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Query: EC:2.7.10.1 (ERK)
 95,504 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The Drosophila slowpoke (slo) gene encodes a subunit of a CAK channel homologous to the vertebrate BK channel. We have examined slo expression throughout development. It is expressed in muscle cells, neurons of the CNS and PNS, mushroom bodies, a limited number of cells in embryonic and larval midgut and in epithelial-derived tracheal cells. The promoter has been cloned and shown to direct expression in the same pattern as the endogenous gene in both neural and epithelial-derived cells. During pupariation and embryogenesis, slo is expressed in muscles many hours prior to the appearance of functional channels.
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PMID:Tissue-specific expression of a Drosophila calcium-activated potassium channel. 766 7

The activity of cyclin-dependent kinases (cdks) depends on the phosphorylation of a residue corresponding to threonine 161 in human p34cdc2. One enzyme responsible for phosphorylating this critical residue has recently been purified from Xenopus and starfish. It was termed CAK (for cdk-activating kinase), and it was shown to contain p40MO15 as its catalytic subunit. In view of the cardinal role of cdks in cell cycle control, it is important to learn if and how CAK activity is regulated during the somatic cell cycle. Here, we report a molecular characterization of a human p40MO15 homologue and its associated CAK activity. We have cloned and sequenced a cDNA coding for human p40MO15, and raised specific polyclonal and monoclonal antibodies against the corresponding protein expressed in Escherichia coli. These tools were then used to demonstrate that p40MO15 protein expression and CAK activity are constant throughout the somatic cell cycle. Gel filtration suggests that active CAK is a multiprotein complex, and immunoprecipitation experiments identify two polypeptides of 34 and 32 kD as likely complex partners of p40MO15. The association of the three proteins is near stoichiometric and invariant throughout the cell cycle. Immunocytochemistry and biochemical enucleation experiments both demonstrate that p40MO15 is nuclear at all stages of the cell cycle (except for mitosis, when the protein redistributes throughout the cell), although the p34cdc2/cyclin B complex, one of the major purported substrates of CAK, occurs in the cytoplasm until shortly before mitosis. The absence of obvious changes in CAK activity in exponentially growing cells constitutes a surprise. It suggests that the phosphorylation state of threonine 161 in p34cdc2 (and the corresponding residue in other cdks) may be regulated primarily by the availability of the cdk/cyclin substrates, and by phosphatase(s).
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PMID:Cell cycle analysis of the activity, subcellular localization, and subunit composition of human CAK (CDK-activating kinase). 792 89

Transitions of the cell cycle are controlled by cyclin-dependent protein kinases (cdks) whose phosphorylation on the Thr residue included in the conserved sequence YTHEVV dramatically increases the activity. A kinase responsible for this specific phosphorylation, called CAK for cdk-activating kinase, has been recently purified from starfish and Xenopus oocytes and shown to contain the MO15 gene product as a catalytic subunit. In the present paper, we have cloned the human homolog of Xenopus p40MO15 by probing a HeLa cell cDNA library with degenerate oligonucleotides deduced from Xenopus and starfish MO15 sequences. Human and Xenopus MO15 displayed a strong homology showing 86% identity with regard to amino acid sequences. Northern blot analysis of RNA extracts from a series of human tissues as well as from cultured rodent fibroblasts revealed a unique 1.4 kb MO15 mRNA. No variation in the amount of MO15 transcript or protein was found along the entire course of the fibroblast cell cycle. Fluorescence in situ hybridization on human lymphocyte metaphases showed two distinct chromosomal locations of human MO15 gene at 5q12-q13 and 2q22-q24. By using gene tagging and mammalian cell transfection, we demonstrate that the KRKR motif located at the carboxy terminal end of MO15 is required for nuclear targeting of the protein. Mutation of KRKR to NGER retains MO15 in the cytoplasmic compartment, whilst the wild-type protein is detected exclusively in the nucleus. Interestingly, we demonstrate that the nuclear targeting of MO15 is necessary to confer the protein its CAK activity. In contrast to the wild-type, the NLS-mutated MO15 expressed in Xenopus oocytes is unable to generate CAK as long as the nuclear envelope is not broken. The nuclear localization of both the MO15 gene product and CAK activity may imply that cdks activation primarily occurs in the cell nucleus.
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PMID:Cloning, expression and subcellular localization of the human homolog of p40MO15 catalytic subunit of cdk-activating kinase. 793 35

p40MO15, a cdc2-related protein, is the catalytic subunit of the kinase (CAK, cdk-activating kinase) responsible for Thr161/Thr160 phosphorylation and activation of cdk1/cdk2. We have found that strong overexpression of p40MO15 only moderately increases CAK activity in Xenopus oocytes, indicating that a regulatory CAK subunit (possibly a cyclin-like protein) limits the ability to generate CAK activity in p40MO15 overexpressing oocytes. This 36 kDa subunit was microsequenced after extensive purification of CAK activity. Production of Xenopus CAK activity was strongly reduced in enucleated oocytes overexpressing p40MO15 and p40MO15 shown to contain a nuclear localization signal required for nuclear translocation and generation of CAK activity. p40MO15 was found to be phosphorylated on Ser170 and Thr176 by proteolytic degradation, radiosequencing of tryptic peptides and mutagenesis. Thr176 phosphorylation is required and Ser170 phosphorylation is dispensable for p40MO15 to generate CAK activity upon association with the 36 kDa regulatory subunit. Finally, Thr176 and Ser170 phosphorylations are not intramolecular autophosphorylation reactions. Taken together, the above results identify protein-protein interactions, nuclear translocation and phosphorylation (by an unidentified kinase) as features of p40MO15 that are required for the generation of active CAK.
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PMID:p40MO15 associates with a p36 subunit and requires both nuclear translocation and Thr176 phosphorylation to generate cdk-activating kinase activity in Xenopus oocytes. 795 80

The eukaryotic cell cycle is regulated by the sequential activation of cyclin-dependent kinases (CDKs). CDK activation is dependent on cyclin binding and phosphorylation of a conserved threonine (T161 in Cdc2) mediated by the CDK-activating kinase CAK. A CDK-related kinase, MO15 (ref. 10), has been identified as the catalytic subunit of CAK (refs 11-13). Here we use a yeast two-hybrid screen to show that a new human cyclin (cyclin H) is a MO15-associated protein. Cyclin H is a major MO15 partner in vivo and enhances the kinase activity of MO15 towards Cdk2/cyclin A. These findings demonstrate that a cyclin/kinase complex can function as a regulator of other cyclin/kinase complexes, and suggest that cyclin/kinase cascades may exist.
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PMID:A cyclin associated with the CDK-activating kinase MO15. 807 87

Phosphorylation of Thr161, a residue conserved in all members of the cdc2 family, has been reported to be absolutely required for the catalytic activity of cdc2, the major regulator of eukaryotic cell cycle. In the present work, we have purified from starfish oocytes a kinase that specifically activates cdc2 in a cyclin-dependent manner through phosphorylation of its Thr161 residue. Our most highly purified preparation contained only two major proteins of apparent M(r) 37 and 40 kDa (p37 and p40), which could not be separated from each other without loss of activity. The purified kinase was found to phosphorylate not only cdc2, but also cdk2 and a divergent cdc2-like protein from Caenorhabditis, in chimeric complexes including both mitotic and G1/S cyclins. Extensive microsequencing of p40 did not reveal any convincing homology with any known protein. In contrast, p37 is the starfish homologue of the M015 gene product, a kinase previously cloned by homology probing from a Xenopus cDNA library. As expected, immunodepletion of the MO15 protein depleted Xenopus egg extracts of CAK (cdk-activating kinase) activity, which was recovered in immunoprecipitates. Taken together, the above results demonstrate that MO15 is a gene conserved throughout evolution (at least from echinoderms to vertebrates) that encodes the catalytic subunit of a protein kinase that activates cdc2-cdks complexes through phosphorylation of Thr161 (or its homologues).
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PMID:The MO15 gene encodes the catalytic subunit of a protein kinase that activates cdc2 and other cyclin-dependent kinases (CDKs) through phosphorylation of Thr161 and its homologues. 834 51

The mitotic inducer p34cdc2 requires association with a cyclin and phosphorylation on Thr161 for its activity as a protein kinase. CAK, the p34cdc2 activating kinase, was previously identified as an enzyme necessary for this activating phosphorylation. We confirm here that CAK is a protein kinase and describe its purification over 13,000-fold from Xenopus egg extracts. We further show that CAK contains a protein identical or closely related to the previously identified Xenopus MO15 gene: p40MO15 copurifies with CAK, and an antiserum to p40MO15 specifically depletes cAK activity. CAK appears to be the only protein in Xenopus egg extracts that can activate complexes of either p34cdc2 or the closely related protein kinase, p33cdk2, with either cyclin A or cyclin B. The sequence similarity between p40MO15 and p34cdc2, and the approximately 200 kDa size of CAK, suggest that p40MO15 may itself be regulated by subunit association and by protein phosphorylations.
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PMID:CAK, the p34cdc2 activating kinase, contains a protein identical or closely related to p40MO15. 834 52

Activation of the cyclin-dependent protein kinases p34cdc2 and p33cdk2 requires binding with a cyclin partner and phosphorylation on the first threonine residue in the sequence THEVVTLWYRAPE. We present evidence that this threonine residue, number 160 in p33cdk2, can be specifically phosphorylated by a cdc2-related protein kinase from Xenopus oocytes called p40MO15. Binding to cyclin A and phosphorylation of this threonine are both required to activate fully the histone H1 kinase activity of p33cdk2. In cell extracts, a portion of p40MO15 is found in a high molecular weight complex that is considerably more active than a lower molecular weight form. Wild-type MO15 protein expressed in bacteria does not possess kinase activity, but acquires p33cdk2-T160 kinase activity after incubation with cell extract and ATP. We conclude that p40MO15 corresponds to CAK (cdc2/cdk2 activating kinase) and speculate that, like p33cdk2 and p34cdc2, p40MO15 requires activation by phosphorylation and association with a companion subunit.
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PMID:The cdc2-related protein p40MO15 is the catalytic subunit of a protein kinase that can activate p33cdk2 and p34cdc2. 839 83

Mammalian CDK7 is a protein kinase identified as the catalytic subunit of cyclin-dependent kinase (CDK)-activating kinase and as an essential component of the transcription factor TFIIH that is involved in transcription initiation and DNA repair. We have identified in human cells a number of CDK7-associated cellular proteins that appear to fall into two classes based on their relative [35S] metabolic labeling intensity. One class of proteins present in CDK7 immunocomplexes as a minor fraction contains components of the TFIIH transcription complex such as p62 and p89ERCC3, whereas the other fraction contains four polypeptides (p35, p37Cyclin H, p75, and p95) that are stoichiometrically associated with CDK7. Whereas the levels of association of p35, p37Cyclin H, and p75 with CDK7 remain unchanged between density-arrested and proliferating Ewing sarcoma EW-1 cells, the association of p95 with CDK7 was significantly decreased as cells reached confluency. Through a large-scale immunopurification of CDK7 complexes and protein microsequencing, we have isolated a cDNA that encodes p35 and have shown that it is the human homologue of Mat1 that is involved in the assembly of CAK. MAT1 contains a highly conserved C3HC4 motif at its NH2 terminus, a characteristic feature shared among RING finger proteins. The human MAT1 gene expresses a single 1.6-kb transcript, the steady-state level of which, like CDK7 and cyclin H, varies significantly in different cell lines and in different terminally differentiated tissues.
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PMID:Molecular cloning of CDK7-associated human MAT1, a cyclin-dependent kinase-activating kinase (CAK) assembly factor. 852 93

The cyclin-dependent kinase (CDK)-activating kinase, CAK, from mammals and amphibians consists of MO15/CDK7 and cyclin H, a complex which has been identified also as a RNA polymerase II C-terminal domain (CTD) kinase. While the Schizosaccharomyces pombe cdc2 gene product also requires an activating phosphorylation, the enzyme responsible has not been identified. We have isolated an essential S.pombe gene, mop1, whose product is closely related to MO15 and to Saccharomyces cerevisiae Kin28. The functional similarity of Mop1 and MO15 is reflected in the ability of MO15 to rescue a mop1 null allele. This suggests that Mop1 would be a CDK, and indeed Mop1 associates with a previously characterized cyclin H-related cyclin Mcs2 of S.pombe. Also, Mop1 and Mcs2 can associate with the heterologous partners human cyclin H and MO15, respectively. Moreover, the rescue of a temperature-sensitive mcs2 strain by expression of mop1+ demonstrates a genetic interaction between mop1 and mcs2. In a functional assay, immunoprecipitated Mop1-Mcs2 acts both as an RNA polymerase II CTD kinase and as a CAK. The CAK activity of Mop1-Mcs2 distinguishes it from the related CDK-cyclin pair Kin28-Ccl1 from S.cerevisiae, and supports the notion that Mop1-Mcs2 may represent a homolog of MO15-cyclin H in S.pombe with apparent dual roles as a RNA polymerase CTD kinase and as a CAK.
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PMID:Schizosaccharomyces pombe Mop1-Mcs2 is related to mammalian CAK. 855 36


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