Phosphorylation and inactivation of the mitotic inhibitor Wee1 by the nim1/cdr1 kinase.
The G2-M phase transition in eukaryotes is regulated by the synergistic and opposing activities of a cascade of distinct protein kinases and phosphatases. This cascade converges on Cdc2, a serine/threonine protein kinase required for entry into mitosis (reviewed in ref. 1). In the fission yeast Schizosaccharomyces pombe, inactivation of the Cdc2/cyclin 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, thereby activating the Cdc2/cyclin B complex. 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/cdr1 protein (Nim1), we have overproduced it in both bacterial and baculoviral expression systems. We report that Nim1 possesses intrinsic serine-kinase, threonine-kinase and tyrosine-kinase activities. Co-expression of the Nim1 and Wee1 kinases in insect cells results in the phosphorylation of Wee1 and therefore a shift in its electrophoretic mobility on SDS-polyacrylamide gels. When Wee1 is phosphorylated, its ability to phosphorylate Cdc2 on tyrosine 15 is inhibited; treatment with phosphatase restores this kinase activity. Furthermore, purified bacterially produced Nim1 kinase directly phosphorylates and inactivates Wee1 in vitro. These results show that nim1/cdr1 functions as a positive regulator of mitosis by directly phosphorylating and inactivating the mitotic inhibitor Wee1.
Beth Israel Hospital, Boston, Massachusetts.
L L
LL
Parker
S A
SA
Walter
P G
PG
Young
H
H
Piwnica-Worms
D000818:Animals; D001419:Bacteria; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D016203:CDC2 Protein Kinase / Q000378:metabolism; D018797:Cell Cycle Proteins; D002478:Cells, Cultured; D003001:Cloning, Molecular; D016213:Cyclins / Q000378:metabolism; D004247:DNA; D005982:Glutathione Transferase / Q000235:genetics; D007313:Insecta; D008938:Mitosis / Q000502:physiology; D008969:Molecular Sequence Data; D009687:Nuclear Proteins; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D029702:Schizosaccharomyces pombe Proteins; D014443:Tyrosine / Q000378:metabolism
D018797:Cell Cycle Proteins; D016213:Cyclins; D009687:Nuclear Proteins; D047428:Protein Kinase Inhibitors; D011993:Recombinant Fusion Proteins; D029702:Schizosaccharomyces pombe Proteins; D014443:Tyrosine; D004247:DNA; D005982:Glutathione Transferase; D011494:Protein Kinases; C080364:cdr1 protein, S pombe; C068689:wee1 protein, S pombe; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D016203:CDC2 Protein Kinase