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 kinase activity of human p34cdc2 is negatively regulated by phosphorylation at Thr-14 and Tyr-15. These residues lie within the putative nucleotide binding domain of p34cdc2. It has been proposed that phosphorylation within this motif ablates the binding of ATP to the active site of p34Cdc2, thereby inhibiting p34cdc2 kinase activity (K. Gould and P. Nurse, Nature [London] 342:39-44, 1989). To understand the mechanism of this inactivation, various forms of p34cdc2 were tested for the ability to bind nucleotide. The active site of p34cdc2 was specifically modified by the MgATP analog 5'-p-fluorosulfonylbenzoyladenosine (FSBA). The apparent Km for modification of wild-type, monomeric p34cd' was 148 ,M FSBA and was not significantly affected by association with cyclin B. Tyrosine-phosphorylated p34cdc2 was modified by FSBA with a slightly higher Km (241 ,uM FSBA). FSBA modification of both tyrosine-phosphorylated and unphosphorylated p34Cdc2 was competitively inhibited by ATP, and half-maximal inhibition in each case occurred at approximately 250 pM ATP. In addition to being negatively regulated by phosphorylation, the kinase activity of p34cdc2 was positively regulated by the cyclin-dependent phosphorylation of Thr-161. Mutation of p34cdc2 at Thr-161 resulted in the formation of an enzymatically inactive p34cdc2/cyclin B complex both in vivo and in vitro. However, mutation of Thr-161 did not significantly affect the ability of p34cdc2 to bind nucleotide (FSBA). Taken together, these results indicate that inhibition of p34cdc2 kinase activity by phosphorylation of Tyr-15 (within the putative ATP binding domain) or by mutation of Thr-161 involves a mechanism other than inhibition of nucleotide binding. We propose instead that the defect resides at the level of catalysis.Entry of eukaryotic cells into mitosis is regulated by a 34-kDa serine/threonine protein kinase designated p34cdc2 whose activity, in turn, is tightly regulated throughout the cell cycle (reviewed in references 31, 34, and 39). The pathway leading to the full activation of p34cdc2 is mechanistically quite complex. One initial step involves the stable association of p34c c2 with cyclin B (8,12,23,27,30,32). While association with cyclin B is necessary for the activation of p34cdc2, it is not sufficient (7,38,45). Multiple posttranslational modifications are required for full activation of the p34Cdc2/cyclin B complex. These modifications include a series of phosphorylations and dephosphorylations that are mediated, in part, by the weel and cdc25 gene products.In the fission yeast Schizosaccharomyces pombe, p34cdc2 is phosphorylated on Tyr-15 (an inhibitory modification) (14). Dephosphorylation of p34cdc2 on Tyr-15 is a key step in the activation of p34cdc2 and entry of cells into mitosis (14, 16). The weel gene, a negative regulator of the cell cycle, encodes a dual-specificity kinase (p107 weel) that phosphorylates p34cdc2 on 35,36,42). The cdc25 gene, a positive regulator of the cell cycle, encodes a protein phosphatase that dephosphorylates ...

In cells of higher eukaryotic organisms the activity of the p34cdc2/cyclin B complex is inhibited by phosphorylation of p34cdc2 at two sites within its amino-terminus (threonine 14 and tyrosine 15). In this study, the cell cycle regulation of the kinases responsible for phosphorylating p34cdc2 on Thr14 and Tyr15 was examined in extracts prepared from both HeLa cells and Xenopus eggs. Both Thr14- and Tyr15- specific kinase activities were regulated in a cell cycle-dependent manner. The kinase activities were high throughout interphase and diminished coincident with entry of cells into mitosis. In HeLa cells delayed in G2 by the DNA-binding dye Hoechst 33342, Thr14- and Tyr15-specific kinase activities remained high, suggesting that a decrease in Thr14- and Tyr15- kinase activities may be required for entry of cells into mitosis. Similar cell cycle regulation was observed for the Thr14/Tyr15 kinase(s) in Xenopus egg extracts. These results indicate that activation of CDC2 and entry of cells into mitosis is not triggered solely by activation of the Cdc25 phosphatase but by the balance between Thr14/Tyr15 kinase and phosphatase activities. Finally, we have detected two activities capable of phosphorylating p34cdc2 on Thr14 and/or Tyr15 in interphase extracts prepared from Xenopus eggs. An activity capable of phosphorylating Tyr15 remained soluble after ultracentrifugation of interphase extracts whereas a second activity capable of phosphorylating both Thr14 and Tyr15 pelleted. The pelleted fraction contained activities that were detergent extractable and that phosphorylated p34cdc2 on both Thr14 and Tyr15. The Thr14- and Tyr15-specific kinase activities co-purified through three successive chromatographic steps indicating the presence of a dual-specificity protein kinase capable of acting on p34cdc2.

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