PHOSPHORYLATION AND INACTIVATION OF THE MITOTIC INHIBITOR WEE1 BY THENIM1 CDR1 KINASE/

THE G2-M phase transition in eukaryotes is regulated by the synergisti c and opposing activities of a cascade of distinct protein kinases and phosphatases. This cascade converges on Cdc2, a serine/threonine prot ein kinase required for entry into mitosis (reviewed in ref. 1). In th e fission yeast Schizosacckaromyces pombe, inactivation of the Cdc2/cy clin B complex is achieved by phosphorylation of tyrosine 15 by Wee1 ( refs 2, 3). The action of the Wee1 kinase is opposed by the action of the Cdc25 phosphatase, which dephosphorylates Cdc2 on tyrosine 15, the reby activating the Cdc2/cyclin B complex4-9. Much less is known about the regulatory signals upstream of cdc25 and wee1. Genetics indicate that the mitotic inducer nim1/cdr1 acts upstream of wee1, possibly as a negative regulator of wee1 (refs 10, 11). To characterize the nim1/c dr1 protein (Nim1), we have overproduced it in both bacterial and bacu loviral expression systems. We report that Nim1 possesses intrinsic se rine-kinase, threonine-kinase and tyrosine-kinase activities. Co-expre ssion of the Nim1 and Wee1 kinases in insect cells results in the phos phorylation of Wee1 and therefore a shift in its electrophoretic mobil ity on SDS-polyacrylamide gels. When Weel is phosphorylated, its abili ty to phosphorylate Cdc2 on tyrosine 15 is inhibited; treatment with p hosphatase restores this kinase activity. Furthermore, purified bacter ially produced Nim1 kinase directly phosphorylates and inactivates Wee l in vitro. These results show that nim1/cdr1 functions as a positive regulator of mitosis by directly phosphorylating and inactivating the mitotic inhibitor Wee1.