Posttranslational regulation of Cox-2 activity

描述（由申请人提供）：环加氧酶（Cox-2）的诱导异构体在许多癌症中上调。选择性Cox-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Cox-2不仅在表达水平上受到调控，而且在催化水平上受到调控，但对Cox-2翻译后调控的研究还不够充分。我们已经证明酪氨酸激酶Fyn可以与Cox-2交联，这表明这两种蛋白在哺乳动物细胞中彼此非常接近。我们的初步数据表明，fyn介导的Cox-2翻译后修饰导致前列腺癌细胞DU145中酶活性增加。通过体外激酶测定和磷酸化酪氨酸抗体的western blot分析，我们已经确定Cox-2可被重组Src家族激酶直接磷酸化。我们利用多级碎片质谱（MS）分析鉴定了Cox-2磷酸化的确切位点。众所周知，细胞通过诱导Cox-2表达来调节前列腺素合成，而酪氨酸磷酸化对Cox-2活性的调节是调节前列腺素生物合成途径的新机制。目前的提案将验证Fyn磷酸化前列腺癌细胞中的Cox-2以调节其酶活性并促进前列腺癌进展的假设。本实验将确定酪氨酸磷酸化是否为Cox-2调控的新机制，并将产生独特的分子工具来评估肿瘤细胞体内Cox-2磷酸化状态。在具体目标1中，我们将分析Cox-2催化域中特定酪氨酸的磷酸化是否对Cox-2酶活性有影响。我们已经生成了可能磷酸化位点发生突变的Cox-2构建体。我们将比较表达这些突变体的前列腺癌细胞与表达野生型Cox-2的细胞中前列腺素的产生。在具体的目的2中，我们将在体内验证Cox-2酪氨酸磷酸化位点，并研究Cox-2酪氨酸磷酸化是否影响前列腺癌细胞的生长和对凋亡的抵抗。我们将生成针对Cox-2酪氨酸磷酸化位点的磷酸化特异性抗体，并分析前列腺癌细胞中Cox-2的磷酸化状态。提出的目标的成功完成将与Cox-2和前列腺素有牵连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Cox-2抑制剂来调节前列腺癌和其他类型癌症的Cox-2活性。

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.