学主修。