Spy1, a Histidine-Containing Phosphotransfer Signaling Protein, Regulates the Fission Yeast Cell Cycle through the Mcs4 Response Regulator

ABSTRACT Common histidine-to-aspartate (His-to-Asp) phosphorelay signaling systems involve three types of signaling components: a sensor His kinase, a response regulator, and a histidine-containing phosphotransfer (HPt) protein. In the fission yeastSchizosaccharomyces pombe, two response regulators, Mcs4 and Prr1, have been identified recently, and it was shown that they are involved in the signal transduction implicated in stress responses. Furthermore, Mcs4 appears to be involved in mitotic cell-cycle control. However, neither the HPt phosphotransmitter nor His kinase has been characterized in S. pombe. In this study, we identified a gene encoding an HPt phosphotransmitter, named Spy1 (S. pombe YPD1-like protein). The spy1+ gene showed an ability to complement a mutational lesion of theSaccharomyces cerevisiae YPD1 gene, which is involved in an osmosensing signal transduction. The result from yeast two-hybrid analysis indicated that Spy1 interacts with Mcs4. To gain insight into the function of Spy1, a series of genetic analyses were conducted. The results provided evidence that Spy1, together with Mcs4, plays a role in regulation of the G2/M cell cycle progression. Spy1-deficient cells appear to be precocious in the entry to M phase. In the proposed model, Spy1 modulates Mcs4 in a negative manner, presumably through a direct His-to-Asp phosphorelay, operating upstream of the Sty1 mitogen-activated protein kinase cascade.

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Cell Cycle Arrest and Reversion of Ras-Induced Transformation by a Conditionally Activated Form of Mitogen-Activated Protein Kinase Kinase Kinase 3

Molecular and Cellular Biology ◽

10.1128/mcb.19.5.3857 ◽

1999 ◽

Vol 19 (5) ◽

pp. 3857-3868 ◽

Cited By ~ 57

Author(s):

Heidrun Ellinger-Ziegelbauer ◽

Kathleen Kelly ◽

Ulrich Siebenlist

Keyword(s):

Cell Cycle ◽

Protein Kinase ◽

Cyclin D1 ◽

Stress Responses ◽

Cell Cycle Progression ◽

Cellular Transformation ◽

Mitogen Activated Protein Kinase ◽

Cycle Progression ◽

Mapk Kinase ◽

Mitogen Activated Protein

ABSTRACT Signal-induced proliferation, differentiation, or stress responses of cells depend on mitogen-activated protein kinase (MAPK) cascades, the core modules of which consist of members of three successively acting kinase families (MAPK kinase kinase [MAP3K], MAPK kinase, and MAPK). It is demonstrated here that the MEKK3 kinase inhibits cell proliferation, a biologic response not commonly associated with members of the MAP3K family of kinases. A conditionally activated form of MEKK3 stably expressed in fibroblasts arrests these cells in early G1. MEKK3 critically blocks mitogen-driven expression of cyclin D1, a cyclin which is essential for progression of fibroblasts through G1. The MEKK3-induced block of cyclin D1 expression and of cell cycle progression may be mediated via p38 MAPK, a downstream effector of MEKK3. The MEKK3-mediated block of proliferation also reverses Ras-induced cellular transformation, suggesting possible tumor-suppressing functions for this kinase. Together, these results suggest an involvement of the MEKK3 kinase in negative regulation of cell cycle progression, and they provide the first insights into biologic activities of this kinase.

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Cdk1-Dependent Phosphorylation ofNPM Overrides G2/M Checkpoint and Increases Leukemic Blasts in Mice

Blood ◽

10.1182/blood.v112.11.1322.1322 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1322-1322

Author(s):

Wei Du ◽

Yun Zhou ◽

Suzette Pike ◽

Qishen Pang

Keyword(s):

Cell Cycle ◽

Cell Cycle Progression ◽

Cell Cycle Control ◽

Xenograft Model ◽

Phosphorylation Sites ◽

M Cell ◽

Cycle Progression ◽

Hematopoietic Stem ◽

Cycle Arrest ◽

Regulation Of Cell Cycle

Abstract An elevated level of nucleophosmin (NPM) is often found in actively proliferative cells including human tumors. To identify the regulatory role for NPM phosphorylation in proliferation and cell cycle control, a series of mutants targeting the consensus cyclin-dependent kinase (CKD) phosphorylation sites was created to mimic or abrogate either single-site or multi-site phosphorylation. Cells expressing the phosphomimetic NPM mutants showed enhanced proliferation and G2/M cell-cycle transition; whereas nonphosphorylatable mutants induced G2/M cell-cycle arrest. Simultaneous inactivation of two CKD phosphorylation sites at Ser10 and Ser70 (S10A/S70A, NPM-AA) induced phosphorylation of Cdk1 at Tyr15 (Cdc2Tyr15) and increased cytoplasmic accumulation of Cdc25C. Strikingly, stress-induced Cdk1Tyr15 and Cdc25C sequestration were completely suppressed by expression of a double phosphomimetic NPM mutant (S10E/S70E, NPM-EE). Further analysis revealed that phosphorylation of NPM at both Ser10 and Ser70 sites were required for proper interaction between Cdk1 and Cdc25C in mitotic cells. Moreover, the NPM-EE mutant directly bound to Cdc25C and prevented phosphorylation of Cdc25C at Ser216 during mitosis. Finally, NPM-EE overrided stress-induced G2/M arrest, increased peripheral-blood blasts and splenomegaly in a NOD/SCID xenograft model, and promoted leukemia development in Fanconi mouse hematopoietic stem/progenitor cells. Thus, these findings reveal a novel function of NPM on regulation of cell-cycle progression, in which Cdk1-dependent phosphorylation of NPM controls cell-cycle progression at G2/M transition through modulation of Cdc25C activity.

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C-terminal domain phosphorylation of ERK3 controlled by Cdk1 and Cdc14 regulates its stability in mitosis

Biochemical Journal ◽

10.1042/bj20091604 ◽

2010 ◽

Vol 428 (1) ◽

pp. 103-111 ◽

Cited By ~ 16

Author(s):

Pierre-Luc Tanguay ◽

Geneviève Rodier ◽

Sylvain Meloche

Keyword(s):

Cell Cycle ◽

Cell Cycle Progression ◽

Mitotic Cell ◽

Mitogen Activated Protein Kinase ◽

Phosphorylation Sites ◽

Dependent Manner ◽

Cell Extracts ◽

Cycle Dependent

ERK3 (extracellular-signal-regulated kinase 3) is an atypical MAPK (mitogen-activated protein kinase) that is suggested to play a role in cell-cycle progression and cellular differentiation. However, it is not known whether the function of ERK3 is regulated during the cell cycle. In the present paper, we report that ERK3 is stoichiometrically hyperphosphorylated during entry into mitosis and is dephosphorylated at the M→G1 transition. The phosphorylation of ERK3 is associated with the accumulation of the protein in mitosis. In vitro phosphorylation of a series of ERK3-deletion mutants by mitotic cell extracts revealed that phosphorylation is confined to the unique C-terminal extension of the protein. Using MS analysis, we identified four novel phosphorylation sites, Ser684, Ser688, Thr698 and Ser705, located at the extreme C-terminus of ERK3. All four sites are followed by a proline residue. We have shown that purified cyclin B-Cdk1 (cyclindependent kinase 1) phosphorylates these sites in vitro and demonstrate that Cdk1 acts as a major Thr698 kinase in vivo. Reciprocally, we found that the phosphatases Cdc14A and Cdc14B (Cdc is cell-division cycle) bind to ERK3 and reverse its C-terminal phosphorylation in mitosis. Importantly, alanine substitution of the four C-terminal phosphorylation sites markedly decreased the half-life of ERK3 in mitosis, thereby linking phosphorylation to the stabilization of the kinase. The results of the present study identify a novel regulatory mechanism of ERK3 that operates in a cell-cycle-dependent manner.

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Cyclin C influences the timing of mitosis in fission yeast

Molecular Biology of the Cell ◽

10.1091/mbc.e16-11-0787 ◽

2017 ◽

Vol 28 (13) ◽

pp. 1738-1744 ◽

Cited By ~ 2

Author(s):

Gabor Banyai ◽

Zsolt Szilagyi ◽

Vera Baraznenok ◽

Olga Khorosjutina ◽

Claes M. Gustafsson

Keyword(s):

Cell Cycle ◽

Fission Yeast ◽

Rna Polymerase Ii ◽

Cell Cycle Progression ◽

Cell Cycle Control ◽

Mediator Complex ◽

Cycle Progression ◽

Cyclin C ◽

M Phase

The multiprotein Mediator complex is required for the regulated transcription of nearly all RNA polymerase II–dependent genes. Mediator contains the Cdk8 regulatory subcomplex, which directs periodic transcription and influences cell cycle progression in fission yeast. Here we investigate the role of CycC, the cognate cyclin partner of Cdk8, in cell cycle control. Previous reports suggested that CycC interacts with other cellular Cdks, but a fusion of CycC to Cdk8 reported here did not cause any obvious cell cycle phenotypes. We find that Cdk8 and CycC interactions are stabilized within the Mediator complex and the activity of Cdk8-CycC is regulated by other Mediator components. Analysis of a mutant yeast strain reveals that CycC, together with Cdk8, primarily affects M-phase progression but mutations that release Cdk8 from CycC control also affect timing of entry into S phase.

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An extensive program of periodic alternative splicing linked to cell cycle progression

eLife ◽

10.7554/elife.10288 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 39

Author(s):

Daniel Dominguez ◽

Yi-Hsuan Tsai ◽

Robert Weatheritt ◽

Yang Wang ◽

Benjamin J Blencowe ◽

...

Keyword(s):

Cell Cycle ◽

Alternative Splicing ◽

Protein Interactions ◽

Cell Cycle Progression ◽

Cell Cycle Control ◽

Mitotic Cell ◽

Protein Protein Interactions ◽

Post Translational Modification ◽

Sr Protein ◽

Cycle Dependent

Progression through the mitotic cell cycle requires periodic regulation of gene function at the levels of transcription, translation, protein-protein interactions, post-translational modification and degradation. However, the role of alternative splicing (AS) in the temporal control of cell cycle is not well understood. By sequencing the human transcriptome through two continuous cell cycles, we identify ~1300 genes with cell cycle-dependent AS changes. These genes are significantly enriched in functions linked to cell cycle control, yet they do not significantly overlap genes subject to periodic changes in steady-state transcript levels. Many of the periodically spliced genes are controlled by the SR protein kinase CLK1, whose level undergoes cell cycle-dependent fluctuations via an auto-inhibitory circuit. Disruption of CLK1 causes pleiotropic cell cycle defects and loss of proliferation, whereas CLK1 over-expression is associated with various cancers. These results thus reveal a large program of CLK1-regulated periodic AS intimately associated with cell cycle control.

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The Saccharomyces SHP1 gene, which encodes a regulator of phosphoprotein phosphatase 1 with differential effects on glycogen metabolism, meiotic differentiation, and mitotic cell cycle progression.

Molecular and Cellular Biology ◽

10.1128/mcb.15.4.2037 ◽

1995 ◽

Vol 15 (4) ◽

pp. 2037-2050 ◽

Cited By ~ 45

Author(s):

S Zhang ◽

S Guha ◽

F C Volkert

Keyword(s):

Cell Cycle ◽

Cell Cycle Progression ◽

State Level ◽

Glycogen Metabolism ◽

Mitotic Cell ◽

M Cell ◽

Cycle Progression ◽

Glycogen Accumulation ◽

Sequencing Project ◽

Phosphoprotein Phosphatase

The phosphoprotein phosphatase 1 (PP1) catalytic subunit encoded by the Saccharomyces GLC7 gene is involved in control of glycogen metabolism, meiosis, translation, chromosome segregation, cell polarity, and G2/M cell cycle progression. It is also lethal when overproduced. We have isolated strains which are resistant to Glc7p overproduction lethality as a result of mutations in the SHP1 (suppressor of high-copy PP1) gene, which was previously encountered in a genomic sequencing project as an open reading frame whose interruption totally blocked sporulation and slightly slowed cell proliferation. These phenotypes also characterized our shp1 mutations, as did deficient glycogen accumulation. Lysates from the shp1 mutants were deficient in PP1 catalytic activity but exhibited no obvious abnormalities in the steady-state level or subcellular localization pattern of a catalytically active Glc7p-hemagglutinin fusion polypeptide. The lower level of PP1 activity in shp1 cells permitted substitution of a galactose-induced GAL10-GLC7 fusion for GLC7; depletion of Glc7p from these cells by growth in glucose medium resulted in G2/M arrest as previously observed for a glc7cs allele but with depletion arrest occurring most frequently at a later stage of mitosis. The higher requirement of glycogen accumulation and sporulation for PP1 activity would permit their regulation via Glc7p activity, independent of its requirement for mitosis.

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Identification of sumoylation targets, combined with inactivation ofSMT3, reveals the impact of sumoylation upon growth, morphology, and stress resistance in the pathogenCandida albicans

Molecular Biology of the Cell ◽

10.1091/mbc.e10-07-0632 ◽

2011 ◽

Vol 22 (5) ◽

pp. 687-702 ◽

Cited By ~ 37

Author(s):

Michelle D. Leach ◽

David A. Stead ◽

Evelyn Argo ◽

Alistair J.P. Brown

Keyword(s):

Cell Cycle ◽

Stress Responses ◽

Cell Cycle Progression ◽

Cell Cycle Control ◽

Nucleocytoplasmic Transport ◽

Environmental Adaptation ◽

Covalent Attachment ◽

Cell Integrity ◽

Growth Morphology ◽

The Impact

Posttranslational modifications of proteins play critical roles in the control of cellular differentiation, development, and environmental adaptation. In particular, the covalent attachment of the small ubiquitin-like modifier, SUMO, to target proteins (sumoylation) regulates cell cycle progression, transcription, nucleocytoplasmic transport, and stress responses. Here we combine proteomic, molecular, and cellular approaches to examine the roles of sumoylation in the major fungal pathogen of humans, Candida albicans. Using an N-terminally FLAG-tagged SUMO, 31 sumoylated proteins were identified in C. albicans with roles in stress responses (e.g., Hsp60, Hsp70 family members, Hsp104), the cytoskeleton and polarized growth (e.g., Tub1, Cct7, Mlc1), secretion, and endocytosis (e.g., Lsp1, Sec24, Sec7). The output from this proteomic screen was entirely consistent with the phenotypes of C. albicans mutants in which the single SUMO-encoding locus (SMT3) was inactivated or down-regulated. C. albicans smt3/smt3 cells displayed defects in growth, morphology, cell separation, nuclear segregation, and chitin deposition, suggesting important roles for sumoylation in cell cycle control. Smt3/smt3 cells also displayed sensitivity to thermal, oxidative, and cell wall stresses as well as to the antifungal drug caspofungin. Mutation of consensus sumoylation sites in Hsp60 and Hsp104 affected the resistance of C. albicans to thermal stress. Furthermore, signaling via the cell integrity pathway was defective in C. albicans smt3/smt3 cells. These observations provide mechanistic explanations for many of the observed phenotypic effects of Smt3 inactivation upon C. albicans growth and environmental adaptation. Clearly sumoylation plays key roles in fundamental cellular processes that underpin the pathogenicity of this medically important fungus.

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GRASP55 Regulates Golgi Ribbon Formation

Molecular Biology of the Cell ◽

10.1091/mbc.e07-11-1200 ◽

2008 ◽

Vol 19 (7) ◽

pp. 2696-2707 ◽

Cited By ~ 101

Author(s):

Timothy N. Feinstein ◽

Adam D. Linstedt

Keyword(s):

Cell Cycle ◽

Golgi Apparatus ◽

Cell Cycle Progression ◽

Control Point ◽

Gene Replacement ◽

Mitogen Activated Protein Kinase ◽

M Cell ◽

Cycle Progression ◽

G2 Phase ◽

Golgi Ribbon

Recent work indicates that mitogen-activated protein kinase kinase (MEK)1 signaling at the G2/M cell cycle transition unlinks the contiguous mammalian Golgi apparatus and that this regulates cell cycle progression. Here, we sought to determine the role in this pathway of Golgi reassembly protein (GRASP)55, a Golgi-localized target of MEK/extracellular signal-regulated kinase (ERK) phosphorylation at mitosis. In support of the hypothesis that GRASP55 is inhibited in late G2 phase, causing unlinking of the Golgi ribbon, we found that HeLa cells depleted of GRASP55 show a fragmented Golgi similar to control cells arrested in G2 phase. In the absence of GRASP55, Golgi stack length is shortened but Golgi stacking, compartmentalization, and transport seem normal. Absence of GRASP55 was also sufficient to suppress the requirement for MEK1 in the G2/M transition, a requirement that we previously found depends on an intact Golgi ribbon. Furthermore, mimicking mitotic phosphorylation of GRASP55 by using aspartic acid substitutions is sufficient to unlink the Golgi apparatus in a gene replacement assay. Our results implicate MEK1/ERK regulation of GRASP55-mediated Golgi linking as a control point in cell cycle progression.

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Advantages of Tyrosine Kinase Anti-Angiogenic Cediranib over Bevacizumab: Cell Cycle Abrogation and Synergy with Chemotherapy

Pharmaceuticals ◽

10.3390/ph14070682 ◽

2021 ◽

Vol 14 (7) ◽

pp. 682

Author(s):

Jianling Bi ◽

Garima Dixit ◽

Yuping Zhang ◽

Eric J. Devor ◽

Haley A. Losh ◽

...

Keyword(s):

Cell Cycle ◽

Endometrial Cancer ◽

Tyrosine Kinase ◽

Cell Viability ◽

Cell Lines ◽

Cell Cycle Progression ◽

Kinase Inhibitor ◽

Cell Cycle Checkpoint ◽

Mitotic Catastrophe ◽

M Cell

Angiogenesis plays a crucial role in tumor development and metastasis. Both bevacizumab and cediranib have demonstrated activity as single anti-angiogenic agents in endometrial cancer, though subsequent studies of bevacizumab combined with chemotherapy failed to improve outcomes compared to chemotherapy alone. Our objective was to compare the efficacy of cediranib and bevacizumab in endometrial cancer models. The cellular effects of bevacizumab and cediranib were examined in endometrial cancer cell lines using extracellular signal-related kinase (ERK) phosphorylation, ligand shedding, cell viability, and cell cycle progression as readouts. Cellular viability was also tested in eight patient-derived organoid models of endometrial cancer. Finally, we performed a phosphoproteomic array of 875 phosphoproteins to define the signaling changes related to bevacizumab versus cediranib. Cediranib but not bevacizumab blocked ligand-mediated ERK activation in endometrial cancer cells. In both cell lines and patient-derived organoids, neither bevacizumab nor cediranib alone had a notable effect on cell viability. Cediranib but not bevacizumab promoted marked cell death when combined with chemotherapy. Cell cycle analysis demonstrated an accumulation in mitosis after treatment with cediranib + chemotherapy, consistent with the abrogation of the G2/M checkpoint and subsequent mitotic catastrophe. Molecular analysis of key controllers of the G2/M cell cycle checkpoint confirmed its abrogation. Phosphoproteomic analysis revealed that bevacizumab and cediranib had both similar and unique effects on cell signaling that underlie their shared versus individual actions as anti-angiogenic agents. An anti-angiogenic tyrosine kinase inhibitor such as cediranib has the potential to be superior to bevacizumab in combination with chemotherapy.

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