The role of the sid1p kinase and cdc14p in regulating the onset of cytokinesis in fission yeast

Coordination of mitosis and cytokinesis is crucial for ensuring proper chromosome segregation and genomic stability. In Schizosaccharomyces pombe, the sid genes (cdc7, cdc11, cdc14, spg1, sid1, sid2 and sid4) define a signaling pathway that regulates septation and cytokinesis. Here we describe the characterization of a novel protein kinase, Sid1p. Sid1p localizes asymmetrically to one spindle pole body (SPB) in anaphase. Sid1p localization is maintained during medial ring constriction and septum synthesis and disappears prior to cell separation. Additionally, we found that Cdc14p is in a complex with Sid1p. Epistasis analysis places Sid1p-Cdc14p downstream of Spg1p-Cdc7p but upstream of Sid2p. Finally, we show that cyclin proteolysis during mitosis is unaffected by inactivating the sid pathway; in fact, loss of Cdc2-cyclin activity promotes Sid1p-Cdc14p association with the SPB, possibly providing a mechanism that couples cytokinesis with mitotic exit.     

p80cdc25 mitotic inducer is the tyrosine phosphatase that activates p34cdc2 kinase in fission yeast.

We have investigated the mechanism by which fission yeast p80cdc25 induces mitosis. The in vivo active domain was localized to the C-terminal 23 kDa of p80cdc25. This domain produced as a bacterial fusion protein (GST-cdc25) caused tyrosyl dephosphorylation and activation of immunoprecipitated p34cdc2. Furthermore, GST-cdc25 dephosphorylated both para-nitrophenyl-phosphate (pNPP) and casein phosphorylated on serine in vitro. Reaction requirements and inhibitor sensitivities were the same as those of phosphotyrosine phosphatases (PTPases). Analysis of cdc25 C-terminal domains from a variety of species revealed a conserved motif having critical residues present at the active site of PTPases. Mutation of the cdc25 Cys480 codon, corresponding to an essential cysteine in the active site of PTPases, abolished the phosphatase activity of GST-cdc25. These data indicate that cdc25 proteins define a novel subclass of eukaryotic PTPases, and strongly argue that cdc25 proteins directly dephosphorylate and activate p34cdc2 kinase to induce M-phase.     

Mitotic hyperphosphorylation of the fission yeast SIN scaffold protein cdc11p is regulated by the protein kinase cdc7p

The fission yeast septation initiation network (SIN) triggers the onset of septum formation and cytokinesis. SIN proteins signal from the spindle pole body (SPB), to which they bind in a cell cycle-dependent manner, via the scaffold proteins sid4p and cdc11p. cdc11p becomes hyperphosphorylated during anaphase, when the SIN is active. We have investigated the phosphorylation state of cdc11p during mitosis in various mutant backgrounds. We show that association of cdc11p with the spindle pole body is required for its phosphorylation and that ectopic activation of the SIN results in hyperphosphorylation of cdc11p. We demonstrate that mitotic hyperphosphorylation of cdc11p requires the activity of cdc7p and that its dephosphorylation at the end of mitosis requires PP2A-par1p. Furthermore, spindle checkpoint arrest prevents cdc11p hyperphosphorylation. Finally, we show that the septation inhibitor byr4p interacts preferentially with hypophosphorylated cdc11p. We conclude that cdc11p hyperphosphorylation correlates with activation of the SIN and that this may be mediated primarily by cdc7p in vivo.     

Hsp90 protein in fission yeast Swo1p and UCS protein Rng3p facilitate myosin II assembly and function

The F-actin-based molecular motor myosin II is involved in a variety of cellular processes such as muscle contraction, cell motility, and cytokinesis. In recent years, a family of myosin II-specific cochaperones of the UCS family has been identified from work with yeasts, fungi, worms, and humans. Biochemical analyses have shown that a complex of Hsp90 and the Caenorhabditis elegans UCS domain protein UNC-45 prevent myosin head aggregation, thereby allowing it to assume a proper structure. Here we demonstrate that a temperature-sensitive mutant of the fission yeast Hsp90 (Swo1p), swo1-w1, is defective in actomyosin ring assembly at the restrictive temperature. Two alleles of swo1, swo1-w1 and swo1-26, showed synthetic lethality with a specific mutant allele of the fission yeast type II myosin head, myo2-E1, but not with two other mutant alleles of myo2 or with mutations affecting 14 other genes important for cytokinesis. swo1-w1 also showed a strong genetic interaction with rng3-65, a gene encoding a mutation in the fission yeast UCS domain protein Rng3p, which has previously been shown to be important for myosin II assembly. A similar deleterious effect was found when myo2-E1, swo1-w1, and rng3-65 were pharmacologically treated with geldanamycin to partially inhibit Hsp90 function. Interestingly, Swo1p-green fluorescent protein is detected at the improperly assembled actomyosin rings in myo2-E1 but not in a wild-type strain. Yeast two-hybrid and coimmunoprecipitation analyses verified interactions between Rng3p and the myosin head domain as well as interactions between Rng3p and Swo1p. Our analyses of Myo2p, Swo1p, and the UCS domain protein Rng3p establish that Swo1p and Rng3p collaborate in vivo to modulate myosin II function.     

14-3-3 protein interferes with the binding of RNA to the phosphorylated form of fission yeast meiotic regulator Mei2p

The switch from mitosis to meiosis is controlled by the Pat1(Ran1) kinase-Mei2p system in Schizosaccharomyces pombe. Mei2p promotes both premeiotic DNA synthesis and meiosis I, and its RNA binding ability is essential for these two processes. Mei2p forms a dot structure in the nucleus prior to meiosis I, aided by a specific RNA species named "meiRNA". Pat1 kinase phosphorylates Mei2p on two positions and downregulates its activity. Pat1 kinase undergoes inactivation under meiotic conditions, as a result of the production of a tethering pseudosubstrate Mei3p, and accumulation of the unphosphorylated form of Mei2p commits cells to meiosis. However, the mechanism of how phosphorylation of Mei2p suppresses its activity to induce meiosis remains largely unknown. Here we show that S. pombe Rad24p, a 14-3-3 protein, functions as a negative factor for meiosis by antagonizing the function of meiRNA to promote the formation of a nuclear Mei2p dot. Rad24p binds preferentially to Mei2p phosphorylated by Pat1 kinase. It inhibits association of meiRNA to the phosphorylated form of Mei2p but not to the unphosphorylated form in vitro. We speculate that Rad24p, bound tightly to the residues phosphorylated by Pat1 kinase, may mask the RNA recognition motifs on Mei2p. This model will explain, at least partly, why phosphorylation by Pat1 kinase inhibits the meiosis-inducing activity of Mei2p.     

Regulation and targeting of the fission yeast formin cdc12p in cytokinesis

Formins are conserved actin nucleators which promote the assembly of actin filaments for the formation of diverse actin structures. In fission yeast Schizosaccharomyces pombe, the formin cdc12p is required specifically in assembly of the actin-based contractile ring during cytokinesis. Here, using a mutational analysis of cdc12p, we identify regions of cdc12p responsible for ring assembly and localization. Profilin-binding residues of the FH1 domain regulate actin assembly and processive barbed-end capping by the FH2 domain. Studies using photobleaching (FRAP) and sensitivity to latrunculin A treatment show that profilin binding modulates the rapid dynamics of actin and cdc12p within the ring in vivo. Visualized by functional GFP-fusion constructs expressed from the endogenous promoter, cdc12p appears in a small number of cytoplasmic motile spot structures that deliver the formin to the ring assembly site, without detectable formation of an intermediate band of "nodes." The FH3/DID region directs interphase spot localization, while an N-terminal region and the FH1-FH2 domains of cdc12p can target its localization to the ring. Mutations in putative DID and DAD regions do not alter regulation, suggesting that cdc12p is not regulated by a canonical autoinhibition mechanism. Our findings provide insights into the regulation of formin activity and the mechanisms of contractile ring dynamics and assembly.     

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.     

HIV-1 Vpr induces G2 cell cycle arrest in fission yeast associated with Rad24/14-3-3-dependent, Chk1/Cds1-independent Wee1 upregulation

Viral protein R (Vpr), an accessory protein of human immunodeficiency virus type 1 (HIV-1), induces the G2 cell cycle arrest in fission yeast for which host factors, such as Wee1 and Rad24, are required. Catalyzing the inhibitory phosphorylation of Cdc2, Wee1 is known to serve as a major regulator of G2/M transition in the eukaryotic cell cycle. It has been reported that the G2 checkpoint induced by DNA damage or incomplete DNA replication is associated with phosphorylation and upregulation of Wee1 for which Chk1 and Cds1 kinase is required. In this study, we demonstrate that the G2 arrest induced by HIV-1 Vpr in fission yeast is also associated with increase in the phosphorylation and amount of Wee1, but in a Chk1/Cds1-independent manner. Rad24 and human 14-3-3 appear to contribute to Vpr-induced G2 arrest by elevating the level of Wee1 expression. It appears that Vpr could cause the G2 arrest through a mechanism similar to, but distinct from, the physiological G2 checkpoint controls. The results may provide useful insights into the mechanism by which HIV-1 Vpr causes the G2 arrest in eukaryotic cells. Vpr may also serve as a useful molecular tool for exploring novel cell cycle control mechanisms.     

Genetic and physical interaction of Ssp1 CaMKK and Rad24 14-3-3 during low pH and osmotic stress in fission yeast

The Ssp1 calmodulin kinase kinase (CaMKK) is necessary for stress-induced re-organization of the actin cytoskeleton and initiation of growth at the new cell end following division in Schizosaccharomyces pombe. In addition, it regulates AMP-activated kinase and functions in low glucose tolerance. ssp1⁻ cells undergo mitotic delay at elevated temperatures and G₂ arrest in the presence of additional stressors. Following hyperosmotic stress, Ssp1-GFP forms transient foci which accumulate at the cell membrane and form a band around the cell circumference, but not co-localizing with actin patches. Hyperosmolarity-induced localization to the cell membrane occurs concomitantly with a reduction of its interaction with the 14-3-3 protein Rad24, but not Rad25 which remains bound to Ssp1. The loss of rad24 in ssp1⁻ cells reduces the severity of hyperosmotic stress response and relieves mitotic delay. Conversely, overexpression of rad24 exacerbates stress response and concomitant cell elongation. rad24⁻ does not impair stress-induced localization of Ssp1 to the cell membrane, however this response is almost completely absent in cells overexpressing rad24.     

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.     

Hpz1 modulates the g1-s transition in fission yeast

Here we characterize a novel protein in S. pombe. It has a high degree of homology with the Zn-finger domain of the human Poly(ADP-ribose) polymerase (PARP). Surprisingly, the gene for this protein is, in many fungi, fused with and in the same reading frame as that encoding Rad3, the homologue of the human ATR checkpoint protein. We name the protein Hpz1 (Homologue of PARP-type Zn-finger). Hpz1 does not possess PARP activity, but is important for resistance to ultraviolet light in the G1 phase and to treatment with hydroxyurea, a drug that arrests DNA replication forks in the S phase. However, we find no evidence of a checkpoint function of Hpz1. Furthermore, absence of Hpz1 results in an advancement of S-phase entry after a G1 arrest as well as earlier recovery from a hydroxyurea block. The hpz1 gene is expressed mainly in the G1 phase and Hpz1 is localized to the nucleus. We conclude that Hpz1 regulates the initiation of the S phase and may cooperate with Rad3 in this function.     

The genomic instability of yeast cdc6-1/cdc6-1 mutants involves chromosome structure and recombination

When diploid cells of Saccharomyces cerevisiae homozygous for the temperature-sensitive cell division cycle mutation cdc6-1 are grown at a semipermissive temperature they exhibit elevated genomic instability, as indicated by enhanced mitotic gene conversion, mitotic intergenic recombination, chromosomal loss, chromosomal gain, and chromosomal rearrangements. Employing quantitative Southern analysis of chromosomes separated by transverse alternating field gel electrophoresis (TAFE), we have demonstrated that 2N-1 cells monosomic for chromosome VII, owing to the cdc6-1 defect, show slow growth and subsequently yield 2N variants that grow at a normal rate in association with restitution of disomy for chromosome VII. Analysis of TAFE gels also demonstrates that cdc6-1/cdc6-1 diploids give rise to aberrant chromosomes of novel lengths. We propose an explanation for the genomic instability induced by the cdc6-1 mutation, which suggests that hyper-recombination, chromosomal loss, chromosomal gain and chromosomal rearrangements reflect aberrant mitotic division by cdc6-1/cdc6-1 cells containing chromosomes that have not replicated fully.     

Molecular evolution of the 14-3-3 protein family

Members of the highly conserved and ubiquitous 14-3-3 protein family modulate a wide variety of cellular processes. To determine the evolutionary relationships among specific 14-3-3 proteins in different plant, animal, and fungal species and to initiate a predictive analysis of isoform-specific differences in light of the latest functional and structural studies of 14-3-3, multiple alignments were constructed from forty-six 14-3-3 sequences retrieved from the GenBank and SwissProt databases and a newly identified second 14-3-3 gene fromCaenorhabditis elegans. The alignment revealed five highly conserved sequence blocks. Blocks 2–5 correlate well with the alpha helices 3, 5, 7, and 9 which form the proposed internal binding domain in the three-dimensional structure model of the functioning dimer. Amino acid differences within the functional and structural domains of plant and animal 14-3-3 proteins were identified which may account for functional diversity amongst isoforms. Protein phylogenic trees were constructed using both the maximum parsimony and neighbor joining methods of the PHYLIP(3.5c) package; 14-3-3 proteins fromEntamoeba histolytica, an amitochondrial protozoa, were employed as an outgroup in our analysis. Epsilon isoforms from the animal lineage form a distinct grouping in both trees, which suggests an early divergence from the other animal isoforms. Epsilons were found to be more similar to yeast and plant isoforms than other animal isoforms at numerous amino acid positions, and thus epsilon may have retained functional characteristics of the ancestral protein. The known invertebrate proteins group with the nonepsilon mammalian isoforms. Most of the current 14-3-3 isoform diversity probably arose through independent duplication events after the divergence of the major eukaryotic kingdoms. Divergence of the seven mammalian isoforms beta, zeta, gamma, eta, epsilon, tau, and sigma (stratifin/ HME1) occurred before the divergence of mammalian and perhaps before the divergence of vertebrate species. A possible ancestral 14-3-3 sequence is proposed. Correspondence to: D.C. Shakes     

A Highlights from MBoC Selection: Multiple protein kinases influence the redistribution of fission yeast Clp1/Cdc14 phosphatase upon genotoxic stress

The Cdc14 phosphatase family antagonizes Cdk1 phosphorylation and is important for mitotic exit. To access their substrates, Cdc14 phosphatases are released from nucleolar sequestration during mitosis. Clp1/Flp1, the Schizosaccharomyces pombe Cdc14 orthologue, and Cdc14B, a mammalian orthologue, also exit the nucleolus during interphase upon DNA replication stress or damage, respectively, implicating Cdc14 phosphatases in the response to genotoxic insults. However, a mechanistic understanding of Cdc14 phosphatase nucleolar release under these conditions is incomplete. We show here that relocalization of Clp1 during genotoxic stress is governed by complex phosphoregulation. Specifically, the Rad3 checkpoint effector kinases Cds1 and/or Chk1, the cell wall integrity mitogen-activated protein kinase Pmk1, and the cell cycle kinase Cdk1 directly phosphorylate Clp1 to promote genotoxic stress–induced nucleoplasmic accumulation. However, Cds1 and/or Chk1 phosphorylate RxxS sites preferentially upon hydroxyurea treatment, whereas Pmk1 and Cdk1 preferentially phosphorylate Clp1 TP sites upon H₂O₂ treatment. Abolishing both Clp1 RxxS and TP phosphosites eliminates any genotoxic stress–induced redistribution. Reciprocally, preventing dephosphorylation of Clp1 TP sites shifts the distribution of the enzyme to the nucleoplasm constitutively. This work advances our understanding of pathways influencing Clp1 localization and may provide insight into mechanisms controlling Cdc14B phosphatases in higher eukaryotes.     

p25rum1 promotes proteolysis of the mitotic B-cyclin p56cdc13 during G1 of the fission yeast cell cycle.

The fission yeast Schizosaccharomyces pombe CDK inhibitor p25rum1 plays a major role in regulating cell cycle progression during G1. Here we show that p25rum1 associates with the CDK p34cdc2/p56cdc13 during G1 in normally cycling cells and is required for the rapid proteolysis of p56cdc13. In vitro binding data indicate that p25rum1 has specificity for the B-cyclin p56cdc13 component of the CDK and can bind the cyclin even in the absence of the cyclin destruction box. At the G1-S-phase transition, p25rum1 levels decrease and p56cd13 levels increase. We also show that on release from a G1 block, the rapid disappearance of p25rum1 requires the activity of the CDK p34cdc2/cig1p and that this same CDK phosphorylates p25rum1 in vitro. We propose that the binding of p25rum1 to p56cdc13 promotes cyclin proteolysis during G1, with p25rum1 possibly acting as an adaptor protein, promoting transfer of p56cdc13 to the proteolytic machinery. At the G1-S-phase transition, p25rum1 becomes targeted for proteolysis by a mechanism which may involve p34cdc2/cig1p phosphorylation. As a consequence, at this point in the cell cycle p56cdc13 proteolysis is inhibited, leading to a rise of p56cdc13 levels in preparation for mitosis.