Posttranslational regulation of Cox-2 activity

Cox-2 活性的翻译后调节

• 批准号: 8765932
• 负责人: ANDREY SOROKIN
• 金额: $ 7.65万
• 依托单位: MEDICAL COLLEGE OF WISCONSIN
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-07-01 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8765932
• 关键词: 2-tyrosine; Adverse effects; Amino Acids; Antibodies; Apoptosis; Attention; Benefits and Risks; Biological Assay; Cancer Cell Growth; Cardiovascular system; Catalysis; Catalytic Domain; Cells; Charge; Chemopreventive Agent; Clinical; DU145; Data; Detection; Diagnostic Procedure; Enzymes; Equilibrium; Family; Generations; Glutamic Acid; In Vitro; Individual; Intervention; Link; Malignant Neoplasms; Malignant neoplasm of prostate; Mammalian Cell; Mass Spectrum Analysis; Mediating; Molecular; Mutate; Pathway interactions; Phenylalanine; Phospho-Specific Antibodies; Phosphorylation; Phosphorylation Site; Phosphotransferases; Phosphotyrosine; Post-Translational Protein Processing; Post-Translational Regulation; Probability; Prostaglandin Production; Prostaglandin-Endoperoxide Synthase; Prostaglandins; Protein Isoforms; Proteins; Proto-Oncogene Proteins c-fyn; Recombinant Proteins; Recombinants; Regulation; Resistance; Side; Signal Transduction; Site; Specificity; Staging; Testing; Therapeutic Agents; Tissue Sample; Tissues; Tyrosine; Tyrosine Phosphorylation; Tyrosine Phosphorylation Site; Western Blotting; base; cancer cell; cancer prevention; cancer therapy; cancer type; cell growth regulation; crosslink; dosage; enzyme activity; in vivo; inhibitor/antagonist; member; mutant; novel; novel diagnostics; prostate cancer cell; protein protein interaction; public health relevance; research study; src-Family Kinases; tool; tumor progression

DESCRIPTION (provided by applicant): An inducible isoform of the cyclooxygenase (Cox-2) is upregulated in many cancers. Selective Cox-2 inhibitors have been developed in anticancer therapy but severe side effects limit their clinical implementation. While experimental data suggest that Cox-2 is regulated not only on the level of expression, but also on the level of catalysis, the posttranslational regulation of Cox-2 was not sufficiently investigated. We have shown that the tyrosine kinase Fyn can be cross-linked to Cox-2 indicating that these two proteins are in close proximity to each other in mammalian cells. Our preliminary data suggest that Fyn-mediated posttranslational modification of Cox-2 results in an increased activity of the enzyme in prostate cancer cells DU145. Using an in vitro kinase assay and western blot analysis with phosphotyrosine antibodies we have established that Cox-2 is subject to direct phosphorylation by recombinant Src family kinases. We utilized multi-stage fragmentation mass spectrometry (MS) analysis to identify the exact Cox-2 phosphosporylation site by Fyn. Whereas cellular regulation of prostaglandin synthesis via induction of Cox-2 expression is well known, the regulation of Cox-2 activity by tyrosine phosphorylation is a new mechanism of modulation of the prostaglandin biosynthetic pathway. The current proposal will test the hypothesis that Fyn phosphorylates Cox-2 in prostate cancer cells to regulate its enzymatic activity and promote the progression of prostate cancer. Proposed experiments will establish whether tyrosine phosphorylation is a new mechanism of Cox-2 regulation and will generate unique molecular tools to evaluate Cox-2 phosphorylation status in vivo in cancer cells. In the specific aim 1 we will analyze whether phosphorylation of particular tyrosines in the catalytic domain of Cox-2 has an effect upon Cox-2 enzymatic activity. We have already generated Cox-2 constructs with probable phosphorylation sites mutated. We will compare prostaglandin production in prostate cancer cells expressing these mutants with prostaglandin production in cells expressing wild type Cox-2. In specific aim 2 we will verify Cox-2 tyrosine phosphorylation sites in vivo and investigate whether Cox-2 tyrosine phosphorylation has an effect upon prostate cancer cell growth and resistance to apoptosis. We will generate phosphospecific antibodies against Cox-2 tyrosine phosphorylation sites and carry out analysis of Cox-2 phosphorylation status in prostate cancer cells. The successful completion of proposed aims will be relevant for all types of cancer in which Cox-2 and prostaglandins have been implicated. If successful, these studies would provide a base for novel diagnostic techniques and will suggest that inhibitors of Src kinases could be an alternative to the currently used Cox-2 inhibitors to modulate Cox-2 activity in prostate cancer and other types of cancers.

描述（由申请人提供）：环氧合酶（考克斯-2）的诱导型同种型在许多癌症中上调。选择性考克斯-2抑制剂已被开发用于抗癌治疗，但严重的副作用限制了其临床应用。虽然实验数据表明考克斯-2不仅在表达水平上受到调节，而且在催化水平上也受到调节，但是对考克斯-2的翻译后调节没有进行充分的研究。我们已经表明，酪氨酸激酶Fyn可以交联的考克斯-2表明，这两个蛋白质是在哺乳动物细胞中彼此接近。我们的初步数据表明，Fyn介导的考克斯-2的翻译后修饰导致前列腺癌细胞DU 145中酶活性增加。使用体外激酶测定和蛋白质印迹分析与磷酸酪氨酸抗体，我们已经建立了考克斯-2是直接磷酸化重组Src家族激酶。我们利用多级裂解质谱（MS）分析，以确定确切的考克斯-2磷酸化位点的Fyn。尽管通过诱导考克斯-2表达来调节前列腺素合成是众所周知的，但是通过酪氨酸磷酸化来调节考克斯-2活性是调节前列腺素生物合成途径的新机制。目前的提案将检验Fyn磷酸化前列腺癌细胞中的考克斯-2以调节其酶活性并促进前列腺癌进展的假设。提出的实验将确定酪氨酸磷酸化是否是考克斯-2调节的新机制，并将产生独特的分子工具来评估体内癌细胞中的考克斯-2磷酸化状态。在具体目标1中，我们将分析考克斯-2催化结构域中特定酪氨酸的磷酸化是否对考克斯-2酶活性有影响。我们已经产生了具有可能的磷酸化位点突变的考克斯-2构建体。我们将比较前列腺癌细胞中表达这些突变体的前列腺素生产与表达野生型考克斯-2的细胞中的前列腺素生产。在具体目标2中，我们将在体内验证考克斯-2酪氨酸磷酸化位点，并研究考克斯-2酪氨酸磷酸化是否对前列腺癌细胞生长和抗凋亡有影响。我们将产生针对考克斯-2酪氨酸磷酸化位点的磷酸特异性抗体，并在前列腺癌细胞中进行考克斯-2磷酸化状态的分析。成功完成所提出的目标将与涉及考克斯-2和前列腺素的所有类型的癌症相关。如果成功，这些研究将为新的诊断技术提供基础，并将表明Src激酶抑制剂可以替代目前使用的考克斯-2抑制剂来调节前列腺癌和其他类型癌症中的考克斯-2活性。