The Arabidopsis functional homolog of the p34cdc2 protein kinase.

The p34cdc2 protein kinase is a key component of the eukaryotic cell cycle, which is required for G1 to S-phase transition and for entry into mitosis. Using a 380-base pair DNA fragment obtained by polymerase chain reaction amplification from an Arabidopsis thaliana flower cDNA library as a probe, we isolated and sequenced a cdc2-homologous cDNA from Arabidopsis. The encoded polypeptide has extensive homology with cdc2-like kinases. Furthermore, when expressed in a CDC28ts Saccharomyces strain, it partially restores the capacity to grow at 36 degrees C, indicating that the plant cDNA is a functional homolog of the p34cdc2 kinase. Genomic hybridization demonstrated that there is one copy of the cdc2 gene per Arabidopsis haploid genome. Using RNA gel blot analysis, we found that cdc2 mRNA is present in all plant organs.     

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.     

Activation of p34cdc2 kinase by cyclin is negatively regulated by cyclic amp-dependent protein kinase in Xenopus oocytes.

Microinjection of a bacterially expressed stable delta 90 sea urchin cyclin B into Xenopus prophase oocytes, in absence or presence of cycloheximide, provokes the activation of histone H1 kinase and the tyrosine dephosphorylation of p34cdc2. Unexpectedly, when prophase oocytes are submitted to a treatment known to elevate the intracellular cAMP level (3-isobutyl-1-methylxanthine and cholera toxin), delta 90 cyclin has no effect and the oocytes remain blocked in prophase. This inhibition is reverted by the microinjection of the inhibitor of cAMP-dependent protein kinase. When delta 90 cyclin is microinjected into oocytes depleted of endogenous cyclins (cycloheximide-treated metaphase I) and in the presence of a high intracellular concentration of cAMP, p34cdc2 kinase is tyrosine rephosphorylated. Altogether, our results indicate that in Xenopus oocyte, cAMP-dependent protein kinase (A-kinase) controls the formation of the cyclin B/p34cdc2 complex which remains inactive and tyrosine phosphorylated.     

Cyclin B targets p34cdc2 for tyrosine phosphorylation.

A universal intracellular factor, the 'M phase-promoting factor' (MPF), triggers the G2/M transition of the cell cycle in all organisms. In late G2, it is present as an inactive complex of tyrosine-phosphorylated p34cdc2 and unphosphorylated cyclin Bcdc13. In M phase, its activation as an active MPF displaying histone H1 kinase (H1K) originates from the concomitant tyrosine dephosphorylation of the p34cdc2 subunit and the phosphorylation of the cylin Bcdc13 subunit. We have investigated the role of cyclin in the formation of this complex and the tyrosine phosphorylation of p34cdc2, using highly synchronous mitotic sea urchin eggs as a model. As cells leave the S phase and enter the G2 phase, a massive tyrosine phosphorylation of p34cdc2 occurs. This large p34cdc2 tyrosine phosphorylation burst does not arise from a massive increase in p34cdc2 concentration. It even appears to affect only a fraction (non-immunoprecipitable by anti-PSTAIR antibodies) of the total p34cdc2 present in the cell. Several observations point to an extremely close association between accumulation of unphosphorylated cyclin and p34cdc2 tyrosine phosphorylation: (i) both events coincide perfectly during the G2 phase; (ii) both tyrosine-phosphorylated p34cdc2 and cyclin are not immunoprecipitated by anti-PSTAIR antibodies; (iii) accumulation of unphosphorylated cyclin by aphidicolin treatment of the cells, triggers a dramatic accumulation of tyrosine-phosphorylated p34cdc2; and (iv) inhibition of cyclin synthesis by emetine inhibits p34cdc2 tyrosine phosphorylation without affecting the p34cdc2 concentration. These results show that, as it is synthesized, cyclin B binds and recruits p34cdc2 for tyrosine phosphorylation; this inactive complex then requires the completion of DNA replication before it can be turned into fully active MPF. These results fully confirm recent data obtained in vitro with exogenous cyclin added to cycloheximide-treated Xenopus egg extracts.     

A cyclin-abundance cycle-independent p34cdc2 tyrosine phosphorylation cycle in early sea urchin embryos.

The activity of the cell cycle control protein p34cdc2 is post-translationally regulated in a variety of cell types. Using anti-phosphotyrosine antibodies, we find that p34cdc2-directed tyrosine kinase activity increases at fertilization in sea urchin eggs, leading to a gradual accumulation of phosphotyrosine on p34 during the early part of the cell cycle. Loss of phosphotyrosine from p34 accompanies entry into mitosis and phosphotyrosine reaccumulates as the embryo enters the next cell cycle. A similar pattern is seen when eggs are parthenogenetically activated with ammonium chloride. Tyrosine phosphorylation and phosphorylation/dephosphorylation cycles are suppressed when embryos are treated with the tyrosine kinase inhibitor genistein. On the other hand, a cycle persists when protein synthesis is inhibited with emetine, indicating that it is independent of the synthesis of another class of cell cycle control proteins, the cyclins. Additional experiments with the phorbol ester, phorbol myristate acetate, demonstrate that activating protein synthesis alone in unfertilized eggs does not result in stimulation of p34cdc2 tyrosine kinase activity. Our results indicate that p34 tyrosine phosphorylation cycles are triggered by the fertilization Cai transient. The first cycle is independent of the fertilization pHi signal, confirming that, in sea urchin embryos, the cycle is not tightly coupled to the cycle of cyclin abundance that is a prominent feature of the eukaryotic cell division cycle.     

Biochemical characterization of the p34cdc2 protein kinase component of purified maturation-promoting factor from Xenopus eggs

Genetic studies in the fission yeast Schizosaccharomyces pombe and biochemical data in oocytes and eggs of Xenopus laevis have implicated the product of the cdc2+ gene as critical for the G2 to M transition in the cell cycle. The product of the cdc2+ gene is a 34-kDa serine/threonine protein kinase, designated p34cdc2, that is a component of purified maturation-promoting factor (MPF) and also of purified mammalian growth-associated histone H1 kinase. The biochemical properties of p34cdc2 H1 kinase activity in the MPF complex were studied. Phosphorylation of the p45cyclin component in the MPF complex by p34cdc2 exhibited kinetics consistent with an intramolecular reaction. On glycerol gradient centrifugation, MPF kinase against several substrates sedimented with an apparent Mr = 45,000-55,000. p34cdc2 was found to utilize ATP, GTP, and adenosine 5'-O-(3-thiotriphosphate) with apparent Km values of 75, 700, and 250 microM, respectively. The kinase activity was inhibited by beta-glycerophosphate, NaF, and zinc, whereas p-nitrophenyl phosphate was slightly stimulatory. The relative rates of phosphorylation of various substrates by MPF and growth-associated H1 kinase were similar. These findings should prove useful in further work on the regulation of MPF kinase activity and characterization of its substrates.     

Protein phosphorylation during meiotic maturation of Xenopus oocytes: cdc2 protein kinase targets

M-Phase specific protein kinase or cdc2 protein kinase is a component of MPF (M-Phase promoting factor). During meiotic maturation of Xenopus oocytes, cdc2 protein kinase is activated in correlation with MPF activity. A protein phosphorylation cascade takes place involving several protein kinases, among which casein kinase II, and different changes associated with meiosis occur such as germinal vesicle breakdown, chromosome condensation, cytoskeletal reorganization and increase in protein synthesis. Our results provide a biochemical link between cdc2 protein kinase and protein synthesis since they show that the kinase phosphorylates in vitro a p47 protein identified as elongation factor EF1 (gamma subunit) and that the in vitro site of p47 corresponds to the site phosphorylated in vivo. Immunofluorescence showed that the elongation factor (EF1-beta gamma) is localized in the oocyte cortex. Furthermore, they show that cdc2 kinase phosphorylates and activates casein kinase II in vitro, strongly supporting the view that casein kinase II is involved in the phosphorylation cascade originated by cdc2 kinase.     

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.     

The identification of a Caenorhabditis elegans homolog of p34cdc2 kinase

We have identified a Caenorhabditis elegans homolog of p34^cdc2 kinase. The C. elegans homolog, ncc-1, is ～-60% identical to p34^cdc2 of Homo sapiens. When expressed from a constitutive yeast promoter, ncc-1 is capable of complementing a conditional lethal mutation in the CDC28 gene of Saccharomyces cerevisiae, indicating that this C. elegans homolog can properly regulate the cell cycle.     

p34cdc2 is physically associated with and phosphorylated by a cdc2-specific tyrosine kinase.

The mammalian homologue of the yeast cdc2 gene encodes a 34-kilodalton serine/threonine kinase that is a subunit of M phase-promoting factor. Recent studies have shown that p34cdc2 is also a major tyrosine-phosphorylated protein in HeLa cells and that its phosphotyrosine content is cell cycle regulated and related to its kinase activity. Here, we show that cdc2 is physically associated with and phosphorylated in vitro by a highly specific tyrosine kinase. Tyrosine phosphorylation of cdc2 in vitro occurs at tyrosine 15, the same site that is phosphorylated in vivo. The association between the two kinases takes place in the cytosolic compartment and involves cyclin B-associated cdc2. Evidence is presented that a substantial fraction of cytosolic cdc2 is hypophosphorylated, whereas nuclear cdc2 is hyperphosphorylated. Finally, we show that the tyrosine kinase associated with cdc2 may be a 67-kilodalton protein and is distinct from src, abl, fms, and other previously reported tyrosine kinases.     

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.     

Cell cycle regulation of the p34cdc2 inhibitory kinases.

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.     

Cell cycle control of DNA replication by p34cdc2.

Commitment to DNA replication is one of the major control points of the eukaryotic cell cycle, and one that has been curiously hard to analyse. However, homologous components of this process are now being identified by genetic analysis of yeast and by biochemical analysis of cell-free systems from higher eukaryotes. This homology suggests that these components are part of a universal mechanism for controlling the eukaryotic cell cycle. The most important component of this mechanism is the cdc2 protein, which controls the initiation of both DNA replication and mitosis. At present, however, its precise role in DNA replication is unclear.     

M-phase-specific phosphorylation and structural rearrangement of the cytoplasmic cross-linking protein plectin involve p34cdc2 kinase.

Plectin, a widespread and abundant cytoskeletal cross-linking protein, serves as a target for protein kinases throughout the cell cycle, without any significant variation in overall phosphorylation level. One of the various phosphorylation sites of the molecule was found to be phosphorylated preferentially during mitosis. By in vivo phosphorylation of ectopically expressed plectin domains in stably transfected Chinese hamster ovary cells, this site was mapped to the C-terminal repeat 6 domain of the polypeptide. The same site has been identified as an in vitro target for p34cdc2 kinase. Mitosis-specific phosphorylation of plectin was accompanied by a rearrangement of plectin structures, changing from a filamentous, largely vimentin-associated state in interphase to a diffuse vimentin-independent distribution in mitosis as visualized by immunofluorescence microscopy. Subcellular fractionation studies showed that in interphase cells up to 80% of cellular plectin was found associated with an insoluble cell fraction mostly consisting of intermediate filaments, while during mitosis the majority of plectin (> 75%) became soluble. Furthermore, phosphorylation of purified plectin by p34cdc2 kinase decreased plectin's ability to interact with preassembled vimentin filaments in vitro. Together, our data suggest that a mitosis-specific phosphorylation involving p34cdc2 kinase regulates plectin's cross-linking activities and association with intermediate filaments during the cell cycle.     

Phosphorylation of caldesmon by p34cdc2 kinase. Identification of phosphorylation sites

It has recently been shown that caldesmon from non-muscle (Yamashiro, S., Yamakita, Y., Hosoya, H., and Matsumura, F. (1991) Nature 349, 169-172) and smooth muscle cells (Mak, A. S., Watson, M. H., Litwin, C. M. E., and Wang, J. H. (1991) J. Biol. Chem. 266, 6678-6681) can be phosphorylated in vitro by p34cdc2 kinase resulting in the inhibition of caldesmon binding to F-actin and Ca(2+)-calmodulin. In this study, we have identified five phosphorylation sites in smooth muscle caldesmon at Ser582, Ser667, Thr673, Thr696, and Ser702. All the sites bear some resemblance to the S(T)-P-X-X motif recognized by p34cdc2. The preferred site of phosphorylation at Thr673 accounts for about 40% of the total phosphorylation. Four of the sites occur in two pairs of closely spaced sites, Ser667/Thr673 and Thr696/Ser702; phosphorylation of one site in each pair inhibits strongly the phosphorylation of the second site in the same pair, presumably due to the close proximity of the two sites. Similar negative cooperativity in phosphorylation of Ser667 and Thr673 was observed using a 22-residue synthetic peptide containing the two sites. Phosphorylation of Ser667/Thr673 and Thr696/Ser702 account for about 90% of the total level of phosphorylation and these sites are located within the 10-kDa CNBr fragment at the COOH-terminal end of caldesmon known to bind actin and Ca(2+)-calmodulin.     

Regulation of p34cdc2 protein kinase during mitosis

The cell-cycle timing of mitosis in fission yeast is determined by the cdc25+ gene product activating the p34cdc2 protein kinase leading to mitotic initiation. Protein kinase activity remains high in metaphase and then declines during anaphase. Activation of the protein kinase also requires the cyclin homolog p56cdc13, which also functions post activation at a later stage of mitosis. The continuing function of p56cdc13 during mitosis is consistent with its high level until the metaphase/anaphase transition. At anaphase the p56cdc13 level falls dramatically just before the decline in p34cdc2 protein kinase activity. The behavior of p56cdc13 is similar to that observed for cyclins in oocytes. p13suc1 interacts closely with p34cdc2; it is required during the process of mitosis and may play a role in the inactivation of the p34cdc2 protein kinase. Therefore, the cdc25+, cdc13+, and suc1+ gene products are important for regulating p34cdc2 protein kinase activity during entry into, progress through, and exit from mitosis.     

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.     

Requirement of mosXe protein kinase for meiotic maturation of Xenopus oocytes induced by a cdc2 mutant lacking regulatory phosphorylation sites.

The p34cdc2 protein kinase is a component of maturation-promoting factor, the master regulator of the cell cycle in all eukaryotes. The activity of p34cdc2 is itself tightly regulated by phosphorylation and dephosphorylation. Predicted regulatory phosphorylation sites of Xenopus p34cdc2 were mutated in vitro, and in vitro-transcribed RNAs were injected into Xenopus oocytes. The cdc2 single mutants Thr-14----Ala and Tyr-15----Phe did not induce germinal vesicle breakdown (BVBD) upon microinjection into oocytes. In contrast, the cdc2 double mutant Ala-14/Phe-15 did induce GVBD. Both the Ala-14 and Ala-14/Phe-15p34cdc2 mutants were shown to coimmunoprecipitate cyclin B1 and to phosphorylate histone H1 in immune complex kinase assays. Microinjection of antisense oligonucleotides to c-mosXe was used to demonstrate the role of mos protein synthesis in the induction of GVBD by the Ala-14/Phe-15 cdc2 mutant. Thr-161 was also mutated. p34cdc2 single mutants Ala-161 and Glu-161 and triple mutants Ala-14/Phe-15/Ala-161 and Ala-14/Phe-15/Glu-161 failed to induce GVBD in oocytes and showed a decreased binding to cyclin B1 in coimmunoprecipitations. Each of the cdc2 mutants was also assayed by coinjection with cyclin B1 or c-mosXe RNA into oocytes. Several of the cdc2 mutants were found to affect the kinetics of cyclin B1 and/or mos-induced GVBD upon coinjection, although none affected the rate of progesterone-induced maturation. We demonstrate here the significance of Thr-14, Tyr-15, and Thr-161 of p34cdc2 in Xenopus oocyte maturation. In addition, these results suggest a regulatory role for mosXe in induction of oocyte maturation by the cdc2 mutant Ala-14/Phe-15.