This is the first competitive renewal of a grant in which the long-term goal is to characterize the biochemical events that underlie the regulation of Mitosis-Promoting Factor (MPF), i.e., the cdc2-cyclin B complex. During interphase, inhibitory kinases Wee1 and Myt-1 suppress the activity of cdc2 by phosphorylating it on Thr14 and Tyr15. When conditions become appropriate for mitosis, the dual specificity phosphatase cdc25 dephosphorylates both Thr14 and Tyr15, thereby activating cdc2 and allowing it to phosphorylate mitotic substrates. In this proposal Dr. Dunphy focusses on the mechanisms underlying the regulation of the activity of this critical phosphatase, cdc25. He has shown that cdc25 activity is low in interphase but is stimulated at the G2/M phase transition, the time at which it catalyzes the dephosphorylation and activation of the cdc2-cyclinB complex. This activation of cdc25 appears to result from phosphorylation catalyzed by at least two kinases, one of which is cdc2-cyclinB, the other a novel enzyme termed Plx-1 that Dr. Dunphy purified and cloned during the initial funding period. In this application he proposes to carry out a comprehensive analysis of the structure and function of Plx-1, using Xenopus oocytes as the model system. He will (I) examine the role of Plx-1 in mitotic control, (ii) characterize the regulation of Plx-1, (iii) identify specific sites in cdc25 that are phosphorylated by Plx-1 and determine the effects of phosphorylation on the function of cdc25, (iv) search for other substrates of Plx-1 and finally (v) broaden the scope of the analysis to examine other mechanisms by which the activity of cdc25 may be regulated, for example through interaction with binding proteins such as 14-3-3.
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