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At least 19 recordsLinked to original sources
PubMed1995

Isolation of a functional homolog of the cell cycle-specific NIMA protein kinase of Aspergillus nidulans and functional analysis of conserved residues.

To investigate the degree of conservation of the cell cycle-specific NIMA protein kinase of Aspergillus nidulans, and to help direct its functional analysis, we cloned a homolog (designated nim-1) from Neurospora crassa. Over the catalytic domain NIM-1 is 75% identical to NIMA, but overall the identity drops to 52%. nim-1 was able to functionally complement nimA5 in A. nidulans. Mutational analysis of potential activating phosphorylation sites found in NIMA, NIM-1, and related protein kinases was performed on NIMA. Mutation of threonine 199 (conserved in all NIMA-related kinases) inhibited NIMA beta-casein kinase activity and abolished its in vivo function. This site conforms to a minimal consensus phosphorylation site for NIMA (FXXT) and is analogous to the autophosphorylation site of cyclic-AMP-dependent protein kinases. However, mutation of a unique cysteine residue found only in the catalytic site of NIMA and NIM-1 had no effect on NIMA kinase activity or function. Three temperature-sensitive alleles of nimA that cause arrest in G2 were sequenced and shown to generate three different amino acid substitutions. None of the mutations prevented accumulation of NIMA protein during G2 arrest, but all prevented the p34cdc2/cyclin B-dependent phosphorylation of NIMA normally seen during mitotic initiation even though p34cdc2/cyclin B H1 kinase activity was fully activated.

Amino Acid Sequence↗
PubMed1995

Substrate specificity and cell cycle regulation of the Nek2 protein kinase, a potential human homolog of the mitotic regulator NIMA of Aspergillus nidulans.

The human Nek2 protein kinase is the closest known mammalian relative of the mitotic regulator NIMA of Aspergillus nidulans. The two kinases share 47% sequence identity over their catalytic domains and display a similar cell cycle-dependent expression peaking at the G2 to M phase transition. Hence, it is attractive to speculate that human Nek2 and fungal NIMA may carry out similar functions at the onset of mitosis. To study the biochemical properties and substrate specificity of human Nek2 and compare them to those reported previously for other NIMA-related protein kinases, we have expressed Nek2 in insect cells. We show that recombinant Nek2 is active as a serine/threonine-specific protein kinase and may undergo autophosphorylation. Both human Nek2 and fungal NIMA phosphorylate a similar, albeit not identical, set of proteins and synthetic peptides, and beta-casein was found to be a suitable substrate for assaying Nek2 in vitro. By exploiting these findings, we have studied the cell cycle regulation of Nek2 activity in HeLa cells. We show that Nek2 activity parallels its abundance, being low during M and G1 but high during S and G2 phase. Taken together, our results suggest that human Nek2 resembles fungal NIMA in its primary structure, cell cycle regulation of expression, and substrate specificity, but that Nek2 may function earlier in the cell cycle than NIMA.

Amino Acid Sequence↗
PubMed1991

Activation of the nimA protein kinase plays a unique role during mitosis that cannot be bypassed by absence of the bimE checkpoint.

Mutation of nimA reversibly arrests cells in late G2 and nimA overexpression promotes premature mitosis. Here we demonstrate that the product of nimA (designated NIMA) has protein kinase activity that can phosphorylate beta-casein but not histone proteins. NIMA kinase activity is cell cycle regulated being 20-fold higher at mitosis when compared to S-phase arrested cells. NIMA activation is normally required in G2 to initiate chromosome condensation, to nucleate spindle pole body microtubules, and to allow an MPM-2 specific mitotic phosphorylation. All three of these mitotic events can occur in the absence of activated NIMA when the bimE gene is mutated (bimE7). However, the bimE7 mutation cannot completely bypass the requirement for nimA during mitosis as entry into mitosis in the absence of NIMA activation results in major mitotic defects that affect both the organization of the nuclear envelope and mitotic spindle. Thus, although nimA plays an essential but limited role during mitosis, mutation of nimA arrests all of mitosis. We therefore propose that mutation of nimA prevents mitotic initiation due to a checkpoint arrest that is negatively mediated by bimE. The checkpoint ensures that mitosis is not initiated until NIMA is mitotically activated.

Amino Acid Sequence↗
PubMed1994

Premature chromatin condensation upon accumulation of NIMA.

The NIMA protein kinase of Aspergillus nidulans is required for the G2/M transition of the cell cycle. Mutants lacking NIMA arrest without morphological characteristics of mitosis, but they do contain an activated p37nimX kinase (the Aspergillus homologue of p34cdc2). To gain a better understanding of NIMA function we have investigated the effects of expressing various NIMA constructs in Aspergillus, fission yeast and human cells. Our experiments have shown that the instability of the NIMA protein requires sequences in the non-catalytic C-terminus of the protein. Removal of this domain results in a stable protein that, once accumulated, promotes a lethal premature condensation of chromatin without any other aspects of mitosis. Similar effects were also observed in fission yeast and human cells accumulating Aspergillus NIMA. This phenotype is independent of cell cycle progression and does not require p34cdc2 kinase activity. As gain of NIMA function by accumulation results in premature chromatin condensation, and loss of NIMA function results in an inability to enter mitosis, we propose that NIMA functions in G2 to promote the condensation of chromatin normally associated with entry into mitosis.

Apoptosis↗
PubMed1992

Cloning of chromosome I DNA from Saccharomyces cerevisiae: analysis of the FUN52 gene, whose product has homology to protein kinases.

A gene whose product has homology to protein kinases and is closely related to the Aspergillus nidulans nimA cell-cycle gene was identified on chromosome I of the yeast, Saccharomyces cerevisiae. This gene has been temporarily designated FUN52, where FUN is the acronym for 'function unknown now'. In A. nidulans, nimA is required to enter mitosis. In addition, overexpression of nimA causes premature onset of mitosis and cell cycle arrest. In contrast, S. cerevisiae cells that were either deleted for FUN52 or were overexpressing it had no detectable growth phenotypes. FUN52 proved to be the same as the previously identified KIN3 gene [Jones and Rosamond, Gene 90 (1990) 87-92] that was reported to map on chromosome VI.

Amino Acid Sequence↗
PubMed1994

Identification of substrate specificity determinants for the cell cycle-regulated NIMA protein kinase.

NIMA is a cell cycle-regulated protein kinase required for the G2/M transition in the filamentous fungus Aspergillus nidulans. Previous biochemical characterization of the recombinant enzyme indicated that NIMA is a protein serine/threonine specific kinase with beta-casein being the best substrate from the many proteins and peptides tested (Lu, K.P., Osmani, S.A., and Means, A.R. (1993) J. Biol. Chem. 268, 8769-8776). However, substrate specificity or physiologically relevant substrates for NIMA remained unknown. In search for a peptide substrate for this enzyme, we screened an assembled library of synthetic peptides that each contained a phosphorylation site for a known protein kinase and found an excellent peptide substrate for NIMA, phospholemman 42-72 (PLM(42-72)). NIMA kinase phosphorylated PLM(42-72) uniquely and stoichiometrically on Ser63 with a Vmax of 1.4 mumol/min/mg and apparent Km of 20.0 microM. These kinetic constants were about 10-fold higher and 3-fold lower than those for beta-casein, respectively. A detailed analysis of substrate specificity determinants using synthetic peptide analogs of PLM(42-72) indicated that Phe-Arg-Xaa-Ser/Thr represents the optimal primary sequence for NIMA kinase phosphorylation. Replacement of the Arg at P-2 with Ala resulted in a 6-fold increase in Km and 2-fold decrease in Vmax, while substitution of the Phe at P-3 with Ala abolished NIMA phosphorylation. These results reveal the unique nature of substrate recognition by the NIMA kinase and should prove valuable in the search for biologically relevant NIMA substrates.

Amino Acid Sequence↗
PubMed1994

Cell cycle-dependent expression of Nek2, a novel human protein kinase related to the NIMA mitotic regulator of Aspergillus nidulans.

The serine/threonine protein kinase NIMA of Aspergillus nidulans is required for entry into mitosis and may function in parallel to the universal mitotic inducer p34cdc2. Here, we report the isolation of complementary DNAs encoding Nek2 and Nek3, two novel human protein kinases structurally related to NIMA. Sequence comparisons revealed several unique features which may define a family of NIMA-related protein kinases. Nek2 was chosen for further study since it represents the closest known mammalian relative of NIMA. Chromosomal mapping of the nek2 gene identified two independent loci on chromosomes 1 and 14, and Northern blot analyses revealed the expression of two distinct mRNAs of approximately 2.4 and 4.7 kilobases in all human cell lines examined. In HeLa cells synchronized by both drug arrest and elutriation, a strikingly cell cycle-dependent pattern of Nek2 expression could be observed; Nek2 protein was almost undetectable during G1 but accumulated progressively throughout S, reaching maximal levels in late G2. These observations demonstrate that Nek2 resembles Aspergillus NIMA, not only in its catalytic domain, but also in its cell cycle-dependent expression. Hence, the human Nek2 protein kinase may also function at the onset of mitosis.

Amino Acid Sequence↗
PubMed1994

Two novel human serine/threonine kinases with homologies to the cell cycle regulating Xenopus MO15, and NIMA kinases: cloning and characterization of their expression pattern.

Using polymerase chain reaction (PCR)-based methods, we have isolated cDNA clones of two new members of serine/threonine kinases, STK1 and STK2, from a cDNA library constructed from the BT-20 human breast cancer cell line. STK1 is transcribed as a 1.4 kilobase (kb) mRNA encoding for a protein of 346 amino acids. Based on amino acid sequence analysis, STK1 is 86% identical to the Xenopus p40mo15, a cdc2-related serine/threonine kinase recently found to be the activating kinase for p34cdc2 and p33cdk2. Thus, STK1 is most likely the human homologue of MO15. An alternatively spliced STK1 message expressed variably in cell lines and in primary carcinomas generates a predicted 58 amino acid protein that lacks the kinase domain. STK2 is transcribed into a 4.0 kb mRNA encoding for an 841 residue protein which exhibits 50% identity in the kinase domain with the mouse nek1 gene product, the relative of the fungal G2-M regulator, nimA. STK1 and STK2 display a variable pattern of expression among a series of primary carcinomas as well as cancer cell lines. Both STK1 and STK2 were expressed at the highest levels in the heart but were also detected in all other organs tested. In embryonal tissues, lower levels of expression were noted. Using cell cycle inhibitors, we have shown that both STK1 and STK2 mRNA levels remain relatively invariant through the cell cycle. Chromosomal assignment has localized STK1 on chromosome 2pcen-2p15, a region implicated in hereditary non-polyposis colorectal carcinoma, and STK2 on chromosome 3p21.1, a region frequently showing chromosomal alterations in renal cells carcinomas.

Amino Acid Sequence↗
PubMed1995

The NIMA protein kinase is hyperphosphorylated and activated downstream of p34cdc2/cyclin B: coordination of two mitosis promoting kinases.

Initiation of mitosis in Aspergillus nidulans requires activation of two protein kinases, p34cdc2/cyclin B and NIMA. Forced expression of NIMA, even when p34cdc2 was inactivated, promoted chromatin condensation. NIMA may therefore directly cause mitotic chromosome condensation. However, the mitosis-promoting function of NIMA is normally under control of p34cdc2/cyclin B as the active G2 form of NIMA is hyperphosphorylated and further activated by p34cdc2/cyclin B when cells initiate mitosis. To see the p34cdc2/cyclin B dependent activation of NIMA, okadaic acid had to be added to isolation buffers to prevent dephosphorylation of NIMA during isolation. Hyperphosphorylated NIMA contained the MPM-2 epitope and, in vitro, phosphorylation of NIMA by p34cdc2/cyclin B generated the MPM-2 epitope, suggesting that NIMA is phosphorylated directly by p34cdc2/cyclin B during mitotic initiation. These two kinases, which are both essential for mitotic initiation, are therefore independently activated as protein kinases during G2. Then, to initiate mitosis, we suggest that each activates the other's mitosis-promoting functions. This ensures that cells coordinately activate p34cdc2/cyclin B and NIMA to initiate mitosis only upon completion of all interphase events. Finally, we show that NIMA is regulated through the cell cycle like cyclin B, as it accumulates during G2 and is degraded only when cells traverse mitosis.

Amino Acid Sequence↗
PubMed1994

Inhibition of G2/M progression in Schizosaccharomyces pombe by a mutant calmodulin kinase II with constitutive activity.

Intracellular signaling by the second messenger Ca2+ through its receptor calmodulin (CaM) regulates cell function via the activation of CaM-dependent enzymes. Previous studies have shown that cell cycle progression at G1/S and G2/M is sensitive to intracellular CaM levels. However, little is known about the CaM-regulated enzymes involved. Protein phosphorylation has been shown to be important for cell-cycle regulation. Because CaM regulates several protein kinases, and at least one protein phosphatase, our studies are focusing on the roles of these enzymes within the cell cycle. As an initial approach to this problem, cDNAs encoding either normal or mutant calcium/calmodulin kinase II (CaMKII) have been expressed in Schizosaccharomyces pombe. The results show that overexpression of a constitutively active mutant CaMKII caused cell-cycle arrest in G2. Arrest was associated with a failure to activate the p34/cdc2 protein kinase. Expression of the mutant CaMKII in strains of S. pombe with altered timing of mitosis revealed that this effect is not mediated either by cdc25+ or wee1+, suggesting that CaMKII may regulate G2/M progression by another mechanism.

Animals↗
PubMed1995

Mitotic destruction of the cell cycle regulated NIMA protein kinase of Aspergillus nidulans is required for mitotic exit.

NIMA is a cell cycle regulated protein kinase required, in addition to p34cdc2/cyclin B, for initiation of mitosis in Aspergillus nidulans. Like cyclin B, NIMA accumulates when cells are arrested in G2 and is degraded as cells traverse mitosis. However, it is stable in cells arrested in mitosis. NIMA, and related kinases, have an N-terminal kinase domain and a C-terminal extension. Deletion of the C-terminus does not completely inactivate NIMA kinase activity but does prevent functional complementation of a temperature sensitive mutation of nimA, showing it to be essential for function. Partial C-terminal deletion of NIMA generates a highly toxic kinase although the kinase domain alone is not toxic. Transient induction experiments demonstrate that the partially truncated NIMA is far more stable than the full length NIMA protein which likely accounts for its toxicity. Unlike full length NIMA, the truncated NIMA is not degraded during mitosis and this affects normal mitotic progression. Cells arrested in mitosis with non-degradable NIMA are able to destroy cyclin B, demonstrating that the arrest is not due to stabilization of p34cdc2/cyclin B activity. The data establish that NIMA degradation during mitosis is required for correct mitotic progression in A. nidulans.

Aspergillus nidulans↗
PubMed1995

Evidence for a NIMA-like mitotic pathway in vertebrate cells.

NIMA is essential for entry into mitosis in Aspergillus nidulans. To examine whether there is a NIMA-like pathway in other eukaryotic cell cycles, we expressed NIMA and its dominant negative mutants in two different eukaryotic systems. In Xenopus oocytes, NIMA induced germinal vesicle breakdown without activating Mos, CDC2, or MAP kinase. In HeLa cells, NIMA induced premature mitotic events without activating CDC2, whereas the mutants caused a specific G2 arrest but did not block mutant CDC2T14AY15F-induced premature entry into mitosis. A sequence essential for both these phenotypes was mapped to a region of approximately 100 amino acids lying just after the catalytic domain of NIMA that shows a significant similarity to protein interaction domains in other proteins. These results provide evidence for the existence of a NIMA-like mitotic pathway in vertebrate cells.

Amino Acid Sequence↗
PubMed1994

Expression of the noncatalytic domain of the NIMA kinase causes a G2 arrest in Aspergillus nidulans.

Temperature-sensitive mutation of the nimA gene of Aspergillus nidulans causes a reversible G2 arrest, whereas overexpression of nimA causes premature entry into mitosis from which the cells cannot exit. The nimA gene encodes a Ser/Thr-specific protein kinase (NIMA) which contains an extended COOH-terminal noncatalytic domain. To evaluate the role of this enzyme in nuclear division control, we introduced various mutant nimA cDNAs under the control of the inducible alcohol dehydrogenase gene promoter into a strain of Aspergillus nidulans containing a temperature-sensitive nimA mutation (nimA5). While expression of the wild type NIMA complemented the nimA5 mutation and induced a premature mitotic arrest when overexpressed, expression of a kinase-negative NIMA containing a single amino acid mutation in the putative ATP-binding site could not rescue the nimA5 mutation but resulted in a specific G2 arrest when overexpressed. An identical phenotype was observed with cells expressing only the noncatalytic COOH-terminal domain of NIMA, whereas overexpression of the inactive kinase domain was without effect. The G2 arrest produced by overexpression of the full-length inactive or COOH-terminal NIMA molecules did not prevent activation of the endogenous NIMA or H1 kinase activity precipitable by p13 beads. We suggest that this dominant-negative phenotype results from competitive inhibition of the association of active NIMA with a cellular target(s) and that appropriate targeting is essential for the mitotic function of the NIMA kinase.

Alcohol Dehydrogenase↗
PubMed1992

NPK1, a nonessential protein kinase gene in Saccharomyces cerevisiae with similarity to Aspergillus nidulans nimA.

A new protein kinase gene [called NPK1 (for nonessential protein kinase)] has been found on chromosome I of Saccharomyces cerevisiae between CDC15 and ADE1. The 435 amino acid/48 kDa gene product is very similar to known protein kinases. It is most closely related to the nimA protein of Aspergillus nidulans, displaying 45.9% identity and 63.5% similarity in the protein kinase domain. A 1.4 kb transcript of the NPK1 gene was detected. Disruption of the NPK1 gene impedes neither growth on glucose or a variety of other carbon sources, nor mating or sporulation.

Amino Acid Sequence↗
PubMed2025

NEK9-mediated Wnt signalling repressor TLE3 rewires Docetaxel resistance in cancer cells by inducing pyroptosis.

BACKGROUND: Docetaxel is the most common chemotherapy regimen for several neoplasms, including advanced OSCC (Oral Squamous Cell Carcinoma). Unfortunately, chemoresistance leads to relapse and adverse disease outcomes. METHODS: We performed CRISPR-based kinome screening to identify potential players of Docetaxel resistance. Immunohistochemistry was performed to examine the expression profile of the target gene across tumour tissues. Global transcriptome analysis was performed to determine the molecular mechanism underlying Docetaxel resistance. NEK9 kinase assay was performed to identify a putative kinase inhibitor. RESULTS: Upon conducting CRISPR-based kinome screening, Never In Mitosis Gene-A Related Kinase-9 (NEK9) was identified as a major player of Docetaxel resistance in OSCC, prostate, and pancreatic cancer lines. NEK9 expression was found to be upregulated in chemotherapy non-responder OSCC patients as compared to responders. NEK9 ablation restores Docetaxel-induced cell death in chemoresistant cells. Mechanistically, we found that NEK9 deletion upregulates Transducin-like enhancer protein 3 (TLE3), which in turn represses Wnt signalling. Fostamatinib was identified as a potent NEK9 inhibitor that overcomes Docetaxel resistance. CONCLUSIONS: Our study demonstrated that NEK9 plays an important role in Docetaxel resistance. The novel combination of NEK9 inhibitor Fostamatinib and Docetaxel needs further clinical investigation in advanced OSCC.

Humans↗