Interaction of cyclin-dependent kinase 2 and the Lyn tyrosine kinase in cells treated with 1-beta-D-arabinofuranosylcytosine

The cyclin dependent kinase 2 (Cdk2) is required for initiation and progression of DNA replication. Activation of Cdk2 involves binding to cyclin E or cyclin A and dephosphorylation of Tyr15. The present studies demonstrate that treatment of U-937 cells with 1-beta-D-arabinofuranosylcytosine (ara-C) is associated with tyrosine phosphorylation of Cdk2 and inhibition of Cdk2 activity. The results also demonstrate that Cdk2 directly associates with the Src-like tyrosine kinase Lyn as a consequence of ara-C-treatment. Confocal microscopy studies show that Lyn is detectable in the nucleus and that it colocalises with Cdk2. Subcellular fractionation and coimmunoprecipitation studies further demonstrate nuclear binding of Lyn and Cdk2. We also show that Lyn phosphorylates Tyr15 of Cdk2 and that incubation of Lyn with Cdk2 results in inhibition of Cdk2 activity. These findings suggest that the association of Lyn and Cdk2 in ara-C-treated cells may contribute to regulation of Cdk2-dependent cell cycle checkpoints.     

Mutant PCNA alleles are associated with cdc phenotypes and sensitivity to DNA damage in fission yeast

Twenty-eight site-directed mutations were introduced into the fission yeast gene (pcn1+) that encodes proliferating cell nuclear antigen (PCNA) and their in vivo effects analyzed in a strain with a null pcn1 background. Mutants defective in enhancing processivity of DNA polymerase delta have previously been identified. In this study, we assessed all of the mutants for their sensitivities to temperature, hydroxyurea, UV irradiation and methyl methanesulfonate (MMS), and specific mutants were also tested for sensitivity to gamma irradiation. One cold-sensitive allele, pcn1-3, was characterized in detail. This mutant had a recessive cold-sensitive cdc phenotype and showed sensitivity to hydroxyurea, UV, and gamma irradiation. At the non-permissive temperature pcn1-3 protein was able to form homotrimers in solution and showed increased stimulation of both synthetic activity and processivity of DNA polymerase delta relative to the wild-type Pcn1+ protein. Epistasis analyses indicated that pcn1-3 is defective in the repair pathway involving rad2+ but not defective in the classical nucleotide excision repair pathway involving rad13+. Furthermore, pcn1-3 is either synthetically or conditionally lethal in null checkpoint rad backgrounds and displays a mitotic catastrophe phenotype in these backgrounds. A model for how pcn1-3 defects may affect DNA repair and replication is presented.     

Interaction between the protein kinase B-Raf and the alpha-subunit of the 11S proteasome regulator

Protein kinases of the Raf family act as signal-transducing elements downstream of activated cell surface receptors and are involved in the regulation of proliferation, differentiation, and cell survival. Whereas the role of c-Raf-1 as a mitogen-activated protein/extracellular signal-regulated kinase activator within the mitogenic cascade is well established, less is known about the mammalian Raf isoforms A-Raf and B-Raf. Here we report that B-Raf binds to PA28alpha, one of two subunits of the 11S regulator of proteasomes. PA28alpha was isolated as a B-Raf-binding protein in a yeast two-hybrid screen of a PC12 cDNA library. Both proteins can be coimmunoprecipitated after transient expression in 293 cells. No association could be found between PA28alpha and A-Raf or c-Raf-1. B-Raf binds to a region in PA28alpha that is important for its proteasome-activating function.     

The fission yeast mitotic regulator win1+ encodes an MAP kinase kinase kinase that phosphorylates and activates Wis1 MAP kinase kinase in response to high osmolarity

The Schizosaccharomyces pombe win1-1 mutant has a defect in the G2-M transition of the cell cycle. Although the defect is suppressed by wis1+ and wis4+, which are components of a stress-activated MAP kinase pathway that links stress response and cell cycle control, the molecular identity of Win1 has not been known. We show here that win1+ encodes a polypeptide of 1436 residues with an apparent molecular size of 180 kDa and demonstrate that Win1 is a MAP kinase kinase kinase that phosphorylates and activates Wis1. Despite extensive similarities between Win1 and Wis4, the two MAP kinase kinase kinases have distinct functions. Wis4 is able to compensate for loss of Win1 only under unstressed conditions to maintain basal Wis1 activity, but it fails to suppress the osmosignaling defect conferred by win1 mutations. The win1-1 mutation is a spontaneous duplication of 16 nucleotides, which leads to a frameshift and production of a truncated protein lacking the kinase domain. We discuss the cell cycle phenotype of the win1-1 cdc25-22 wee1-50 mutant and its suppression by wis genes.     

sud1(+) targets cyclin-dependent kinase-phosphorylated Cdc18 and Rum1 proteins for degradation and stops unwanted diploidization in fission yeast

In the fission yeast Schizosaccharomyces pombe, S phase is limited to a single round per cell cycle through cyclin-dependent kinase phosphorylation of critical replication factors, including the Cdc18 replication initiator protein. Because defects in Cdc18 phosphorylation lead to a hyperstable and hyperactive form of Cdc18 that promotes high levels of overreplication in vivo, we wished to identify the components of the Cdc18 proteolysis pathway in fission yeast. In this paper we describe one such component, encoded by the sud1(+) gene. sud1(+) shares homology with the budding yeast CDC4 gene and is required to prevent spontaneous re-replication in fission yeast. Cells lacking sud1(+) accumulate high levels of Cdc18 and the CDK inhibitor Rum1, because they cannot degrade these two key cell cycle regulators. Through genetic analysis we show that hyperaccumulation of Rum1 contributes to re-replication in Deltasud1 cells, but is not the cause of the defect in Cdc18 proteolysis. Rather, Sud1 itself is associated with the ubiquitin pathway in fission yeast and binds to Cdc18 in vivo. Most importantly, Sud1-Cdc18 binding requires prior phosphorylation of the Cdc18 polypeptide at CDK consensus sites. These results provide a biochemical mechanism for the phosphorylation-dependent degradation of Cdc18 and other cell cycle regulators, including Rum1. Evolutionary conservation of the Sud1/CDC4 pathway suggests that phosphorylation-coupled proteolysis may be a general feature of nearly all eukaryotic cell cycles.     

Activation of the yeast SSK2 MAP kinase kinase kinase by the SSK1 two-component response regulator

Exposure of yeast cells to increased extracellular osmolarity induces the HOG1 mitogen-activated protein kinase (MAPK) cascade, which is composed of SSK2, SSK22 and STE11 MAPKKKs, PBS2 MAPKK and HOG1 MAPK. The SSK2/SSK22 MAPKKKs are activated by a 'two-component' osmosensor composed of SLN1, YPD1 and SSK1. The SSK1 C-terminal receiver domain interacts with an N-terminal segment of SSK2. Upon hyperosmotic treatment, SSK2 is autophosphorylated rapidly, and this reaction requires the interaction of SSK1 with SSK2. Autophosphorylation of SSK2 is an intramolecular reaction, suggesting similarity to the mammalian MEKK1 kinase. Dephosphorylation of SSK2 renders the kinase inactive, but it can be re-activated by addition of SSK1 in vitro. A conserved threonine residue (Thr1460) in the activation loop of SSK2 is important for kinase activity. Based on these observations, we propose the following two-step activation mechanism of SSK2 MAPKKK. In the first step, the binding of SSK1 to the SSK1-binding site in the N-terminal domain of SSK2 causes a conformational change in SSK2 and induces its latent kinase activity. In the second step, autophosphorylation of SSK2 renders its activity independent of the presence of SSK1. A similar mechanism might be applicable to other MAPKKKs from both yeast and higher eukaryotes.     

Localization of the 26S proteasome during mitosis and meiosis in fission yeast

The 26S proteasome is a large multisubunit complex involved in degrading both cytoplasmic and nuclear proteins. We have investigated the localization of this complex in the fission yeast, Schizosaccharomyces pombe. Immunofluorescence microscopy shows a striking localization pattern whereby the proteasome is found predominantly at the nuclear periphery, both in interphase and throughout mitosis. Electron microscopic analysis revealed a concentration of label near the inner side of the nuclear envelope. The localization of green fluorescent protein (GFP)-tagged 26S proteasomes was analyzed in live cells during mitosis and meiosis. Throughout mitosis the proteasome remained predominantly at the nuclear periphery. During meiosis the proteasome was found to undergo dramatic changes in its localization. Throughout the first meiotic division, the signal is more dispersed over the nucleus. During meiosis II, there was a dramatic re-localization, and the signal became restricted to the area between the separating DNA until the end of meiosis when the signal dispersed before returning to the nuclear periphery during spore formation. These findings strongly imply that the nuclear periphery is a major site of protein degradation in fission yeast both in interphase and throughout mitosis. Furthermore they raise interesting questions as to the spatial organization of protein degradation during meiosis.     

Regulation of exocytosis by cyclin-dependent kinase 5 via phosphorylation of Munc18

Munc18a, a mammalian neuronal homologue of Saccharomyces cerevisiae Sec1p protein, is essential for secretion, likely as a result of its high affinity interaction with the target SNARE protein syntaxin 1a (where SNARE is derived from SNAP receptor (the soluble N-ethylmaleimide-sensitive fusion protein)). However, this interaction inhibits vesicle SNARE interactions with syntaxin that are required for secretory vesicles to achieve competency for membrane fusion. As such, regulation of the interaction between Munc18a and syntaxin 1a may provide an important mechanism controlling secretory responsiveness. Cyclin-dependent kinase 5 (Cdk5), a member of the Cdc2 family of cell division kinases, co-purifies with Munc18a from rat brain, interacts directly with Munc18a in vitro, and utilizes Munc18a as a substrate for phosphorylation. We have now demonstrated that Cdk5 is capable of phosphorylating Munc18a in vitro within a preformed Munc18a.syntaxin 1a heterodimer complex and that this results in the disassembly of the complex. Using site-directed mutagenesis, the Cdk5 phosphorylation site on Munc18a was identified as Thr574. Stimulation of secretion from neuroendocrine cells produced a corresponding rapid translocation of cytosolic Cdk5 to a particulate fraction and an increase of Cdk5 kinase activity. Inhibition of Cdk5 with olomoucine decreased evoked norepinephrine secretion from chromaffin cells, an effect not observed with the inactive analogue iso-olomoucine. The effects of olomoucine were independent of calcium influx as evidenced by secretory inhibition in permeabilized chromaffin cells and in cells under whole-cell voltage clamp. Furthermore, transfection and expression in chromaffin cells of a neural specific Cdk5 activator, p25, led to a strong increase in nicotinic agonist-induced secretory responses. Our data suggest a model whereby Cdk5 acts to regulate Munc18a interaction with syntaxin 1a and thereby modulates the level of vesicle SNARE interaction with syntaxin 1a and secretory responsiveness.     

Suppressors of cdc25p overexpression identify two pathways that influence the G2/M checkpoint in fission yeast

Checkpoints maintain the order of cell-cycle events. At G2/M, a checkpoint blocks mitosis in response to damaged or unreplicated DNA. There are significant differences in the checkpoint responses to damaged DNA and unreplicated DNA, although many of the same genes are involved in both responses. To identify new genes that function specifically in the DNA replication checkpoint pathway, we searched for high-copy suppressors of overproducer of Cdc25p (OPcdc25(+)), which lacks a DNA replication checkpoint. Two classes of suppressors were isolated. One class includes a new gene encoding a putative DEAD box helicase, suppressor of uncontrolled mitosis (sum3(+)). This gene negatively regulates the cell-cycle response to stress when overexpressed and restores the checkpoint response by a mechanism that is independent of Cdc2p tyrosine phosphorylation. The second class includes chk1(+) and the two Schizosaccharomyces pombe 14-3-3 genes, rad24(+) and rad25(+), which appear to suppress the checkpoint defect by inhibiting Cdc25p. We show that rad24Delta mutants are defective in the checkpoint response to the DNA replication inhibitor hydroxyurea at 37 degrees and that cds1Delta rad24Delta mutants, like cds1Delta chk1Delta mutants, are entirely checkpoint deficient at 29 degrees. These results suggest that chk1(+) and rad24(+) may function redundantly with cds1(+) in the checkpoint response to unreplicated DNA.     

The Mcs4 response regulator coordinately controls the stress-activated Wak1-Wis1-Sty1 MAP kinase pathway and fission yeast cell cycle

The fission yeast Sty1 MAP kinase is required for cell cycle control, initiation of sexual differentiation, and protection against cellular stress. Like the mammalian JNK/SAPK and p38/CSBP1 MAP kinases, Sty1 is activated by a range of environmental insults including osmotic stress, hydrogen peroxide, menadione, heat shock, and the protein synthesis inhibitor anisomycin. We have identified an upstream regulator that mediates activation of the Sty1 MAP kinase by multiple environmental stresses as the product of the mitotic catastrophe suppressor, mcs4. Mcs4 is structurally and functionally homologous to the budding yeast SSK1 response regulator, suggesting that the eukaryotic stress-activated MAP kinase pathway is controlled by a conserved two-component system. Mcs4 acts upstream of Wak1, a homolog of the SSK2 and SSK22 MEK kinases, which transmits the stress signal to the Wis1 MEK. We show that the Wis1 MEK is controlled by an additional pathway that is independent of both Mcs4 and the Wak1 MEK kinase. Furthermore, we demonstrate that Mcs4 is required for the correct timing of mitotic initiation by mechanisms both dependent and independent on Sty1, indicating that Mcs4 coordinately controls cell cycle progression with the cellular response to environmental stress.     

Cell cycle phase specificity of putative cyclin-dependent kinase variants in synchronized alfalfa cells

OBJECTIVE: Impaired beta-adrenergic signal transduction has been proposed as a mechanism contributing to myocardial depression after cardiac surgery. This study determined the changes in the beta-adrenergic system in a model of postoperative myocardial dysfunction induced by myocardial ischaemia and reperfusion under cardiopulmonary bypass (CPB). Those changes were then related to contractility and responsiveness to beta-adrenergic stimulation. METHODS: Four groups of dog hearts were studied: 7 hearts harvested immediately after anaesthesia induction (control group representing the preoperative cardiac condition); 6 hearts harvested after three hours of chest opening by sternotomy (open chest group serving as control for the effects of anaesthesia and surgery); 7 hearts harvested during CPB after 30 minutes of global ischaemia (ischaemia group); and 10 hearts from dogs submitted to one hour of CPB involving 30 minutes of global cardiac ischaemia, harvested 30 minutes after CPB (ischaemia-reperfusion group). Myocardial membranes were prepared to assess: (1) beta-adrenergic receptor density using the radioligand [125I]iodocyanopindolol; (2) GTP-sensitive adenylate cyclase activity and its regulation by isoprenaline and forskolin; (3) G protein levels, using an immunoblotting technique. Ventricular trabeculae or papillary muscles served to assess contractility and responsiveness to isoprenaline. RESULTS: The control and open chest groups had comparable beta-adrenergic receptor density, adenylate cyclase activity and cardiac contractility. In the ischaemia group, the left ventricular membranes had a 55% decrease in receptor density as compared to the controls (P < 0.005), similar GTP-sensitive adenylate cyclase activity and significantly lower adenylate cyclase responses to stimulation with isoprenaline and forskolin. In the ischaemia-reperfusion group, a 144% increase in the left ventricular receptor density was found as compared to the controls (P < 0.005), with a 70% increase in GTP-sensitive adenylate cyclase activity (P < 0.05), a similar adenylate cyclase response to isoprenaline and a 61% increase in response to forskolin (P < 0.005). As compared to the controls, the ischaemia and ischaemia-reperfusion groups had comparable Gs alpha levels, but markedly decreased Gi alpha-2 and Gi alpha-3 levels. The baseline tension of the isolated muscles in the ischaemia and ischaemia-reperfusion groups was comparable, but was 61% and 47% lower than the controls, respectively (P < 0.05). The maximal isoprenaline stimulated tension in the ischaemia and ischaemia-reperfusion groups was 66% and 36% lower than the controls, respectively (P < 0.05 between all groups). CONCLUSIONS: The beta-adrenergic system is severely depressed during global cardiac ischaemia under CPB, but recovers to supranormal values after CPB. However the increased cAMP generation by myocardial membranes after CPB is associated with decreased tension generation by corresponding cardiac muscles. Thus decreased contractility after CPB may be better explained by cellular alterations distal to cAMP generation rather than by changes in the beta-adrenergic system.     

The Win1 mitotic regulator is a component of the fission yeast stress-activated Sty1 MAPK pathway

Women's anger experience has been poorly understood and insufficiently researched. Yet the emotion of anger is vitally important to women's physical and mental health, and to the quality of their relationships. This phenomenological study was undertaken as an expansion and extension of the Women's Anger Study, the first large survey of the genesis, manifestations and correlates of anger in American women. Although the earlier study contributed to understanding of anger, a deeper examination of the context and meanings of anger experience was sought. Twenty-nine Caucasian women ranging in age from 21 to 66 years were interviewed. Illustrative occupations ranged from homemaker, student, waitress to business executive, professor, and human service professional. Analysis involved thematizing by the researchers independently and within a multidisciplinary phenomenological research group. The thematic structure of women's anger involved a building over time of a confusing mixture of feelings (hurt, frustration, disillusionment) precipitated by a violation of the core values of the self. The precipitant of anger was unfair and/or disrespectful treatment or lack of reciprocity in relationships. When the anger was confined within self, the woman felt helpless and powerless. However, powerlessness was also evident when anger was externalized in an outburst. To the study participants, an angry outburst meant a loss of control rather than ability to achieve control. Women reported a sense of power when using anger to restore justice, respect, and relationship reciprocity. Clinicians can assist women to reflect on their core values and use the power of their anger effectively. Further studies are in progress to examine the relevance of these findings for women of other races and cultural contexts.     

Purification of Hsk1, a minichromosome maintenance protein kinase from fission yeast

Members of the Cdc7 family of protein kinases are essential for the initiation of DNA replication in all eukaryotes, but their precise biochemical function is unclear. We have purified the fission yeast Cdc7 homologue Hsk1 approximately 30,000-fold, to near homogeneity. Purified Hsk1 has protein kinase activity on several substrates and is capable of autophosphorylation. Point mutations in highly conserved regions of Hsk1 inactivate the kinase in vitro and in vivo. Overproduction of two of the mutant hsk1 alleles blocks initiation of DNA replication and deranges the mitotic checkpoint, a phenotype consistent with a role for Hsk1 in the early stages of initiation. The purified Hsk1 kinase can be separated into two active forms, a Hsk1 monomer and a heterodimer consisting of Hsk1 complexed with a co-purifying polypeptide, Dfp1. Association with Dfp1 stimulates phosphorylation of exogenous substrates but has little effect on autokinase activity. We have identified Dfp1 as the fission yeast homologue of budding yeast Dbf4. Purified Hsk1 phosphorylates the Cdc19 (Mcm2) subunit of the six-member minichromosome maintenance protein complex purified from fission yeast. Since minichromosome maintenance proteins have been implicated in the initiation of DNA replication, the essential function of Hsk1 at the G1/S transition may be mediated by phosphorylation of Cdc19. Furthermore, the phosphorylation of critical substrates by Hsk1 kinase is likely regulated by association with a Dbf4-like co-factor.     

Role of polo kinase and Mid1p in determining the site of cell division in fission yeast

The fission yeast Schizosaccharomyces pombe divides symmetrically using a medial F-actin- based contractile ring to produce equal-sized daughter cells. Mutants defective in two previously described genes, mid1 and pom1, frequently divide asymmetrically. Here we present the identification of three new temperature-sensitive mutants defective in localization of the division plane. All three mutants have mutations in the polo kinase gene, plo1, and show defects very similar to those of mid1 mutants in both the placement and organization of the medial ring. In both cases, ring formation is frequently initiated near the cell poles, indicating that Mid1p and Plo1p function in recruiting medial ring components to the cell center. It has been reported previously that during mitosis Mid1p becomes hyperphosphorylated and relocates from the nucleus to a medial ring. Here we show that Mid1p first forms a diffuse cortical band during spindle formation and then coalesces into a ring before anaphase. Plo1p is required for Mid1p to exit the nucleus and form a ring, and Pom1p is required for proper placement of the Mid1p ring. Upon overexpression of Plo1p, Mid1p exits the nucleus prematurely and displays a reduced mobility on gels similar to that of the hyperphosphorylated form observed previously in mitotic cells. Genetic and two-hybrid analyses suggest that Plo1p and Mid1p act in a common pathway distinct from that involving Pom1p. Plo1p localizes to the spindle pole bodies and spindles of mitotic cells and also to the medial ring at the time of its formation. Taken together, the data indicate that Plo1p plays a role in the positioning of division sites by regulating Mid1p. Given its previously known functions in mitosis and the timing of cytokinesis, Plo1p is thus implicated as a key molecule in the spatial and temporal coordination of cytokinesis with mitosis.     

Fission yeast cdc21, a member of the MCM protein family, is required for onset of S phase and is located in the nucleus throughout the cell cycle

The fission yeast cdc21 protein belongs to the MCM family, implicated in the once per cell cycle regulation of chromosome replication. In budding yeast, proteins in this family are eliminated from the nucleus during S phase, which has led to the suggestion that they may serve to distinguish unreplicated from replicated DNA, as in the licensing factor model. We show here that, in contrast to the situation in budding yeast, cdc21 remains in the nucleus after S phase, as is found for related proteins in mammalian cells. We suggest that regulation of nuclear import of these proteins may not be an essential aspect of their function in chromosome replication. To determine the function of cdc21+, we have analysed the phenotype of a gene deletion. cdc21+ is required for entry into S phase and, unexpectedly, a proportion of cells depleted of the gene product are able to enter mitosis in the absence of DNA replication. These results are consistent with the view that individual proteins in the MCM family are required for all initiation events, and defective initiation may impair the coordination between mitosis and S phase.     

Correlation of p34cdc2 cyclin-dependent kinase overexpression, CD44s downregulation, and HER-2/neu oncogene amplification with recurrence in prostatic adenocarcinomas

PURPOSE: To test whether p34cdc2 overexpression, CD44s downregulation, and HER-2/neu amplification correlate with disease recurrence after radical prostatectomy, and to evaluate a possible biologic association between p34cdc2 and HER-2/neu expression. MATERIALS AND METHODS: Immunohistochemical (IHC) detection of both p34cdc2 cyclin-dependent kinase (CDK) and CD44s expression and fluorescence in situ hybridization (FISH)-based analysis of HER-2/neu gene status were performed on formalin-fixed, paraffin-embedded sections of 106 prostatic adenocarcinomas (PACs). Findings were correlated with Gleason grade, pathologic stage, DNA ploidy, and postsurgical biochemical disease recurrence. RESULTS: CDK overexpression correlated with tumor grade (P = .001), DNA ploidy (P = .001), pathologic stage (P = .04), and disease recurrence (P = .01). CD44s downregulation correlated with grade (P = .03), ploidy (P = .01), and recurrence (P = .02). HER-2/neu amplification correlated with grade (P = .001), ploidy (P = .001), and recurrence (P = .01). On multivariate analysis, CDK overexpression independently predicted recurrence (P = .001) after prostatectomy. CDK expression correlated with HER-2/neu status with 32 of 65 (49%) tumors that overexpressed CDK and showed concomitant HER-2/neu amplification (P = .04). CONCLUSION: This study showed that p34cdc2, CD44s, and HER-2/neu are variably expressed or amplified in prostatic carcinoma and that such alteration may affect tumor behavior. In addition, CDK overexpression and HER-2/neu amplification may be biologically related.