Inhibition of endocytic vesicle fusion in vitro by the cell-cycle control protein kinase cdc2

Nucleolin is a ubiquitous multifunctional protein involved in preribosome assembly and associated with both nucleolar chromatin in interphase and nucleolar organizer regions on metaphasic chromosomes in mitosis. Extensive nucleolin phosphorylation by a casein kinase (CKII) occurs on serine in growing cells. Here we report that while CKII phosphorylation is achieved in interphase, threonine phosphorylation occurs during mitosis. We provide evidence that this type of in vivo phosphorylation involves a mammalian homolog of the cell cycle control Cdc2 kinase. In vitro M-phase H1 kinase from starfish oocytes phosphorylated threonines in a TPXK motif present nine times in the amino-terminal part of the protein. The same sites which matched the p34cdc2 consensus phosphorylation sequence were used in vivo during mitosis. We propose that successive Cdc2 and CKII phosphorylation could modulate nucleolin function in controlling cell cycle-dependent nucleolar function and organization. Our results, along with previous studies, suggest that while serine phosphorylation is related to nucleolin function in the control of rDNA transcription, threonine phosphorylation is linked to mitotic reorganization of nucleolar chromatin.

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The Xenopus protein kinase pEg2 associates with the centrosome in a cell cycle-dependent manner, binds to the spindle microtubules and is involved in bipolar mitotic spindle assembly

Journal of Cell Science ◽

10.1242/jcs.111.5.557 ◽

1998 ◽

Vol 111 (5) ◽

pp. 557-572 ◽

Cited By ~ 19

Author(s):

C. Roghi ◽

R. Giet ◽

R. Uzbekov ◽

N. Morin ◽

I. Chartrain ◽

...

Keyword(s):

Cell Cycle ◽

Protein Kinase ◽

Mitotic Spindle ◽

Cultured Cells ◽

Dependent Manner ◽

Egg Extracts ◽

Pericentriolar Material ◽

Spindle Microtubules ◽

Cycle Dependent

By differential screening of a Xenopus laevis egg cDNA library, we have isolated a 2,111 bp cDNA which corresponds to a maternal mRNA specifically deadenylated after fertilisation. This cDNA, called Eg2, encodes a 407 amino acid protein kinase. The pEg2 sequence shows significant identity with members of a new protein kinase sub-family which includes Aurora from Drosophila and Ipl1 (increase in ploidy-1) from budding yeast, enzymes involved in centrosome migration and chromosome segregation, respectively. A single 46 kDa polypeptide, which corresponds to the deduced molecular mass of pEg2, is immunodetected in Xenopus oocyte and egg extracts, as well as in lysates of Xenopus XL2 cultured cells. In XL2 cells, pEg2 is immunodetected only in S, G2 and M phases of the cell cycle, where it always localises to the centrosomal region of the cell. In addition, pEg2 ‘invades’ the microtubules at the poles of the mitotic spindle in metaphase and anaphase. Immunoelectron microscopy experiments show that pEg2 is located precisely around the pericentriolar material in prophase and on the spindle microtubules in anaphase. We also demonstrate that pEg2 binds directly to taxol stabilised microtubules in vitro. In addition, we show that the presence of microtubules during mitosis is not necessary for an association between pEg2 and the centrosome. Finally we show that a catalytically inactive pEg2 kinase stops the assembly of bipolar mitotic spindles in Xenopus egg extracts.

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Protein Kinase C Signaling Mediates a Program of Cell Cycle Withdrawal in the Intestinal Epithelium

The Journal of Cell Biology ◽

10.1083/jcb.151.4.763 ◽

2000 ◽

Vol 151 (4) ◽

pp. 763-778 ◽

Cited By ~ 81

Author(s):

Mark R. Frey ◽

Jennifer A. Clark ◽

Olga Leontieva ◽

Joshua M. Uronis ◽

Adrian R. Black ◽

...

Keyword(s):

Cell Cycle ◽

Protein Kinase C ◽

Protein Kinase ◽

Intestinal Epithelium ◽

Molecular Mechanisms ◽

Model Systems ◽

Intestinal Epithelial ◽

Kinase C

Members of the protein kinase C (PKC) family of signal transduction molecules have been widely implicated in regulation of cell growth and differentiation, although the underlying molecular mechanisms involved remain poorly defined. Using combined in vitro and in vivo intestinal epithelial model systems, we demonstrate that PKC signaling can trigger a coordinated program of molecular events leading to cell cycle withdrawal into G0. PKC activation in the IEC-18 intestinal crypt cell line resulted in rapid downregulation of D-type cyclins and differential induction of p21waf1/cip1 and p27kip1, thus targeting all of the major G1/S cyclin-dependent kinase complexes. These events were associated with coordinated alterations in expression and phosphorylation of the pocket proteins p107, pRb, and p130 that drive cells to exit the cell cycle into G0 as indicated by concomitant downregulation of the DNA licensing factor cdc6. Manipulation of PKC isozyme levels in IEC-18 cells demonstrated that PKCα alone can trigger hallmark events of cell cycle withdrawal in intestinal epithelial cells. Notably, analysis of the developmental control of cell cycle regulatory molecules along the crypt–villus axis revealed that PKCα activation is appropriately positioned within intestinal crypts to trigger this program of cell cycle exit–specific events in situ. Together, these data point to PKCα as a key regulator of cell cycle withdrawal in the intestinal epithelium.

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Microtubule-entrained kinase activities associated with the cortical cytoskeleton during cytokinesis

Journal of Cell Science ◽

10.1242/jcs.110.12.1373 ◽

1997 ◽

Vol 110 (12) ◽

pp. 1373-1386 ◽

Cited By ~ 2

Author(s):

G.R. Walker ◽

C.B. Shuster ◽

D.R. Burgess

Keyword(s):

Cell Cycle ◽

Protein Kinase ◽

Protein Phosphorylation ◽

Immunoblot Analysis ◽

Negative Regulator ◽

Mitogen Activated Protein Kinase ◽

Mitotic Apparatus ◽

Cortical Cytoskeleton

Research over the past few years has demonstrated the central role of protein phosphorylation in regulating mitosis and the cell cycle. However, little is known about how the mechanisms regulating the entry into mitosis contribute to the positional and temporal regulation of the actomyosin-based contractile ring formed during cytokinesis. Recent studies implicate p34cdc2 as a negative regulator of myosin II activity, suggesting a link between the mitotic cycle and cytokinesis. In an effort to study the relationship between protein phosphorylation and cytokinesis, we examined the in vivo and in vitro phosphorylation of actin-associated cortical cytoskeletal (CSK) proteins in an isolated model of the sea urchin egg cortex. Examination of cortices derived from eggs or zygotes labeled with 32P-orthophosphate reveals a number of cortex-associated phosphorylated proteins, including polypeptides of 20, 43 and 66 kDa. These three major phosphoproteins are also detected when isolated cortices are incubated with [32P]ATP in vitro, suggesting that the kinases that phosphorylate these substrates are also specifically associated with the cortex. The kinase activities in vivo and in vitro are stimulated by fertilization and display cell cycle-dependent activities. Gel autophosphorylation assays, kinase assays and immunoblot analysis reveal the presence of p34cdc2 as well as members of the mitogen-activated protein kinase family, whose activities in the CSK peak at cell division. Nocodazole, which inhibits microtubule formation and thus blocks cytokinesis, significantly delays the time of peak cortical protein phosphorylation as well as the peak in whole-cell histone H1 kinase activity. These results suggest that a key element regulating cortical contraction during cytokinesis is the timing of protein kinase activities associated with the cortical cytoskeleton that is in turn regulated by the mitotic apparatus.

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Quantitative Characterization of a Mitotic Cyclin Threshold Regulating Exit from Mitosis

Molecular Biology of the Cell ◽

10.1091/mbc.e04-10-0897 ◽

2005 ◽

Vol 16 (5) ◽

pp. 2129-2138 ◽

Cited By ~ 32

Author(s):

Frederick R. Cross ◽

Lea Schroeder ◽

Martin Kruse ◽

Katherine C. Chen

Keyword(s):

Cell Cycle ◽

Budding Yeast ◽

Cell Cycle Control ◽

Predictive Ability ◽

Eukaryotic Cell ◽

Model Predictions ◽

Mitotic Cyclin ◽

Constitutive Overexpression

Regulation of cyclin abundance is central to eukaryotic cell cycle control. Strong overexpression of mitotic cyclins is known to lock the system in mitosis, but the quantitative behavior of the control system as this threshold is approached has only been characterized in the in vitro Xenopus extract system. Here, we quantitate the threshold for mitotic block in budding yeast caused by constitutive overexpression of the mitotic cyclin Clb2. Near this threshold, the system displays marked loss of robustness, in that loss or even heterozygosity for some regulators becomes deleterious or lethal, even though complete loss of these regulators is tolerated at normal cyclin expression levels. Recently, we presented a quantitative kinetic model of the budding yeast cell cycle. Here, we use this model to generate biochemical predictions for Clb2 levels, asynchronous as well as through the cell cycle, as the Clb2 overexpression threshold is approached. The model predictions compare well with biochemical data, even though no data of this type were available during model generation. The loss of robustness of the Clb2 overexpressing system is also predicted by the model. These results provide strong confirmation of the model's predictive ability.

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Cell cycle regulation of the yeast Cdc7 protein kinase by association with the Dbf4 protein.

Molecular and Cellular Biology ◽

10.1128/mcb.13.5.2899 ◽

1993 ◽

Vol 13 (5) ◽

pp. 2899-2908 ◽

Cited By ~ 153

Author(s):

A L Jackson ◽

P M Pahl ◽

K Harrison ◽

J Rosamond ◽

R A Sclafani

Keyword(s):

Cell Cycle ◽

Protein Kinase ◽

Protein Interactions ◽

Kinase Activity ◽

Mitotic Cell ◽

Common Point ◽

Temperature Sensitive ◽

Cdc7 Kinase

Yeast Cdc7 protein kinase and Dbf4 protein are both required for the initiation of DNA replication at the G1/S phase boundary of the mitotic cell cycle. Cdc7 kinase function is stage-specific in the cell cycle, but total Cdc7 protein levels remained unchanged. Therefore, regulation of Cdc7 function appears to be the result of posttranslational modification. In this study, we have attempted to elucidate the mechanism responsible for achieving this specific execution point of Cdc7. Cdc7 kinase activity was shown to be maximal at the G1/S boundary by using either cultures synchronized with alpha factor or Cdc- mutants or with inhibitors of DNA synthesis or mitosis. Therefore, Cdc7 kinase is regulated by a posttranslational mechanism that ensures maximal Cdc7 activity at the G1/S boundary, which is consistent with Cdc7 function in the cell cycle. This cell cycle-dependent regulation could be the result of association with the Dbf4 protein. In this study, the Dbf4 protein was shown to be required for Cdc7 kinase activity in that Cdc7 kinase activity is thermolabile in vitro when extracts prepared from a temperature-sensitive dbf4 mutant grown under permissive conditions are used. In vitro reconstitution assays, in addition to employment of the two-hybrid system for protein-protein interactions, have demonstrated that the Cdc7 and Dbf4 proteins interact both in vitro and in vivo. A suppressor mutation, bob1-1, which can bypass deletion mutations in both cdc7 and dbf4 was isolated. However, the bob1-1 mutation cannot bypass all events in G1 phase because it fails to suppress temperature-sensitive cdc4 or cdc28 mutations. This indicates that the Cdc7 and Dbf4 proteins act at a common point in the cell cycle. Therefore, because of the common point of function for the two proteins and the fact that the Dbf4 protein is essential for Cdc7 function, we propose that Dbf4 may represent a cyclin-like molecule specific for the activation of Cdc7 kinase.

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Ras signalling is required for inactivation of the tumour suppressor pRb cell-cycle control protein

Current Biology ◽

10.1016/s0960-9822(97)70094-0 ◽

1997 ◽

Vol 7 (3) ◽

pp. 219-221 ◽

Cited By ~ 88

Author(s):

Sibylle Mittnacht ◽

Hugh Paterson ◽

Michael F Olson ◽

Christopher J Marshall

Keyword(s):

Cell Cycle ◽

Cell Cycle Control ◽

Tumour Suppressor ◽

Control Protein ◽

Ras Signalling

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Overexpression of Bcl-w in the Testis Disrupts Spermatogenesis: Revelation of a Role of BCL-W in Male Germ Cell Cycle Control

Molecular Endocrinology ◽

10.1210/me.2002-0389 ◽

2003 ◽

Vol 17 (9) ◽

pp. 1868-1879 ◽

Cited By ~ 22

Author(s):

Wei Yan ◽

Jun-Xing Huang ◽

Anna-Stina Lax ◽

Lauri Pelliniemi ◽

Eeva Salminen ◽

...

Keyword(s):

Cell Cycle ◽

Germ Cell ◽

Sertoli Cell ◽

Germ Cells ◽

Cell Cycle Progression ◽

Cell Cycle Control ◽

Testicular Development ◽

Dna Ladder

Abstract To explore physiological roles of BCL-W, a prosurvival member of the BCL-2 protein family, we generated transgenic (TG) mice overexpressing Bcl-w driven by a chicken β-actin promoter. Male Bcl-w TG mice developed normally but were infertile. The adult TG testes displayed disrupted spermatogenesis with various severities ranging from thin seminiferous epithelium containing less germ cells to Sertoli cell-only appearance. No overpopulation of any type of germ cells was observed during testicular development. In contrast, the developing TG testes displayed decreased number of spermatogonia, degeneration, and detachment of spermatocytes and Sertoli cell vacuolization. The proliferative activity of germ cells was significantly reduced during testicular development and spermatogenesis, as determined by in vivo and in vitro 5′-bromo-2′deoxyuridine incorporation assays. Sertoli cells were structurally and functionally normal. The degenerating germ cells were TUNEL-negative and no typical apoptotic DNA ladder was detected. Our data suggest that regulated spatial and temporal expression of BCL-W is required for normal testicular development and spermatogenesis, and overexpression of BCL-W inhibits germ cell cycle entry and/or cell cycle progression leading to disrupted spermatogenesis.

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