p13suc1 acts in the fission yeast cell division cycle as a component of the p34cdc2 protein kinase.

cdc2+ encodes a protein kinase that is required during both G1 and G2 phases of the cell division cycle in fission yeast. suc1+ is an essential gene that was originally identified as a plasmid-borne sequence that could rescue certain temperature-sensitive cdc2 mutants. To investigate the role of the suc1+ gene product in the cell cycle p13suc1 has been expressed in Escherichia coli and purified. An immunoaffinity purified anti-p13suc1 polyclonal serum has been prepared and used to identify p13suc1 in fission yeast. The abundance of this protein did not alter either during the cell cycle or during entry into stationary phase. p13suc1 was found in yeast lysates in a complex with the cdc2+ gene product. Approximately 5% of cellular p34cdc2 was associated with p13suc1, and this fraction of p34cdc2 was active as a protein kinase. The stability of the complex was disrupted in yeast strains carrying temperature-sensitive alleles of cdc2 that are suppressible by overexpression of suc1+. The level of association between p13suc1 and p34cdc2 was not affected by cell cycle arrest in adverse nutritional conditions. p13suc1 is not a substrate of the p34cdc2 protein kinase. We propose instead that it acts as a regulatory component of p34cdc2 that facilitates interaction with other proteins.     

Regulation of cdc2 activity in Schizosaccharomyces pombe: the role of phosphorylation.

The cdc2 protein kinase, first identified as a cell cycle gene required for transition into the S- and M-phases of budding and fission yeast, has been shown to act as a key component in the regulation of the eukaryotic cell cycle. The periodic activation of cdc2 kinase, which is required for entry into M-phase, is regulated by subunit association with cyclin B, the cdc25, wee1, mik1 gene products and differential phosphorylation of the cdc2 protein. Phosphorylation at Tyr 15 inhibits activation of the cdc2/cdc13 complex whereas phosphorylation of Thr 167 is required for kinase activity.     

Sce3, a suppressor of the Schizosaccharomyces pombe septation mutant cdc11, encodes a putative RNA-binding protein.

In the fission yeast Schizosaccharomyces pombe, the cdc11 gene is required for the initiation of septum formation at the end of mitosis. The sce3 gene was cloned as a multi-copy suppressor of the heat-sensitive mutant cdc11-136. When over-expressed, it rescues all mutants of cdc11 and also a heat-sensitive allele of cdc14, but not the cdc14 null mutant. Deletion shows that sce3 is not essential for cell proliferation. It encodes a putative RNA-binding protein which shows homology to human eIF4B. Immunolocalisation indicates that Sce3p is located predominantly in the cytoplasm. Elevated expression of sce3 increases the steady-state level of cdc14 mRNA. Possible mechanisms of its action are discussed.     

Highly conserved toxicity of Saccharomyces cerevisiae Rap1p

Budding yeast ( Saccharomyces cerevisiae ) Rap1p has been expressed in fission yeast ( Schizosaccharo-myces pombe ) under the control of the regulatable fructose bisphosphatase ( fbp ) promoter. When the fbp promoter was derepressed, cells containing the complete RAP1 gene failed to show any significant growth, suggesting that Rap1p is toxic. A derivative of Rap1p that has a temperature-sensitive mutation in the DNA-binding domain was not toxic in cells grown at 37°C, a temperature at which DNA binding by rap1p ^ts is severely inhibited. Removal of a short region downstream of the DNA-binding domain, including a region previously shown to be essential for Rap1p toxicity in budding yeast, also abolished the toxic effect. The toxic effect of Rap1p has therefore been conserved between two distantly related yeasts. In budding yeast, overexpression of Rap1p also caused changes to the lengths of the telomeric repeats. No effects on telomeres were detected in fission yeast.     

Isolation and characterization of a second protein tyrosine phosphatase gene, PTP2, from Saccharomyces cerevisiae.

A putative protein tyrosine phosphatase (PTPase) gene, PTP2, was cloned from Saccharomyces cerevisiae. The complete yeast PTP2 gene encodes a 750-amino acid residue protein with a predicted mass of 86 kDa. The conserved PTPase domain was localized in the C-terminal half of the protein. Amino acid sequence alignment of the yeast PTPase domain with other phosphatases indicated approximately 20-25% sequence identity with the mammalian PTPase and a similar degree of identity with the PTPase encoded by the yeast PTP1 gene. The PTP2 gene is closely linked to the yeast RET1 and STE4 genes and is localized on the right arm of chromosome 15. Gene disruption experiments demonstrated that neither PTP2 alone nor PTP2 in combination with PTP1 was essential for growth under the conditions tested. The ability of PTP2 to complement the cdc25-22 mutant of Schizosaccharomyces pombe was also examined, and unlike the human T-cell PTPase, which was able to complement the cdc25-22 mutant, the S. cerevisiae PTP2 was unable to complement the cdc25-22 mutant of S. pombe.     

The fission yeast cell cycle control gene cdc2: isolation of a sequence suc1 that suppresses cdc2 mutant function

Summary A DNA fragment called suc1 has been found to rescue cells mutated in the cell cycle control gene cdc2 of the fission yeast Schizosaccharomyces pombe. The suppressing activity of suc1 is observed when it is present on a multicopy number plasmid. The gene does not hybridise to cdc2 and maps elsewhere in the genome. Its effect is cdc2 allele specific suggesting that it interacts directly with the cdc2 gene function.     

Expression of the murine homologue of the cell cycle control protein p34cdc2 in T lymphocytes.

The mammalian homologue of the cdc2 gene of the fission yeast Schizosaccharomyces pombe encodes a p34cdc2 cyclin-dependent kinase that regulates the cell cycle of a wide variety of cell types. Resting murine T lymphocytes contained no detectable p34cdc2 protein, histone kinase activity, or specific mRNA for the cdc2 gene. Activation of the T cells by immobilized anti-CD3 resulted in the expression of specific mRNA late in the G1 phase of the cell cycle, and p34cdc2 protein was detectable at or near G1/S. At this point in the cell cycle, the protein was phosphorylated at tyrosine and displayed no H1 histone kinase activity. As the cells progressed through the cycle, the amount of specific mRNA and p34cdc2 increased, and H1 histone kinase activity was detectable when the cells were blocked at G2/M by nocodazole. The activation of T cells by phorbol dibutyrate induced the expression of IL-2R but failed to induce the synthesis of IL-2 or the expression of cdc2-specific mRNA. Under these conditions, the activated cells failed to enter the S phase of the cell cycle. Because the presence of IL-2 added exogenously during activation by phorbol dibutyrate resulted in the expression of cdc2-specific mRNA and progression through the cell cycle, either IL-2 or the interaction with IL-2R may be involved in the expression of cdc2 and regulation of the G1/S transition.     

A Monoclonal Antibody Against the PSTAIR Sequence of p34cdc2, Catalytic Subunit of Maturation-Promoting Factor and Key Regulator of the Cell Cycle

A homolog of the serine/threonine protein kinase (p34^cdc2), encoded by the cdc2 ⁺ gene of the fission yeast ( Schizosaccharomyces pombe ), is a catalytic subunit of maturation-promoting factor and a key regulator of the cell cycle. We have raised a monoclonal antibody against the most conserved amino acid sequence, the PSTAIR sequence (EGVPSTAIREISLLKE) of p34^cdc2 This antibody recognizes 31–34 kDa proteins by immunoblotting in all species examined so far. The proteins recognized by the anti-PSTAIR antibody are probably either p34^cdc2 itself or proteins highly homologous to p34^cdc2 in the given species, since, in all species studies to date, they are all precipitated with p13^suc1, the fission yeast suc 1⁺ gene product, which binds to p34^cdc2 with high specificity. The anti-PSTAIR immunoprecipitate had no histone H1 kinase activity and did not contain cyclin B, suggesting that the PSTAIR region is masked when p34^cdc2 forms a complex with cyclin B as an active kinase. Immunoblotting with the anti-PSTAIR antibody demonstrated that the fastest-migrating form of p34^cdc2 homologues becomes abundant, when oocytes mature or the cell enters M phase. The possible significance of this observation is discussed in relation to the phosphorylation and activity state of p34^cdc2 The observed broad cross-reactivity of the anti-PSTAIR antibody against p34^cdc2 homologues in various species should permit us to examine the role of p34^cdc2 homologues in the regulation of the cell cycle in a variety of organisms.     

Interaction between cdc13+ and cdc2+ in the control of mitosis in fission yeast; dissociation of the G1 and G2 roles of the cdc2+ protein kinase

A cold-sensitive (cs) allele of cdc2, a gene that acts in both the G1 and G2 phases of the fission yeast cell cycle, has been isolated by classical mutagenesis. Further mutagenesis of a cdc2cs strain yielded an extragenic suppressor that rescued the cs cell cycle defect but simultaneously conferred a temperature-sensitive (ts) cdc phenotype. This suppressor mutation was shown to be an allele of cdc13, a previously identified gene. A variety of allele-specific interactions between cdc2 and cdc13 were discovered. These included suppression of cdc13ts alleles by introduction of the cdc2+ gene on a multi-copy plasmid vector. cdc13+ is required in G2 for mitotic initiation and was shown to play no role in the G1 phase of the cell cycle. cdc2+, however, is essential in G1 for DNA replication and in G2 for mitosis. The newly isolated cs allele of cdc2 that is rescued by a ts allele of cdc13 is defective only in its G2 function. cdc13+ cooperates with cdc2+ in the initiation of mitosis but not in the regulation of DNA replication. We propose that the cdc13+ gene product might be a G2-specific substrate of the cdc2+ protein kinase.     

Specific activation of cdc25 tyrosine phosphatases by B-type cyclins: evidence for multiple roles of mitotic cyclins.

Two previously unidentified human cdc25 genes have been isolated, cdc25A and cdc25B. Both genes rescue a cdc25ts mutant of fission yeast. Microinjection of anti-cdc25A antibodies into HeLa cells causes their arrest in mitosis. cdc25A and cdc25B display endogenous tyrosine phosphatase activity that is stimulated several-fold, in the absence of cdc2, by stoichiometric addition of either cyclin B1 or B2 but not A or D1. Association between cdc25A and cyclin B1/cdc2 was detected in the HeLa cells. These findings indicate that B-type cyclins are multifunctional proteins that not only act as M phase regulatory subunits of the cdc2 protein kinase, but also activate the cdc25 tyrosine phosphatase, of which cdc2 is the physiological substrate. A region of amino acid similarity between cyclins and tyrosine PTPases has been detected. This region is absent in cdc25 phosphatases. The motif may represent an activating domain that has to be provided to cdc25 by intermolecular interaction with cyclin B.     

Requirement for p34cdc2 kinase is restricted to mitosis in the mammalian cdc2 mutant FT210

The mouse FT210 cell line is a temperature-sensitive cdc2 mutant. FT210 cells are found to arrest specifically in G2 phase and unlike many alleles of cdc2 and cdc28 mutants of yeasts, loss of p34cdc2 at the nonpermissive temperature has no apparent effect on cell cycle progression through the G1 and S phases of the division cycle. FT210 cells and the parent wild-type FM3A cell line each possess at least three distinct histone H1 kinases. H1 kinase activities in chromatography fractions were identified using a synthetic peptide substrate containing the consensus phosphorylation site of histone H1 and the kinase subunit compositions were determined immunochemically with antisera prepared against the "PSTAIR" peptide, the COOH-terminus of mammalian p34cdc2 and the human cyclins A and B1. The results show that p34cdc2 forms two separate complexes with cyclin A and with cyclin B1, both of which exhibit thermal lability at the non-permissive temperature in vitro and in vivo. A third H1 kinase with stable activity at the nonpermissive temperature is comprised of cyclin A and a cdc2-like 34-kD subunit, which is immunoreactive with anti-"PSTAIR" antiserum but is not recognized with antiserum specific for the COOH-terminus of p34cdc2. The cyclin A-associated kinases are active during S and G2 phases and earlier in the division cycle than the p34cdc2-cyclin B1 kinase. We show that mouse cells possess at least two cdc2-related gene products which form cell cycle regulated histone H1 kinases and we propose that the murine homolog of yeast p34cdc/CDC28 is essential only during the G2-to-M transition in FT210 cells.