Mutations of p34cdc2 phosphorylation sites induce premature mitotic events in HeLa cells: evidence for a double block to p34cdc2 kinase activation in vertebrates.

In vertebrates, entry into mitosis is accompanied by dephosphorylation of p34cdc2 kinase on threonine 14 (Thr14) and tyrosine 15 (Tyr15). To examine the role of these residues in controlling p34cdc2 kinase activation, and hence the onset of mitosis, we replaced Thr14 and/or Tyr15 by non-phosphorylatable residues and transfected wild-type and mutant chicken p34cdc2 cDNAs into HeLa cells. While expression of wild-type p34cdc2 did not interfere with normal cell cycle progression, p34cdc2 carrying mutations at both Thr14 and Tyr15 displayed increased histone H1 kinase activity and rapidly induced premature mitotic events, including chromosome condensation and lamina disassembly. No phenotype was observed in response to mutation of only Thr14, and although single-site mutation at Tyr15 did induce premature mitotic events, effects were partial and their onset was delayed. These results identify both Thr14 and Tyr15 as sites of negative regulation of vertebrate p34cdc2 kinase, and they suggest that dephosphorylation of p34cdc2 represents the rate-limiting step controlling entry of vertebrate cells into mitosis.     

Human Wee1 kinase inhibits cell division by phosphorylating p34cdc2 exclusively on Tyr15.

In fission yeast, the M-phase inducing kinase, a complex of p34cdc2 and cyclin B, is maintained in an inhibited state during interphase due to the phosphorylation of Cdc2 at Tyr15. This phosphorylation is believed to be carried out primarily by the Wee1 kinase. In human cells the negative regulation of p34cdc2/cyclin B is more complex, in that Cdc2 is phosphorylated at two inhibitory sites, Thr14 and Tyr15. The identities of the kinases that phosphorylate these sites are unknown. Since fission yeast Wee1 kinase behaves as a dual-specificity kinase in vitro, a popular hypothesis is that a human Wee1 homolog might phosphorylate p34cdc2 at both sites. We report here that a human gene, identified as a possible Wee1 homologue, blocks cell division when overexpressed in HeLa cells. This demonstrates functional conservation of the Wee1 mitotic inhibitor. Contrary to the dual-specificity kinase hypothesis, purified human Wee1 phosphorylates p34cdc2 exclusively on Tyr15 in vitro; no Thr14 phosphorylation was detected. Human and fission yeast Wee1 also specifically phosphorylate synthetic peptides at sites equivalent to Tyr15. Mutation of a critical lysine codon (Lys114) believed to be essential for kinase activity abolished both the in vivo mitotic inhibitor function and in vitro kinase activities of human Wee1. These results conclusively prove that Wee1 kinases inhibit mitosis by directly phosphorylating p34cdc2 on Tyr15, and strongly indicate that human cells have independent kinase pathways directing the two inhibitor phosphorylations of p34cdc2.     

Membrane localization of the kinase which phosphorylates p34cdc2 on threonine 14.

The key regulator of entry into mitosis is the serine/threonine kinase p34cdc2. This kinase is regulated both by association with cyclins and by phosphorylation at several sites. Phosphorylation at Tyr 15 and Thr 14 are believed to inhibit the kinase activity of cdc2. In Schizosaccharomyces pombe, the wee1 (and possibly mik1) protein kinase catalyzes phosphorylation of Tyr 15. It is not clear whether these or other, as yet unidentified, protein kinases phosphorylate Thr 14. In this report we show, using extracts of Xenopus eggs, that the Thr 14-directed kinase is tightly membrane associated. Specifically, we have shown that a purified membrane fraction, in the absence of cytoplasm, can promote phosphorylation of cdc2 on both Thr 14 and Tyr 15. In contrast, the cytoplasm can phosphorylate cdc2 only on Tyr 15, suggesting the existence of at least two distinctly localized subpopulations of cdc2 Tyr 15-directed kinases. The membrane-associated Tyr 15 and Thr 14 kinase activities behaved similarly during salt or detergent extraction and were similarly regulated during the cell cycle and by the checkpoint machinery that delays mitosis while DNA is being replicated. This suggests the possibility that a dual-specificity membrane-associated protein kinase may catalyze phosphorylation of both Tyr 15 and Thr 14.     

Regulatory phosphorylation of the p34cdc2 protein kinase in vertebrates.

The p34cdc2 protein kinase is a conserved regulator of the eukaryotic cell cycle. Here we show that residues Thr14 and Tyr15 of mouse p34cdc2 become phosphorylated as mouse fibroblasts proceed through the cell cycle. We have mutated these residues and measured protein kinase activity of the p34cdc2 variants in a Xenopus egg extract. Phosphorylation of residues 14 and 15, which lie within the presumptive ATP-binding region of p34cdc2, normally restrains the protein kinase until it is specifically dephosphorylated and activated at the G2/M transition. Regulation by dephosphorylation of Tyr15 is conserved from fission yeast to mammals, while an extra level of regulation of mammalian p34cdc2 involves Thr14 dephosphorylation. In the absence of phosphorylation on these two residues, the kinase still requires cyclin B protein for its activation. Inhibition of DNA synthesis inhibits activation of wild-type p34cdc2 in the Xenopus system, but a mutant which cannot be phosphorylated at residues 14 and 15 escapes this inhibition, suggesting that these phosphorylation events form part of the pathway linking completion of DNA replication to initiation of mitosis.     

Elimination of cdc2 phosphorylation sites in the cdc25 phosphatase blocks initiation of M-phase.

The cdc25 phosphatase is a mitotic inducer that activates p34cdc2 at the G2/M transition by dephosphorylation of Tyr15 in p34cdc2. cdc25 itself is also regulated through periodic changes in its phosphorylation state. To elucidate the mechanism for induction of mitosis, phosphorylation of cdc25 has been investigated using recombinant proteins. cdc25 is phosphorylated by both cyclin A/p34cdc2 and cyclin B/p34cdc2 at similar sets of multiple sites in vitro. This phosphorylation retards its electrophoretical mobility and activates its ability to increase cyclin B/p34cdc2 kinase activity three- to fourfold in vitro, as found for endogenous Xenopus cdc25 in M-phase extracts. The threonine and serine residues followed by proline that are conserved between Xenopus and human cdc25 have been mutated. Both the triple mutation of Thr48, Thr67, and Thr138 and the quintuple mutation of these three threonine residues plus Ser205 and Ser285, almost completely abolish the shift in electrophoretic mobility of cdc25 after incubation with M-phase extracts or phosphorylation by p34cdc2. These mutations inhibit the activation of cdc25 by phosphorylation with p34cdc2 by 70 and 90%, respectively. At physiological concentrations these mutants cannot activate cyclin B/p34cdc2 in cdc25-immunodepleted oocyte extracts, suggesting that a positive feed-back loop between cdc2 and cdc25 is necessary for the full activation of cyclin B/p34cdc2 that induces abrupt entry into mitosis in vivo.     

Mitosis-specific phosphorylation of p60c-src by p34cdc2-associated protein kinase

As cells enter mitosis, the protein-tyrosine kinase, p60c-src, is known to be extensively phosphorylated on threonine in its amino-terminal region. In the present work, extracts of mitotic cells were searched for the protein kinase responsible for this phosphorylation. HeLa cells and Xenopus eggs were found to contain a mitosis-specific protein kinase activity capable of phosphorylating highly purified p60c-src in vitro on threonine residues. Tryptic phosphopeptide maps indicate that the mitotic HeLa kinase phosphorylates the same sites in vitro as those used during mitosis in vivo. In addition, this mitotic HeLa kinase comigrates on gel filtration with p34cdc2-associated histone H1 kinase, a well known regulator of mitotic events. Finally, antibodies to the C-terminal peptide of human p34cdc2 specifically deplete p60c-src-phosphorylating activity from mitotic extracts. These results suggest that p60c-src may act as an effector of p34cdc2 in certain mitotic processes.     

Regulation of the cdc25 protein during the cell cycle in Xenopus extracts.

The cdc25 protein is a highly specific tyrosine phosphatase that triggers mitosis by dephosphorylating the cdc2 protein kinase. Using Xenopus extracts, we have found that the cdc25 protein is active at a low level throughout interphase. Near the onset of mitosis, the cdc25 protein undergoes a marked elevation in phosphatase activity that coincides with an extensive phosphorylation of the protein in its N-terminal region. In vitro dephosphorylation of this hyperphosphorylated form of cdc25 reduces its phosphatase activity back to the interphase level. Moreover, treatment of interphase Xenopus extracts with okadaic acid, a phosphatase inhibitor that accelerates the entry into mitosis, elicits both the premature hyperphosphorylation of cdc25 and the stimulation of its cdc2-specific tyrosine phosphatase activity. These experiments demonstrate the existence of a cdc25 regulatory system consisting of both a stimulatory kinase that phosphorylates a putative regulatory domain of the cdc25 protein and an inhibitory serine/threonine phosphatase that counteracts this kinase activity.     

Phosphorylation at Thr167 is required for Schizosaccharomyces pombe p34cdc2 function.

Eukaryotic cell cycle progression requires the periodic activation and inactivation of a protein-serine/threonine kinase which in fission yeast is encoded by the cdc2+ gene. The activity of this gene product, p34cdc2, is controlled by numerous interactions with other proteins and by its phosphorylation state. In fission yeast, p34cdc2 is phosphorylated on two sites, one of which has been identified as Tyr15. Dephosphorylation of Tyr15 regulates the initiation of mitosis. To understand more completely the regulation of p34cdc2 kinase activity, we have identified the second site of phosphorylation as Thr167, a residue conserved amongst all p34cdc2 homologues. By analysing the phenotypes of cells expressing various position 167 mutations and performing in vitro experiments, we establish that Thr167 phosphorylation is required for p34cdc2 kinase activity at mitosis and is involved in the association of p34cdc2 with cyclin B. Dephosphorylation of Thr167 might also play a role in the exit from mitosis.     

The G2/M DNA damage checkpoint inhibits mitosis through Tyr15 phosphorylation of p34cdc2 in Aspergillus nidulans.

It is possible to cause G2 arrest in Aspergillus nidulans by inactivating either p34cdc2 or NIMA. We therefore investigated the negative control of these two mitosis-promoting kinases after DNA damage. DNA damage caused rapid Tyr15 phosphorylation of p34cdc2 and transient cell cycle arrest but had little effect on the activity of NIMA. Dividing cells deficient in Tyr15 phosphorylation of p34cdc2 were sensitive to both MMS and UV irradiation and entered lethal premature mitosis with damaged DNA. However, non-dividing quiescent conidiospores of the Tyr15 mutant strain were not sensitive to DNA damage. The UV and MMS sensitivity of cells unable to tyrosine phosphorylate p34cdc2 is therefore caused by defects in DNA damage checkpoint regulation over mitosis. Both the nimA5 and nimT23 temperature-sensitive mutations cause an arrest in G2 at 42 degrees C. Addition of MMS to nimT23 G2-arrested cells caused a marked delay in their entry into mitosis upon downshift to 32 degrees C and this delay was correlated with a long delay in the dephosphorylation and activation of p34cdc2. Addition of MMS to nimA5 G2-arrested cells caused inactivation of the H1 kinase activity of p34cdc2 due to an increase in its Tyr15 phosphorylation level and delayed entry into mitosis upon return to 32 degrees C. However, if Tyr15 phosphorylation of p34cdc2 was prevented then its H1 kinase activity was not inactivated upon MMS addition to nimA5 G2-arrested cells and they rapidly progressed into a lethal mitosis upon release to 32 degrees C. Thus, Tyr15 phosphorylation of p34cdc2 in G2 arrests initiation of mitosis after DNA damage in A. nidulans.     

Full activation of p34CDC28 histone H1 kinase activity is unable to promote entry into mitosis in checkpoint-arrested cells of the yeast Saccharomyces cerevisiae.

In most cells, mitosis is dependent upon completion of DNA replication. The feedback mechanisms that prevent entry into mitosis by cells with damaged or incompletely replicated DNA have been termed checkpoint controls. Studies with the fission yeast Schizosaccharomyces pombe and Xenopus egg extracts have shown that checkpoint controls prevent activation of the master regulatory protein kinase, p34cdc2, that normally triggers entry into mitosis. This is achieved through inhibitory phosphorylation of the Tyr-15 residue of p34cdc2. However, studies with the budding yeast Saccharomyces cerevisiae have shown that phosphorylation of this residue is not essential for checkpoint controls to prevent mitosis. We have investigated the basis for checkpoint controls in this organism and show that these controls can prevent entry into mitosis even in cells which have fully activated the cyclin B (Clb)-associated forms of the budding yeast homolog of p34cdc2, p34CDC28, as assayed by histone H1 kinase activity. However, the active complexes in checkpoint-arrested cells are smaller than those in cycling cells, suggesting that assembly of mitosis-inducing complexes requires additional steps following histone H1 kinase activation.     

Differential phosphorylation of vertebrate p34cdc2 kinase at the G1/S and G2/M transitions of the cell cycle: identification of major phosphorylation sites.

The cdc2 kinase is a key regulator of the eukaryotic cell cycle. The activity of its catalytic subunit, p34cdc2, is controlled by cell cycle dependent interactions with other proteins as well as by phosphorylation--dephosphorylation reactions. In this paper, we examine the phosphorylation state of chicken p34cdc2 at various stages of the cell cycle. By peptide mapping, we detect four major phosphopeptides in chicken p34cdc2; three phosphorylation sites are identified as threonine (Thr) 14, tyrosine (Tyr) 15 and serine (Ser) 277. Analysis of synchronized cells demonstrates that phosphorylation of all four sites is cell cycle regulated. Thr 14 and Tyr 15 are phosphorylated maximally during G2 phase but dephosphorylated abruptly at the G2/M transition, concomitant with activation of p34cdc2 kinase. This result suggests that phosphorylation of Thr 14 and/or Tyr 15 inhibits p34cdc2 kinase activity, in line with the location of these residues within the putative ATP binding site of the kinase. During M phase, p34cdc2 is also phosphorylated, but phosphorylation occurs on a threonine residue distinct from Thr 14. Finally, phosphorylation of Ser 277 peaks during G1 phase and drops markedly as cells progress through S phase, raising the possibility that this modification may contribute to control the proposed G1/S function of the vertebrate p34cdc2 kinase.     

In vitro cell cycle arrest induced by using artificial DNA templates.

In cell extracts of Xenopus eggs which oscillate between S and M phases of the cell cycle, the onset of mitosis is blocked by the presence of incompletely replicated DNA. In this report, we show that several artificial DNA templates (M13 single-stranded DNA and double-stranded plasmid DNA) can trigger this feedback pathway, which inhibits mitosis. Single-stranded M13 DNA is much more effective than double-stranded plasmid DNA at inhibiting the onset of mitosis. Furthermore, we have shown that low levels of M13 single-stranded DNA and high levels of double-stranded plasmid DNA can elevate the tyrosine kinase activity responsible for phosphorylating p34cdc2, thereby inactivating maturation-promoting factor and inhibiting entry into mitosis. This constitutes a simplified system with which to study the signal transduction pathway from the DNA template to the tyrosine kinase responsible for inhibiting p34cdc2 activity.     

A link between MAP kinase and p34(cdc2)/cyclin B during oocyte maturation: p90(rsk) phosphorylates and inactivates the p34(cdc2) inhibitory kinase Myt1.

M-phase entry in eukaryotic cells is driven by activation of MPF, a regulatory factor composed of cyclin B and the protein kinase p34(cdc2). In G2-arrested Xenopus oocytes, there is a stock of p34(cdc2)/cyclin B complexes (pre-MPF) which is maintained in an inactive state by p34(cdc2) phosphorylation on Thr14 and Tyr15. This suggests an important role for the p34(cdc2) inhibitory kinase(s) such as Wee1 and Myt1 in regulating the G2-->M transition during oocyte maturation. MAP kinase (MAPK) activation is required for M-phase entry in Xenopus oocytes, but its precise contribution to the activation of pre-MPF is unknown. Here we show that the C-terminal regulatory domain of Myt1 specifically binds to p90(rsk), a protein kinase that can be phosphorylated and activated by MAPK. p90(rsk) in turn phosphorylates the C-terminus of Myt1 and down-regulates its inhibitory activity on p34(cdc2)/cyclin B in vitro. Consistent with these results, Myt1 becomes phosphorylated during oocyte maturation, and activation of the MAPK-p90(rsk) cascade can trigger some Myt1 phosphorylation prior to pre-MPF activation. We found that Myt1 preferentially associates with hyperphosphorylated p90(rsk), and complexes can be detected in immunoprecipitates from mature oocytes. Our results suggest that during oocyte maturation MAPK activates p90(rsk) and that p90(rsk) in turn down-regulates Myt1, leading to the activation of p34(cdc2)/cyclin B.     

cdc25 is one of the MPM-2 antigens involved in the activation of maturation-promoting factor.

MPM-2 antigens, a discrete set of phosphoproteins that contain similar phosphoepitopes recognized by the monoclonal antibody MPM-2, are phosphorylated during M-phase induction. Our previous studies suggested that certain MPM-2 antigens are involved in the appearance of maturation-promoting factor (MPF) activity. Because the central mitotic regulator cdc2 kinase has been shown to exhibit MPF activity, we explored the possibility that certain MPM-2 antigens are regulators of cdc2 kinase. We found that MPM-2 binding of its antigens would inhibit the autoamplification of cdc2 kinase in Xenopus oocytes and interfere with cyclin-activation of cdc2 kinase in Xenopus interphase egg extract. Immunodepletion of MPM-2 antigens from cyclin-induced M-phase egg extract caused the inactivation of cdc2 kinase, which was accompanied by an inhibitory phosphorylation of p34cdc2 on Thr 14 and Tyr 15, indicating that at least one MPM-2 antigen is a positive regulator of p34cdc2 dephosphorylation. We then showed that cdc25 from M-phase arrested egg extract is an MPM-2 antigen. These results suggest that phosphorylation of the epitope recognized by MPM-2 may be a crucial event in the activation of cdc25 and that the kinase(s) that phosphorylates this MPM-2 epitope may be an important regulator of cdc2 kinase activation.     

Structural basis of specific and efficient phosphorylation of peptides derived from p34cdc2 by a pp60src-related protein tyrosine kinase.

Cys-cdc2(8-20), a synthetic peptide derived from p34cdc2, was previously reported to be a specific and efficient substrate of a pp60c-src-related tyrosine kinase isolated from bovine spleen (the spleen tyrosine kinase) (Litwin, C.M.E., Cheng, H.-C., and Wang, J.H. (1991) J. Biol. Chem. 266, 2557-2566). The longer peptide, cdc2(1-24), was found to be phosphorylated by the kinase with similar efficiency, and Tyr15 was the only amino acid residue phosphorylated. This indicated that the amino acid sequence of cdc2(8-20) peptide, EKI-GEGTYGVVYK, contained the structural features important for protein tyrosine kinase substrate activity. A stepwise procedure using synthetic peptides was employed to investigate such structural features. First, a computer search of protein sequences homologous to cdc2(8-20) uncovered five protein kinases containing homologous sequence with tyrosine at a position corresponding to Tyr15 of p34cdc2. Second, a peptide derived from ribosomal S6 protein kinase (rsk(436-456] was synthesized. The rsk(436-456) peptide contained a segment, ETIGVGSYSVCKR, which is highly homologous to that of cdc2(8-20). It was found to be a very poor substrate of the spleen tyrosine kinase. Third, peptide analogs of cdc2(6-20) with single substitutions of amino acid residues Lys9, Glu12, Thr14, Gly16, Val18, and Tyr19 by amino acid residues at corresponding positions of rsk were synthesized and tested as spleen tyrosine kinase substrates. Only Glu12 and Thr14 substituted peptide analogs showed decreased substrate activities. (The substrate activity of a peptide is the ability of the peptide to serve as the substrate of the spleen tyrosine kinase. It was determined of the spleen tyrosine kinase. It was determined either by the kinetic parameters (Km and Vmax) of phosphorylation of the peptide or by the initial phosphorylation rate of the peptide by the spleen tyrosine kinase.) An analog with double substitution at Glu12 An analog with double substitution at Glu12 and Thr14 was found to be almost as poor a substrate as the rsk peptide. In addition, peptide analogs with Tyr15 substituted by Phe or D-Tyr had poor substrate activities as well as weak inhibitory activities. Thus, Glu12, Thr14, and Tyr15 residues of p34cdc2 contained structural components essential for the efficient phosphorylation of the peptides derived from p34cdc2 by the pp60c-src-related spleen tyrosine kinase.     

Regulation of microtubule organization during interphase and M phase

Microtubule (MT) dynamics and organization change markedly during interphase-M phase transition of the cell cycle. This mini review focuses first on p220, a ubiquitous MT-associated protein of Xenopus. p220 is phosphorylated by p34cdc2 kinase and MAP kinase in M phase, and concomitantly loses its MT-binding and MT-stabilizing activities. A cDNA encoding p220 was cloned, which identified p220 as a Xenopus homolog of MAP4, and p220 was therefore termed XMAP4. To examine the physiological relevance of XMAP4 phosphorylation during mitosis, Xenopus A6 cells were transfected with cDNA encoding wild-type or various XMAP4 mutants fused with a green fluorescent protein (GFP). Mutations of serine and threonine within potential phosphorylation sites for p34cdc2 kinase to nonphosphorylatable alanine interfered with mitosis-associated reduction in MT-affinity of XMAP4 and their overexpression affected chromosome movement during anaphase A. These results indicated that phosphorylation of XMAP4 by p34cdc2 kinase is responsible for the decrease in its MT-binding and MT-stabilizing activities during mitosis which are important for chromosome movement during anaphase A. The second focus is on a novel monoclonal antibody W8C3, which recognizes alpha-tubulin. W8C3 stained spindle MTs but not interphase MTs of Xenopus A6 cells, although tubulin dimers in M phase and interphase were equally recognized by this antibody. The difference in MT staining pattern may be because the W8C3-recognition site on alpha-tubulin is sterically hidden in interphase MTs but not in spindle MTs.     

Cdc25 regulates the phosphorylation and activity of the Xenopus cdk2 protein kinase complex.

The Xenopus cdk2 gene encodes a 32-kDa protein kinase with sequence similarity to the 34-kDa product of the cdc2 gene. Previous studies have shown that the kinase activity of the protein product of the cdk2 gene oscillates in the Xenopus embryonic cell cycle with a high in M-phase and a low in interphase. In the present study cdk2 was found not to be associated with any newly synthesized proteins during the cell cycle, but the enzyme did undergo periodic changes in phosphorylation. Upon exit from metaphase, cdk2 became increasingly phosphorylated on both tyrosine and serine residues, and labeling on these residues increased progressively until entry into mitosis, when tyrosine residues were markedly dephosphorylated. Phosphopeptide mapping of cdk2 demonstrated the major sites of phosphorylation were in a phosphopeptide with a pI of 3.7 that contained both phosphoserine and phosphotyrosine. This phosphopeptide accumulated in egg extracts blocked in S-phase with aphidicolin and was not evident in cdc2 immunoprecipitated under the same conditions. Under the same conditions cdc2 was phosphorylated primarily on a phosphopeptide containing both phosphothreonine and phosphotyrosine residues, most likely threonine 14 and tyrosine 15. Affinity-purified human GST-cdc25 was able to dephosphorylate and activate cdk2 isolated from interphase cells. Phosphopeptide mapping demonstrated that the phosphate was specifically removed from the same phosphopeptide identified as the major in vivo site of phosphorylation. These results demonstrate that cdk2 is regulated in the cell cycle by phosphorylation and dephosphorylation on both serine and tyrosine residues. Moreover, the increased phosphorylation of cdk2 in aphidicolin-blocked extracts and the ability of cdc25 to mediate cdk2 dephosphorylation in vitro suggest the possibility that cdk2 is part of the mechanism ensuring mitosis is not initiated until completion of DNA replication. It also implies cdc25 may have other functions in addition to the regulation of cdc2 kinase activity.     

Fission yeast p13 blocks mitotic activation and tyrosine dephosphorylation of the Xenopus cdc2 protein kinase

It has been demonstrated that the Xenopus homolog of the fission yeast cdc2 protein is a component of M phase promoting factor (MPF). We show that the Xenopus cdc2 protein is phosphorylated on tyrosine in vivo, and that this tyrosine phosphorylation varies markedly with the stage of the cell cycle. Tyrosine phosphorylation is high during interphase (in Xenopus oocytes and activated eggs) but absent during M phase (in unfertilized eggs). In vitro activation of pre-MPF from Xenopus oocytes results in tyrosine dephosphorylation of the cdc2 protein and switching-on of its kinase activity. The product of the fission yeast suc1 gene (p13), which inhibits the entry into mitosis in Xenopus extracts, completely blocks tyrosine dephosphorylation and kinase activation. However, p13 has no effect on the activated form of the cdc2 kinase. These findings suggest that p13 controls the activation of the cdc2 kinase, and that tyrosine dephosphorylation is an important step in this process.     

CAK, the p34cdc2 activating kinase, contains a protein identical or closely related to p40MO15.

The mitotic inducer p34cdc2 requires association with a cyclin and phosphorylation on Thr161 for its activity as a protein kinase. CAK, the p34cdc2 activating kinase, was previously identified as an enzyme necessary for this activating phosphorylation. We confirm here that CAK is a protein kinase and describe its purification over 13,000-fold from Xenopus egg extracts. We further show that CAK contains a protein identical or closely related to the previously identified Xenopus MO15 gene: p40MO15 copurifies with CAK, and an antiserum to p40MO15 specifically depletes cAK activity. CAK appears to be the only protein in Xenopus egg extracts that can activate complexes of either p34cdc2 or the closely related protein kinase, p33cdk2, with either cyclin A or cyclin B. The sequence similarity between p40MO15 and p34cdc2, and the approximately 200 kDa size of CAK, suggest that p40MO15 may itself be regulated by subunit association and by protein phosphorylations.     

The cdc2 kinase is a nuclear protein that is essential for mitosis in mammalian cells

A homolog of the fission yeast cdc2-encoded protein kinase (p34) is a component of M phase promoting factor in Xenopus oocytes. The homologous kinase in human HeLa cells is maximally active during mitosis, suggesting a mitotic role in mammalian somatic cells. This has been directly investigated by microinjection of anti-p34 antibodies into serum-stimulated rat fibroblasts. DNA synthesis was unaffected but cell division was quantitatively blocked in injected cells. Injection of antibodies against p13suc1, a component of the p34 kinase complex, did not block mitosis but caused mitotic abnormalities resulting in cells containing multiple micronuclei in the subsequent interphase. p34 localized in the nucleus during interphase. During mitosis, a fraction tightly associated with centrosomes. p13 was more evenly distributed between the nucleus and cytoplasm. These observations demonstrate that cdc2 is a nuclear and centrosomal protein that is required for mitosis in mammalian cells.