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.
Cyclooxygenases are key enzymes in the production of prostaglandins (PG). An inducible isoform of cyclooxygenase (Cox-2) is upregulated in many cancers and targeting the PG synthesis is potentially a critical intervention for the treatment and prevention of cancer. We propose a novel mechanism of modulation of the PG biosynthetic pathway: the regulation of Cox-2 activity by tyrosine phosphorylation by the cytoplasmic tyrosine kinase Fyn, a member of the Src family of kinases. This novel mechanism of cellular regulation of Cox-2 activity should take place predominately in cancer cells and particularly in prostate cancer cells which are characterized by enhanced expression of Fyn in contrast to other members of the Src family. These studies are important because they will uncover signaling mechanisms causative to cancer progression, provide new diagnostic tools and may offer novel targets for inhibition of Cox-2 and treatment of prostate cancer.
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