| The activity of cyclin B/cdc2, or mitosis promoting factor (MPF), which is the key regulator of mitosis in all eucaryotic cells, is highly controlled across the cell cycle. To understand the underlying molecular mechanisms, three aspects of the regulation of cyclin B/cdc2 were investigated by using concentrated cell-free systems that reproduce in vivo processes. First, this work demonstrates that a small domain in cyclin B, the P box, which distantly resembles a region in some protein tyrosine phosphatases (PTPase) but is absent in cdc25, the cdc2 specific PTPase, is essential for cdc2 dephosphorylation, the last step in the cyclin B/cdc2 activation pathway. This result establishes the dependency of cdc2 dephosphorylation/activation on cyclin B. Second, association of cyclin B with cdc2 does not regulate the destruction of cyclin B. Binding to an active cdc2 is not necessary for the M phase destruction of cyclin B, nor does binding to an inactive cdc2 inhibit cyclin B degradation. These results suggest that modulation of the protease system involved may instead be the key regulatory mechanism for M phase cyclin destruction. Finally, this work demonstrates that the inhibitory phosphorylation on cdc2 at thr-14 is due to a distinct kinase activity from the tyr-15 kinase. These results also indicate that thr-14 and tyr-15 phosphorylations cooperatively inhibit cdc2 kinase activity during interphase. |
