Type of Document	Dissertation
Author	Tomko Jr., Robert Joseph
URN	etd-06172008-171118
Title	Cellular and Biochemical Regulation of Cdc25A Phosphatase by Nitrosative Stress
Degree	Doctor of Philosophy
Program	Molecular Pharmacology
School	School of Medicine
Advisory Committee	Advisor Name Title Donald B. DeFranco, Ph.D. Committee Chair Billy W. Day, Ph.D. Committee Member Bruce R. Pitt, Ph.D. Committee Member John S. Lazo, Ph.D. Committee Member Valerian E. Kagan, Ph.D. Committee Member
Keywords	eIF2 Cdc25A nitrosylation phosphatase Nitric oxide apoptosis signal-regulating kinase-1 ASK-1 nitrosation nitrosative stress
Date of Defense	2008-04-24
Availability	restricted
Abstract Numerous reports correlate nitric oxide (•NO) production with stalled S-phase progression, but the molecular mechanism(s) of cell cycle arrest remains elusive. Paradoxically numerous human tumors are exposed to vast quantities of nitric oxide and its reactive byproducts in situ, yet they continue to grow and proliferate. The dual-specificity phosphatase Cdc25A promotes cell cycle progression by dephosphorylating and activating cyclin-dependent kinases. Deregulation of Cdc25A is characteristic of human tumors, accelerates the cell cycle, and confers resistance to apoptosis, highlighting the importance of stringent Cdc25A control. Biochemical and structural analyses of Cdc25A indicate the potential for inhibition by S-nitrosation of the catalytic cysteine, providing a linkage between •NO and cytostatic signaling. Thus, the overall hypothesis examined in this dissertation was that Cdc25A is a target and transducer of signaling by •NO and •NO-derived reactive species. The specific aims were to: 1) probe the susceptibility of Cdc25A to enzymatic regulation by •NO-derived reactive species; 2) examine regulation of Cdc25A protein in nitrosatively challenged cells; and 3) determine whether Cdc25A activity was limiting for S-phase progression in nitrosatively-challenged tumor cells. My studies identified novel mechanisms controlling Cdc25A abundance and activity. S-Nitrosothiols rapidly S-nitrosated and inactivated Cdc25A in vitro, and Cdc25A activity was restored by reductants. Generation of nitrosative stress in cells either by iNOS-derived •NO or the cell-permeable S-nitrosating agent S-nitrosocysteine ethyl ester (SNCEE) caused translational inhibition of Cdc25A via hyperphosphorylation and inhibition of the eukaryotic translational regulator eIF2á. Although iNOS-derived •NO and SNCEE inhibited DNA synthesis coincident with Cdc25A loss, restoration of Cdc25A activity in nitrosatively-challenged cells did not alter DNA synthesis inhibition, distinguishing nitrosative inhibition of DNA synthesis from the canonical intra-S-phase checkpoint. SNCEE decoupled Cdc25A from ASK-1 and sensitized cells to chemotherapeutic-induced apoptosis, suggesting that Cdc25A suppression by nitrosative stress may lower the apoptotic threshold in nitrosatively-challenged cells by priming ASK-1 for activation. In summary, these studies describe novel regulation of Cdc25A translation and activity, and a model wherein selective inhibition of Cdc25A phosphatase-dependent and independent activities can occur under nitrosative stress, and implicate Cdc25A as a regulator of apoptotic threshold following nitrosative insult via priming of ASK-1.
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