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Query: EC:2.7.11.22 (cdc2)
 8,319 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Activation of conditional alleles of Myc can induce proliferation in quiescent cells. We now report that induction of Myc in density-arrested fibroblasts triggers rapid hyperphosphorylation of the retinoblastoma protein and activation of both cyclin D1- and cyclin E-associated kinase activities in the absence of significant changes in the amounts of cyclin-cdk complexes. Kinase activation by Myc is blocked by inhibitors of transcription and requires intact DNA binding and heterodimerization domains of Myc. Activation of cyclin E-cdk2 kinase in serum-starved cells occurs in two steps. The first is induced by Myc and involves the release of a 120 kDa cyclin E-cdk2 complex from a 250 kDa inactive complex that is present in starved cells. This is necessary, but not sufficient, to generate full kinase activity, as cdc25 phosphatase activity is limiting in the absence of external growth factors. In vivo cdc25 activity can be supplied by the addition of growth factors. In vitro recombinant cdc25a strongly activates the 120 kDa, but only poorly activates the 250 kDa cyclin E-cdk2 complex. Our data show that two distinct signals, one of which is supplied by Myc, are necessary for consecutive steps during growth factor-induced formation of active cyclin E-cdk2 complexes in G(o)-arrested rodent fibroblasts.
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PMID:Identification of a Myc-dependent step during the formation of active G1 cyclin-cdk complexes. 758 11

Assembly and disassembly of the nucleus at mitosis in eukaryotes involves the reversible interaction of chromatin with the nuclear membrane. Previously we have shown that this interaction is regulated by the antagonistic activities of a kinase and a phosphatase. The kinase promotes membrane release while the phosphatase stimulates binding. In this report we describe four steps in the purification of the kinase needed for release of membranes from chromatin. We also show that the release kinase and the mitotic initiation kinase, cdc2, are distinct and are separated from each other during the second purification step. Reconstitution experiments using these two kinases demonstrate that the release kinase and cdc2 kinase work in concert to cause membrane release from chromatin. In phosphorylation experiments, protein targets that are substrates for the regulatory release kinase are identified on the membranes. These phosphorylated proteins ae candidates for regulated proteins mediating membrane-chromatin interaction. Finally, we find that membrane release activity can also be extracted from membranes by high salt treatment, indicating a possible dual localization of this activity.
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PMID:Assembly/disassembly of the nuclear envelope membrane. Characterization of the membrane-chromatin interaction using partially purified regulatory enzymes. 764 70

We identified a novel human nucleolar phosphoprotein p130 (130 kDa) using a strategy for selecting monoclonal antibodies against nuclear proteins which oscillate in the cell cycle. p130 is localized in interphase nucleoli in a dotted manner. Complete extraction of p130 required a high concentration of salt (0.5 M NaCl) indicating that it binds firmly to the nucleolar components via ionic interaction. p130 is heavily phosphorylated, since alkaline phosphatase treatment converted the purified p130 into a 95 kDa product; this was further supported by the in vitro demonstration that cellular phosphatase and casein kinase II activities were responsible for the interchange of these two forms. Extracts of mitotic cells had lower concentrations of p130 compared to those of interphase cells suggesting that a proportion of p130 might be degraded during mitosis. Moreover, all the remaining p130 in mitotic cells was further phosphorylated, likely by a cdc2 kinase, resulting in increase in its solubility, and its dispersion throughout the entire cytoplasm. Thus, p130 in metaphase and anaphase cells was unable to be detected by immunofluorescence microscopy. At telophase, p130 reappeared and aggregated into a granular structure, resembling the prenucleolar bodies. These granules migrated from the nucleoplasm to the nucleoli in early G1-phase. Actinomycin D was able to induce segregation of p130-containing granules into the nucleoplasm, similar to the well-known behavior of the fibrillarin-containing granules, indicating that p130 is localized in the dense fibrillar component, a subnucleolar region for pre-rRNA synthesis and processing. The cDNA sequence of p130 revealed a remarkable feature, that a serine-rich stretch interspersed with acidic residues is repeated ten times. Such a characteristic is shared with a rat nucleolar phosphoprotein Nopp140, which is thought to shuttle between the nucleolus and the cytoplasm. Although p130 shows 74% identity to Nopp140, our observations suggest that during mitosis the functions of p130 are related to nucleologenesis.
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PMID:Cell-cycle-dependent alterations of a highly phosphorylated nucleolar protein p130 are associated with nucleologenesis. 765 14

To elucidate the role of phosphorylation of p53 we used the baculovirus expression system to obtain high yields of protein eventually in distinct phosphorylation states. Initially, we obtained only marginal phosphorylation, despite high levels of expression. Two-dimensional phosphopeptide maps exhibited the same pattern as known from rat cells although some sites were underrepresented. Coexpression of simian virus 40 (SV40) large T antigen or cyclin-dependent kinases, cdc2 or cdk2, had only marginal effects on the phosphorylation state of p53. However, when we employed the phosphatase inhibitor okadaic acid, overall phosphorylation of p53 was drastically enhanced in a dose-dependent manner and resembled that of p53 from SV40-transformed rat cells. This hyperphosphorylation resulted in enhanced binding of a consensus oligonucleotide as revealed by electrophoretic mobility shift assays. To assess the role of individual phosphorylation sites, we generated a set of mutants at putative or identified sites. All mutants retained the ability to bind wild-type conformation-specific antibody Pab1620, to complex with SV40 large T antigen, and to bind to the consensus oligonucleotide. Moreover, most mutants exhibited enhanced DNA binding upon okadaic acid treatment, except for a mutant at the cdk site which failed to do so. These data show that: (a) insect cells contain all the protein kinases necessary for phosphorylation of a mammalian protein, p53; (b) in insect cells the ratio of kinase/phosphatase activities differs from that in mammalian cells so that underphosphorylation of recombinant proteins in this system may result from high phosphatase activities rather than saturation of kinases with recombinant substrate; (c) the system can be manipulated to obtain subpopulations of recombinant protein in a desired phosphorylation state, and (d) phosphorylation may regulate the DNA-binding activity of p53.
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PMID:Phosphorylation studies on rat p53 using the baculovirus expression system. Manipulation of the phosphorylation state with okadaic acid and influence on DNA binding. 773 56

Neurofilament (NF) protein [high molecular mass (NF-H)] is extensively phosphorylated in vivo. The phosphorylation occurs mainly in its characteristic KSP (Lys-Ser-Pro) repeat motifs. There are two major types of KSP motifs in the NF-H tail domain: KSPXKX and KSPXXX. Recent studies by two different laboratories have demonstrated the presence of a cdc2-like kinase [cyclin-dependent kinase-5 (cdk5)] in nervous tissue that selectively phosphorylates KSPXKX and XS/TXK motifs in NF-H and lysine-rich histone (H1). This article describes the identification of phosphatases dephosphorylating three different substrates: histone (H1), NF-H in a NF preparation, and a bacterially expressed C-terminal tail domain of NF-H, each containing KSPXKX repeats phosphorylated in vitro by cdk5. Among various phosphatases identified, protein phosphatase (PP) 2A from rabbit skeletal muscle appeared to be the most effective phosphatase in in vitro assays. Three phosphatase activity peaks--P1, P2, and P3--were partially purified from frozen rat spinal cord by ion exchange and size exclusion column chromatography and then characterized on the basis of biochemical, pharmacological, and immunochemical studies. One of the three peaks was identified as PP2A, whereas the others were mixtures of both PP2A and PP1. These three peaks could dephosphorylate cdk5-phosphorylated 32P-histone (H1), 32P-NF-H in the NF preparation, and 32P-NF-H tail fusion protein. These studies suggest the involvement of PP2A or a PP2A-like activity in the regulation of the phosphorylation state of KSPXKX motifs in NF-H.
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PMID:Neuronal cyclin-dependent kinase-5 phosphorylation sites in neurofilament protein (NF-H) are dephosphorylated by protein phosphatase 2A. 776 48

The M-phase inducer, Cdc25C, is a dual-specificity phosphatase that directly phosphorylates and activates the cyclin B/Cdc2 kinase complex, leading to initiation of mitosis. Cdc25 itself is activated at the G2/M transition by phosphorylation on serine and threonine residues. Previously, it was demonstrated that Cdc2 kinase is capable of phosphorylating and activating Cdc25, suggesting the existence of a positive feedback loop. In the present study, kinases other than Cdc2 that can phosphorylate and activate Cdc25 were investigated. Cdc25 was found to be phosphorylated and activated by cyclin A/Cdk2 and cyclin E/Cdk2 in vitro. However, in interphase Xenopus egg extracts with no detectable Cdc2 and Cdk2, treatment with the phosphatase inhibitor microcystin activated a distinct kinase that could phosphorylate and activate Cdc25. Microcystin also induced other mitotic phenomena such as chromosome condensation and nuclear envelope breakdown in extracts containing less than 5% of the mitotic level of Cdc2 kinase activity. These findings implicate a kinase other than Cdc2 and Cdk2 that may initially activate Cdc25 in vivo and suggest that this kinase may also phosphorylate M-phase substrates even in the absence of Cdc2 kinase.
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PMID:Phosphorylation and activation of the Xenopus Cdc25 phosphatase in the absence of Cdc2 and Cdk2 kinase activity. 778 47

A growing family of kinases and phosphatases controls the activity of the cyclin-dependent kinase cdc2. The past year has seen the identification of the cdk activating kinase as well as considerable elucidation of the cdc25/wee1 regulatory pathways. Both cdc25 and wee1 appear to be regulated by upstream kinase/phosphatase networks. In addition, it is likely that other regulatory mechanisms cooperate with the wee1/cdc25 phosphorylation systems to control the action of cdc2. Together, these elaborate checks and balances ensure that cdc2 triggers mitosis at the appropriate time.
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PMID:Cdc2 regulatory factors. 788 May 37

Oocyte maturation is triggered by the activation in the oocyte cytoplasm of maturation-promoting factor (MPF), which consists of cdc2 (a catalytic subunit) and cyclin B (a regulatory subunit). Immature goldfish oocytes contain only inactive monomeric 35-kDa cdc2 and do not stockpile cyclin B. In maturing oocytes, activation of cdc2 is associated with its Thr161 phosphorylation and mobility shift on SDS-PAGE from 35 to 34 kDa after binding to cyclin B. Using mutant cdc2, we show that Thr161 phosphorylation is required for both the downward shift and the kinase activation. Since cdc2 Tyr15 is not phosphorylated after binding to cyclin B, it does not require dephosphorylation. This situation is obviously different from that in immature Xenopus oocytes, in which the cdc2-cyclin B complex preexists with cdc2 phosphorylated on both Tyr15 and Thr161, thereby requiring Tyr15 dephosphorylation catalyzed by cdc25 phosphatase for MPF activation. These results indicate that these species employ different mechanisms of MPF activation during oocyte maturation, although the final molecular structure of the active MPF (cdc2 bound to cyclin B and phosphorylated on Thr161) is identical.
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PMID:Molecular mechanisms of the activation of maturation-promoting factor during goldfish oocyte maturation. 788 79

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

G2 arrest induced by nitrogen mustard in human lymphoma CA46 cells is associated with a failure to activate hyperphosphorylated cdc2/cyclin B1 complexes. We investigated the possibility that this might be due to a suppression of cdc25C phosphatase activity. cdc25C from interphase cells migrated as a 54- to 57-kDa doublet in SDS gels and exhibited basal phosphatase activity. cdc25C from mitotic cells migrated as a 66-kDa hyperphosphorylated species and exhibited elevated phosphatase activity. cdc25C hyperphosphorylation and activation were mediated by cdc2, supporting the view of a cdc2-cdc25C autocatalytic feedback loop. Immunofluorescence and cell fractionation studies suggested cdc2-cdc25C interaction occurred within the cytoplasm. Cells arrested in G2 phase following nitrogen mustard treatment or cells arrested in S phase with aphidicolin failed to dephosphorylate and activate cdc2, and this correlated with failure to convert cdc25C into the most active hyperphosphorylated species. Our findings suggest that checkpoints guarding against mitotic entry in the presence of unreplicated or damaged DNA suppress formation of the cdc2-cdc25C autocatalytic feedback loop that normally brings about rapid activation of cdc2.
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PMID:Role of the cdc25C phosphatase in G2 arrest induced by nitrogen mustard. 793 93


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