Entry into mitosis in Schizosaccharomyces pombe is negatively regulated by the wee1+ gene, which encodes a protein kinase with serine-, theonine-, and tyrosine-phosphorylating activities. The wee1+ kinase negatively regulates mitosis by phosphorylating p34cdc2 on tyrosine 15, thereby inactivating the p34cdc2-cyclin B complex. The human homolog of the wee1+ gene (WEE1Hu) was overproduced in bacteria and assayed in an in vitro system. Unlike its fission yeast homolog, the product of the WEE1Hu gene encoded a tyrosine-specific protein kinase. The human WEE1 kinase phosphorylated the p34cdc2-cyclin B complex on tyrosine 15 but not on threonine 14 in vitro and inactivated the p34cdc2-cyclin B kinase. This inhibition was reversed by the human Cdc25C protein, which catalyzed the dephosphorylation of p34cdc2. These results indicate that the product of the WEE1Hu gene directly regulates the p34cdc2-cyclin B complex in human cells and that a kinase other than that encoded by WEE1Hu phosphorylates p34cdc2 on threonine 14.

The regulation of p34cdc2 was investigated by overproducing p34cdc2, cyclin (A and B) and the weel + gene product (p107wee1) using a baculoviral expression system. p34cdc2 formed a functional complex with both cyclins as judged by co-precipitation, phosphorylation of cyclin in vitro, and activation of p34cdc2 histone Hi kinase activity. Co-production of p34cdc2 and p107wl in insect cells resulted in a minor population of p34cdc2 that was phosphorylated on tyrosine and displayed an altered electrophoretic mobility. When p34c c2 and p107w`el were co-produced with cyclin (A or B) in insect cells, there was a dramatic increase in the population of p34cd2 that was phosphorylated on tyrosine and that displayed a shift in electrophoretic mobility. The phosphorylation of p34cdc2 on tyrosine was absolutely dependent upon the presence of kinase-active plO7'e. Tyrosine-specific as well as serine/threonine-specific protein kinase activities co-immunoprecipitated with plrO'. These results suggest that cyclin functions to facilitate tyrosine phosphorylation of p34cdc2 and that p107w`l functions to regulate p34C2, either directly or indirectly, by tyrosine phosphorylation.

ABSTRACTp1O7w"l is a protein kinase that functions as a dose-dependent inhibitor of mitosis through its interactions with p34CdC2 in Schizosaccharomyces pombe. To characterize the kinase activity of p1O7"'', its carboxyl-terminal catalytic domain was purified to homogeneity from overproducing insect cells. The apparent molecular mass of the purified protein (p37WCelKD) was determined to be -37 kDa by gel filtration, consistent with it being a monomer. Serine and tyrosine kinase activities cofiltered with p37w"lKD, demonstrating that p1O7w`I is a dual-specificity kinase. In vitro, p1O7'1' phosphorylated p34cIc2 on Tyr-15 only when p34cdc2 was complexed with cyclin. Neither monomeric p34c nor a peptide containing Tyr-15 was able to substitute for the p34>d2/cyclin complex in this assay. Furthermore, the phosphorylation of p342 by p1O7"'1 in vitro inhibited the histone Hi kinase activity of p34cdc2. These results indicate that p107"'' functions as a mitotic inhibitor by directly phosphorylating p34Oc2 on and that the preferred substrate for phosphorylation is the p34cdc2/cyclin complex.The cell cycle regulator p107weel was first identified in the fission yeast Schizosaccharomyces pombe as a dosedependent inhibitor of mitosis (1, 2). Genetic analysis suggests that p107weel functions by inhibiting the activity of p34cdc2, a serine/threonine protein kinase required for entry of cells into mitosis (1, 3). Sequence analysis revealed a significant degree of homology between p107wee1 and the serine/threonine class of protein kinases (4). However, recent evidence has implicated p107wee1 in the phosphorylation of p34cdc2 on Tyr-15 (an inhibitory phosphorylation), despite its lack of homology to known tyrosine kinases. First, coexpression of p34cdc2 with cyclin and plO7weel in insect cells leads to the stoichiometric phosphorylation of p34cdc2 on Tyr-15. In this system, the tyrosine phosphorylation of p34cdc2 is absolutely dependent upon the kinase activity of p107weel, as a kinase-deficient mutant of p107wee1 does not induce the tyrosine phosphorylation of p34cdc2 (5). Second, although deletion of the weel+ gene of S. pombe leads to no detectable change in the phosphorylation state of p34cdc2 (6), deletion of weel+ and a related gene, mikl+, leads to a rapid decrease in tyrosine phosphorylated p34cdc2 and mitotic lethality (3). These genetic findings suggest that weel+ and mikl+ encode partially redundant proteins that cooperate to control the tyrosine phosphorylation of p34cdc2. However, to date, reconstitution of these interactions in vitro has not been reported.Several proteins have recently been identified that possess intrinsic serine/threonine as well as tyrosine kinase activities. These proteins belong to a class of dual-specificity kinases and include such proteins as ERK 1 and ERK 2, STY1/clk, MCK1 (previously named YPK1), and SPK1 (7-15). Recent evidence has indicated that p107wee1 may also belong to this class of kinases. First, immunoprecipitates of p107W'1 from insect cells overproducing p107wee1 or from bu...

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.