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E A Nigg

Publications and source records attributed to E A Nigg.

At least 37 records · Page 2Linked to original sources
PubMed1998

C-Nap1, a novel centrosomal coiled-coil protein and candidate substrate of the cell cycle-regulated protein kinase Nek2.

Nek2 (for NIMA-related kinase 2) is a mammalian cell cycle-regulated kinase structurally related to the mitotic regulator NIMA of Aspergillus nidulans. In human cells, Nek2 associates with centrosomes, and overexpression of active Nek2 has drastic consequences for centrosome structure. Here, we describe the molecular characterization of a novel human centrosomal protein, C-Nap1 (for centrosomal Nek2-associated protein 1), first identified as a Nek2-interacting protein in a yeast two-hybrid screen. Antibodies raised against recombinant C-Nap1 produced strong labeling of centrosomes by immunofluorescence, and immunoelectron microscopy revealed that C-Nap1 is associated specifically with the proximal ends of both mother and daughter centrioles. On Western blots, anti-C-Nap1 antibodies recognized a large protein (>250 kD) that was highly enriched in centrosome preparations. Sequencing of overlapping cDNAs showed that C-Nap1 has a calculated molecular mass of 281 kD and comprises extended domains of predicted coiled-coil structure. Whereas C-Nap1 was concentrated at centrosomes in all interphase cells, immunoreactivity at mitotic spindle poles was strongly diminished. Finally, the COOH-terminal domain of C-Nap1 could readily be phosphorylated by Nek2 in vitro, as well as after coexpression of the two proteins in vivo. Based on these findings, we propose a model implicating both Nek2 and C-Nap1 in the regulation of centriole-centriole cohesion during the cell cycle.

Amino Acid Sequence↗
PubMed1998

The polo-like kinase Plx1 is required for M phase exit and destruction of mitotic regulators in Xenopus egg extracts.

Polo-like kinases (Plks), named after the Drosophila gene product polo, have been implicated in the regulation of multiple aspects of mitotic progression, including the activation of the Cdc25 phosphatase, bipolar spindle formation and cytokinesis. Genetic analyses performed in yeast and Drosophila suggest a function for Plks at late stages of mitosis, but biochemical data to support such a function in vertebrate organisms are lacking. Here we have taken advantage of Xenopus egg extracts for exploring the function of Plx1, a Xenopus Plk, during the cell cycle transition from M phase to interphase (I phase). We found that the addition of a catalytically inactive Plx1 mutant to M phase-arrested egg extracts blocked their Ca2+-induced release into interphase. Concomitantly, the proteolytic destruction of several targets of the anaphase-promoting complex and the inactivation of the Cdc2 protein kinase (Cdk1) were prevented. Moreover, the M to I phase transition could be abolished by immunodepletion of Plx1, but was restored upon the addition of recombinant Plx1. These results demonstrate that the exit of egg extracts from M phase arrest requires active Plx1, and they strongly suggest an important role for Plx1 in the activation of the proteolytic machinery that controls the exit from mitosis.

Anaphase-Promoting Complex-Cyclosome↗
PubMed1998

A centrosomal function for the human Nek2 protein kinase, a member of the NIMA family of cell cycle regulators.

Nek2, a mammalian protein kinase of unknown function, is closely related to the mitotic regulator NIMA of Aspergillus nidulans. Here we show by both immunofluorescence microscopy and biochemical fractionation that human Nek2 localizes to the centrosome. Centrosome association occurs throughout the cell cycle, including all stages of mitosis, and is independent of microtubules. Overexpression of active Nek2 induces a striking splitting of centrosomes, whereas prolonged expression of either active or inactive Nek2 leads to dispersal of centrosomal material and loss of a focused microtubule-nucleating activity. Surprisingly, this does not prevent entry into mitosis, as judged by the accumulation of mitotically arrested cells induced by co-expression of a non-destructible B-type cyclin. These results bear on the dynamic function of centrosomes at the onset of mitosis. Moreover, they indicate that one function of mammalian Nek2 relates to the centrosome cycle and thus provide a new perspective on the role of NIMA-related kinases.

Amino Acid Sequence↗
PubMed1998

Rigor-type mutation in the kinesin-related protein HsEg5 changes its subcellular localization and induces microtubule bundling.

HsEg5 is a human kinesin-related motor protein essential for the formation of a bipolar mitotic spindle. It interacts with the mitotic centrosomes in a phosphorylation-dependent manner. To investigate further the mechanisms involved in targetting HsEg5 to the spindle apparatus, we expressed various mutants of HsEg5 in HeLa cells. All these mutants share a mutation of Thr-112 in the N-terminal motor domain, resulting in the inactivation of the ATP binding domain. In vitro, the HsEg5-T112N mutant motor domain showed a nucleotide-independent microtubule association, typical of a kinesin protein binding to microtubules in a rigor state. In vivo, overexpression of the HsEg5 rigor mutant in HeLa cells induced, in interphase, microtubule bundling, and, in mitosis, the formation of monopolar mitotic spindles similar to those observed after microinjection of anti-HsEg5 antibodies. Localization of the HsEg5 rigor mutant on cytoplasmic microtubules did not require the C-terminal tail domain but was lost when the stalk domain was also deleted. Sucrose gradient centrifugation experiments showed that microtubule bundling was most likely caused by the binding of HsEg5 mutants in a dimeric state. These results demonstrate that the precise subcellular localization of HsEg5 in vivo is regulated not only by the phosphorylation of the tail domain but also by the oligomeric state of the protein.

Adenosine Triphosphate↗
PubMed1998

GFP tagging reveals human Polo-like kinase 1 at the kinetochore/centromere region of mitotic chromosomes.

Polo-like kinases (Plks) have been implicated in various aspects of M-phase progression in organisms ranging from yeast to man. In vertebrates, Plks participate in centrosome maturation and spindle assembly, as well as the activation of the Cdk1/cyclin B complex. Moreover, Plks are required for the destruction of mitotic cyclins, indicating that they play an important role in the regulation of the ubiquitin-dependent proteolytic degradation machinery that controls exit from M-phase. Here, we have fused Green Fluorescent Protein (GFP) to the N-terminus of human Plk1, and expressed this chimeric construct in human cells. We found that GFP-Plk1 associates with centrosomes, the equatorial spindle midzone and the postmitotic bridge of dividing cells, confirming and extending previous results obtained with conventional immunofluorescence microscopy. In addition, however, we observed fluorescence emanating from the midbody between dividing cells, and from discrete dots associated with mitotic chromosomes. This latter staining pattern being reminiscent of centromeres, we performed double-labeling experiments with antibodies against the centromeric marker CENP-B, and reexamined the subcellular localization of endogenous Plk1 using different fixation procedures. Our data clearly show that both GFP-tagged Plk1 and endogenous Plk1 associate with the kinetochore/centromere region of human mitotic chromosomes. This novel localization of Plk1 suggests that substrates and/or regulators of Plks may be found among kinetochore-associated proteins with important functions in chromosome segregation and/or spindle checkpoint mechanisms.

Cell Cycle Proteins↗
PubMed1998

Polo-like kinases: positive regulators of cell division from start to finish.

Present in organisms ranging from yeast to man, homologues of the Drosophila Polo kinase control multiple stages of cell division. At the onset of mitosis, Polo-like kinases (Plks) function in centrosome maturation and bipolar spindle formation, and they contribute to the activation of cyclin-dependent kinase (Cdk)1-cyclin B. Subsequently, they are required for the inactivation of Cdk1 and exit from mitosis. In the absence of Plk function, mitotic cyclins fail to be destroyed, indicating that Plks are important regulators of the anaphase-promoting complex/cyclosome (APC/C), a key component of the ubiquitin-dependent proteolytic degradation pathway. Finally, recent evidence implicates Plks in the temporal and spatial coordination of cytokinesis.

Animals↗
PubMed1997

The in vivo expression pattern of mouse Nek2, a NIMA-related kinase, indicates a role in both mitosis and meiosis.

The human protein kinase Nek2 is related to the NIMA cell cycle regulatory kinase of Aspergillus nidulans. Whereas NIMA has been shown to be essential for cell cycle progression into mitosis in this fungus, the function of mammalian Nek2 remains to be elucidated. In this study, we isolated a cDNA coding for a mouse ortholog of human Nek2 and analyzed the expression of this kinase in different organs. RNase protection assays performed on RNAs from mouse adult organs showed very high expression of Nek2 in testis. Lower levels of transcripts were detected in intestine, thymus, and skin, three mitotically active organs, and whole-mount in situ hybridization performed on 10.5-day embryos allowed the detection of Nek2 transcripts in the brain. In situ hybridization analysis of testis sections revealed that the transcription of Nek2 occurred in a stage-specific manner during spermatogenesis. The strongest signals were seen in cells undergoing meiosis, but Nek2 transcripts could also be detected in haploid cells (stage I and II spermatids). Extending these results, in situ hybridization performed on ovary sections revealed strong signals in meiotically active oocytes. In addition, some Nek2 transcription was observed in actively dividing follicle cells surrounding the oocytes and in the oviduct. Finally, indirect immunofluorescence staining of testis sections with Nek2-specific antibodies confirmed that this kinase is highly expressed in spermatocytes and, to a lesser extent, in early spermatids. Taken together, these results indicate that Nek2 may play an important role not only during mitosis but also during meiosis.

Amino Acid Sequence↗
PubMed1997

On the regulation and function of human polo-like kinase 1 (PLK1): effects of overexpression on cell cycle progression.

The human protein kinase Plk1, a member of the polo-like kinase family, is known to function at mitosis. Here we show that the relative specific activity of Plk1 increases in mitosis, that Plk1 is specifically phosphorylated during mitosis, and that phosphatase treatment reduces mitotic Plk1 activity to interphase levels. To identify domains involved in the regulation of Plk1 activity, deletion mutants of Plk1 were constructed and their activities examined. Deletion of the extreme C-terminus of Plk1 substantially increased kinase activity, indicating that the C-terminus harbors an inhibitory domain. Finally, the consequences of over-production of wild-type and mutant Plk1 protein were analyzed, using transient transfection assays. Cells overexpressing Plk1 protein were able to enter mitosis and establish an apparently normal bipolar spindle. In contrast, progression through mitosis was transiently delayed, and cytokinesis appeared to be disturbed, as reflected by a significant increase in large cells with multiple, often fragmented nuclei. These results are relevant to recently proposed roles for Plks during both entry into and exit from mitosis.

Cell Cycle↗
PubMed1997

Phosphorylation by p34cdc2 protein kinase regulates binding of the kinesin-related motor HsEg5 to the dynactin subunit p150.

The kinesin-related motor HsEg5 is essential for centrosome separation, and its association with centrosomes appears to be regulated by phosphorylation of tail residue threonine 927 by the p34(cdc2) protein kinase. To identify proteins able to interact with the tail of HsEg5, we performed a yeast two-hybrid screen with a HsEg5 stalk-tail construct as bait. We isolated a cDNA coding for the central, alpha-helical region of human p150(Glued), a prominent component of the dynactin complex. The interaction between HsEg5 and p150(Glued) was enhanced upon activation of p34(CDC28), the budding yeast homolog of p34(cdc2), provided that HsEg5 had a phosphorylatable residue at position 927. Phosphorylation also enhanced the specific binding of p150(Glued) to the tail domain of HsEg5 in vitro, indicating that the two proteins are able to interact directly. Immunofluorescence microscopy revealed co-localization of HsEg5 and p150(Glued) during mitosis but not during interphase, consistent with a cell cycle-dependent association between the two proteins. Taken together, these results suggest that HsEg5 and p150(Glued) may interact in mammalian cells in vivo and that p34(cdc2) may regulate this interaction. Furthermore, they imply that the dynactin complex may functionally interact not only with dynein but also with kinesin-related motors.

Amino Acid Sequence↗
PubMed1997

Nucleocytoplasmic transport: signals, mechanisms and regulation.

In eukaryotic organisms, DNA replication and RNA biogenesis occur in the cell nucleus, whereas protein synthesis occurs in the cytoplasm. Integration of these activities depends on selective transport of proteins and ribonucleoprotein particles between the two compartments. Transport across the nuclear envelope occurs through large multiprotein structures, termed nuclear pore complexes. It is signal-mediated and requires both energy and soluble factors, including shuttling carriers. Here I summarize current understanding of nucleocytoplasmic transport and illustrate the importance of regulated transport for signal transduction.

Amino Acid Sequence↗
PubMed1996

Dynamic changes in nuclear architecture during mitosis: on the role of protein phosphorylation in spindle assembly and chromosome segregation.

During mitosis, the vertebrate cell nucleus undergoes profound changes in architecture. At the onset of mitosis, the nuclear envelope breaks down, the nuclear lamina is depolymerized, and interphase chromatin is condensed to chromosomes. Concomitantly, cytoplasmic microtubules are reorganized into a mitotic spindle apparatus, a highly dynamic structure required for the segregation of sister chromatids. Many of the above events are controlled by reversible phosphorylation. Hence, our laboratory is interested in characterizing the kinases involved in promoting progression through mitosis and in identifying their relevant substrates. Prominent among the kinases responsible for regulating entry into mitosis is the Cdc2 kinase, the first member of the cyclin dependent kinase (Cdk) family. Recently, we found that Cdc2 phosphorylates HsEg5, a human kinesin-related motor protein associated with centrosomes and the spindle apparatus. Our results indicate that phosphorylation regulates the association of HsEg5 with the mitotic spindle and that the function of this plus-end directed motor is essential for centrosome separation and bipolar spindle formation. Another kinase implicated in regulating progression through mitosis is Plk1 (polo-like kinase 1), the human homologue of the Drosophila gene product "polo." By antibody microinjection we have found that Plk1 is required for the functional maturation of centrosomes and hence for entry into mitosis. Furthermore, we found that microinjected anti-Plk1 antibodies caused a more severe block to cell cycle progression in diploid fibroblasts than in immortalized tumor cells. This observation hints at the existence of a checkpoint linking Cdc2 activation to the presence of functional centrosomes.

Animals↗
PubMed1996

Viral transactivators E1A and VP16 interact with a large complex that is associated with CTD kinase activity and contains CDK8.

Previously, we showed that the viral transactivator proteins E1A and VP16 specifically interact with a cellular CTD kinase activity in vitro. We now report that E1A and VP16 complexes contain human CDK8, a newly identified member of the cyclin-dependent kinase family that has been shown to be a component of the RNA polymerase II (RNAP II) holoenzyme complex. The presence of CDK8 in the E1A- and VP16-containing complexes is specific for a functional activation domain of these viral transactivators, strongly suggesting that this association is relevant for the transactivation function of E1A and VP16. We show that CDK8 is associated with CTD kinase activity and that CDK8 co-fractionates with E1A- and VP16-associated CTD kinase activity over several chromatography columns. Therefore, CDK8 is likely responsible for the E1A- and VP16-associated CTD kinase activity. Gel filtration chromatography indicates that the E1A- and VP16-associated CTD kinase activity has a molecular size of approximately 1.5 MDa and contains cyclin C and the human homolog of SRB7 in addition to CDK8. This implies that E1A and VP16 associate with the RNAP II holoenyzme. We also looked at the transcriptional activity of CDK8 and found that CDK8 can function as a transcriptional activator when fused to the DNA binding domain of GAL4. Surprisingly, the ability of GAL4-CDK8 to activate transcription in this assay was not dependent on the kinase activity of CDK8, since a kinase-deficient mutant of CDK8 stimulated transcription nearly as well as wild-type GAL4-CDK8. This suggests that CDK8 may play a role in transcription that is distinct from its ability to function as a CTD kinase.

Adenovirus E1A Proteins↗
PubMed1996

MAT1, cdk7 and cyclin H form a kinase complex which is UV light-sensitive upon association with TFIIH.

MAT1, cyclin H and cdk7 are part of TFIIH, a class II transcription factor which possesses numerous subunits of which several have been shown to be involved in processes other than transcription. Two of them, XPD (ERCC2) and XPB (ERCC3), are helicases involved in nucleotide excision repair (NER), whereas cdk7, cyclin H and MAT1 are thought to participate in cell cycle regulation. MAT1, cyclin H and cdk7 exist as a ternary complex either free or associated with TFIIH from which the latter can be dissociated at high salt concentration. MAT1 is strongly associated with cdk7 and cyclin H. Although not strictly required for the formation and activity of the complex, it stimulates its kinase activity. The kinase activity of TFIIH, which is constant during the cell cycle, is reduced after UV light irradiation.

Amino Acid Sequence↗
PubMed1996

The family of polo-like kinases.

Here we discuss members of a new family of serine/threonine protein kinases with a likely role in cell cycle control. These kinases are referred to as polo-like kinases, after the prototypic founding member of the family, the polo gene product of Drosophila melanogaster. The polo kinase was originally identified in mutants that display abnormal mitotic spindle organization. Subsequently, potential homologues of Drosophila polo have been identified in yeasts (Cdc5p in Saccharomyces cerevisiae; plo1+ in Schizosaccharmoyces pombe) and in mammals (polo-like kinase 1; Plk1). Genetic and biochemical studies suggest that polo, Cdc5p and plo1+ may be required for mitotic spindle organization and, possibly, for cytokinesis. Likewise, the patterns of expression, activity and subcellular localization of Plk1 strongly suggest that this mammalian kinase functions also during mitosis, possibly in spindle assembly and function. In addition to Plk1, however, more distantly related members of the polo-like kinase family have been identified in mammalian cells, and the available data are consistent with the idea that some of these may act earlier in the cell cycle, possibly during G1. If this hypothesis is correct, different members of the polo-like kinase family would act at several points during the cell cycle, reminiscent of the behaviour of Cdk/cyclin complexes.

Amino Acid Sequence↗
PubMed1996

Cyclin-dependent kinase 7: at the cross-roads of transcription, DNA repair and cell cycle control?

Cyclin-dependent kinase (CDK) 7 was originally implicated in cell cycle control by virtue of its ability to phosphorylate and activate other CDKs. Subsequently, both CDK7 and its partner, cyclin H, were found to be associated with the general transcription factor TFIIH, suggesting additional roles for CDK7 in transcription and DNA repair. During the past year, a third subunit associated with CDK7 and cyclin H has been characterized, and the functional link between CDK7 and RNA polymerase II has been strengthened.

Animals↗
PubMed1996

Antibody microinjection reveals an essential role for human polo-like kinase 1 (Plk1) in the functional maturation of mitotic centrosomes.

Mammalian polo-like kinase 1 (Plk1) is structurally related to the polo gene product of Drosophila melanogaster, Cdc5p of Saccharomyces cerevisiae, and plo1+ of Schizosaccharomyces pombe, a newly emerging family of serine-threonine kinases implicated in cell cycle regulation. Based on data obtained for its putative homologues in invertebrates and yeasts, human Plk1 is suspected to regulate some fundamental aspect(s) of mitosis, but no direct experimental evidence in support of this hypothesis has previously been reported. In this study, we have used a cell duplication, microinjection assay to investigate the in vivo function of Plk1 in both immortalized (HeLa) and nonimmortalized (Hs68) human cells. Injection of anti-Plk1 antibodies (Plk1+) at various stages of the cell cycle had no effect on the kinetics of DNA replication but severely impaired the ability of cells to divide. Analysis of Plk1(+)-injected, mitotically arrested HeLa cells by fluorescence microscopy revealed abnormal distributions of condensed chromatin and monoastral microtubule arrays that were nucleated from duplicated but unseparated centrosomes. Most strikingly, centrosomes in Plk1(+)-injected cells were drastically reduced in size, and the accumulation of both gamma-tubulin and MPM-2 immunoreactivity was impaired. These data indicate that Plk1 activity is necessary for the functional maturation of centrosomes in late G2/early prophase and, consequently, for the establishment of a bipolar spindle. Additional roles for Plk1 at later stages of mitosis are not excluded, although injection of Plk1+ after the completion of spindle formation did not interfere with cytokinesis. Injection of Plk1+ into nonimmortalized Hs68 cells produced qualitatively similar phenotypes, but the vast majority of the injected Hs68 cells arrested as single, mononucleated cells in G2. This latter observation hints at the existence, in nonimmortalized cells, of a centrosome-maturation checkpoint sensitive to the impairment of Plk1 function.

Antibodies↗