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by Megan Fredericks Genzale B.S., 1998, University of Scranton Thesis Advisor: Carlos A. Molina, Ph.D. Assistant Professor Departments of Obstetrics, Gynecology and Women`s Health and Biochemistry and Molecular Biology MSB E-609b Thursday, October 9, 2003 2:00 p.m. Abstract Inducible cAMP early repressor (ICER) protein exhibits several characteristics of a tumor suppressor protein. It was previously determined that ICER protein levels are markedly reduced in prostate epithelial cells and forced expression in various human and rat prostate cancer cell lines rendered the cells unable to grow in soft agar and incapable of forming tumors in nude mice. These observations suggest that deregulation of ICER expression may be related to carcinogenesis of the prostate gland. Tumor suppressor proteins aid in the regulation of the cell cycle through the control of proliferation and survival of normal cells; consequently, their inactivation can initiate or drive cancer progression. Therefore, determining the regulation of the putative tumor suppressor ICER in the mammalian somatic cell cycle provides a comprehensive understanding of ICER and imparts insight as to why this tumor suppressor is necessary to maintain normal cell growth. In this study, a rat prostate cancer cell line termed At6.3 cells, previously engineered to ectopically express ICER-II?, was treated with nocodazole to block cells in mitosis. In this report, we found that ICER was subject to phosphorylation by Cdc2 in mitosis. An interaction was observed between Cdc2 and ICER protein, this interaction was enhanced in mitotic cells. Mutation of ICER at serines 35, 41 and 104 exhibited reduced phosphorylation in mitotic HeLa cells, indicating putative phosphorylation sites. Concomitant with this, the phosphorylation of serine residues 35 and 41 were reduced in kinase assays after immunoprecipitation with cyclin B. Evidence has shown that phosphorylation in mitosis is sensitive to chemical inhibitors of Cdc2, but not to inhibitors of MEK 1/ 2 or PI-3 kinase. We report for the first time that in AtICER9 cells, ICER undergoes subcellular localization to the cytosol where it is ubiquitinated. ICER is classically nuclear, however, subcellular localization to the cytosol seems to be contingent upon Cdc2 phosphorylation of ICER in the nucleus. These results provide support that the ubiquitinated, cytosolic ICER may not targeted to proteasomal degradation. The data proposes that the cytosolic localization of ICER is transient and the protein translocates back to the nucleus as cells enter G1. It is evident that the At6.3 parental cell line exhibits mislocalization of ICER to the cytosol. The mistargeting of ICER to the cytosol presumably causes loss of function for the ICER protein, potentially enhancing the progression of cancer in these cells. Our laboratory has recently shown that ICER is phosphorylated by MAPK. As a result, phosphorylation targets ICER to ubiquitin mediated proteasomal degradation. In this report, we found that ERK activity and the proteasome regulate the expression of ICER protein during the somatic cell cycle. ICER was found to be expressed in early G1, and down-regulated in mitosis and late G1. The expression of ICER mRNA does not fluctuate during the cell cycle, suggesting that ICER regulation is post-translational. Indeed, pharmacological inhibition of the MAPK or the proteasome impeded ICER protein down-regulation during the cell cycle. Cell cycle analysis showed that when MAPK was inhibited, 20-30% more ICER expressing cell clones were halted in G1 compared to the parental cells. These results provide novel evidence that the MAPK pathway targets ICER for proteasomal degradation during the somatic mammalian cell cycle and that the decline of ICER is important for the G1/S transition. |
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