The C-terminal domain of the Cdc2 inhibitory kinase Myt1 interacts with Cdc2 complexes and is required for inhibition of G(2)/M progression

Activation of Cdc2, is the universal event controlling the onset of mitosis , In higher eukaryotes, Cdc2 activity is in part regulated by inhibitory ph osphorylation of Thr14 and Tyr15, catalyzed by Wee1 and Myt1, which prevent s catastrophic premature entry into mitosis, In this study we defined the f unction of Myt1 by overexpression studies in both S, pombe and a human oste osarcoma cell line. Similar to Wee1, overexpression of human Myt1 prevented entry into mitosis in both cell types; however, Myt1 catalytic activity wa s not essential for the cell cycle delay observed with human cells. Myt1 ex pression was restricted to proliferating cells, Furthermore, we detected no major decline in Myt1 protein abundance prior to the entry into mitosis, w hich coincides with the loss of Myt1 activity. We localized mitotic phospho epitopes, recognized by the monoclonal antibody MPM-2, to the C-terminal do main of Myt1, The mitotic peptidyl-prolyl isomerase, Pin1, was able to asso ciate with this domain in a phosphorylation-dependent manner. Truncation of the C-terminal domain of Myt1 prevented its ability to induce G(2)/M phase arrest in overexpression studies in human cells and dramatically reduced i ts ability to phosphorylate Cdc2 in vitro. We demonstrate that the C-termin al domain of Myt1 was required for recruitment of Cdc2, and,ve infer that t his domain lies in the cytoplasm because it can interact with and is phosph orylated by Cdc2, In conclusion, we propose that Myt1 can negatively regula te Cdc2/cyclin B1 and inhibit G(2)/M progression by two means, both of whic h require the C-terminal domain; first, Myt1 can bind and sequester Cdc2/cy clin B1 in the cytoplasm preventing entry into the nucleus, and, second, it can phosphorylate associated Cdc2/cyclin B1 at Thr14 and Tyr15 thus inhibi ting its catalytic activity.