Gene/Protein Disease Symptom Drug Enzyme Compound
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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)

The RNA polymerase II large subunit contains an essential carboxy-terminal domain (CTD) believed to be involved in the response to regulators during transcription initiation. The CTD is phosphorylated on a portion of RNA polymerase II molecules in vivo and it can be phosphorylated by the general transcription factor TFIIH in vitro. A highly purified TFIIH from rat liver has been described; this, like human and yeast TFIIH, contains associated CTD kinase and helicase activities. We report here that two polypeptides of the purified mammalian TFIIH are the MO15/Cdk7 kinase and cyclin H subunits of the Cdk-activating kinase Cak, previously identified as a positive regulator of Cdc2 and Cdk2. TFIIH and Cak preparations are each capable of phosphorylating recombinant CTD and recombinant Cdk2 proteins. The presence of Cak in TFIIH indicates that Cak may have roles in transcriptional regulation and in cell-cycle control.
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PMID:Association of Cdk-activating kinase subunits with transcription factor TFIIH. 788 50

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

The assembly of functional holoenzymes composed of regulatory D-type cyclins and cyclin-dependent kinases (cdks) is rate limiting for progression through the G1 phase of the mammalian somatic cell cycle. Complexes between D-type cyclins and their major catalytic subunit, cdk4, are catalytically inactive until cyclin-bound cdk4 undergoes phosphorylation on a single threonyl residue (Thr-172). This step is catalyzed by a cdk-activating kinase (CAK) functionally analogous to the enzyme which phosphorylates cdc2 and cdk2 at Thr-161/160. Here, we demonstrate that the catalytic subunit of mouse cdc2/cdk2 CAK (a 39-kDa protein designated p39MO15) can assemble with a regulatory protein present in either insect or mammalian cells to generate a CAK activity capable of phosphorylating and enzymatically activating both cdk2 and cdk4 in complexes with their respective cyclin partners. A newly identified 37-kDa cyclin-like protein (cyclin H [R. P. Fisher and D. O. Morgan, Cell 78:713-724, 1994]) can assemble with p39MO15 to activate both cyclin A-cdk2 and cyclin D-cdk4 in vitro, implying that CAK is structurally reminiscent of cyclin-cdk complexes themselves. Antisera produced to the p39MO15 subunit can completely deplete mammalian cell lysates of CAK activity for both cyclin A-cdk2 and cyclin D-cdk4, with recovery of activity in the resulting immune complexes. By using an immune complex CAK assay, CAK activity for cyclin A-cdk2 and cyclin D-cdk4 was detected both in quiescent cells and invariantly throughout the cell cycle. Therefore, although it is essential for the enzymatic activation of cyclin-cdk complexes, CAK appears to be neither rate limiting for the emergence of cells from quiescence nor subject to upstream regulatory control by stimulatory mitogens.
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PMID:Activation of cyclin-dependent kinase 4 (cdk4) by mouse MO15-associated kinase. 793 41

Cyclic AMP (cAMP) blocks the mitogenic effects of colony-stimulating factor 1 (CSF-1) in macrophages, inducing cell cycle arrest in mid-G1 phase. Complexes between cyclin D1 and cyclin-dependent kinase 4 (cdk4) assemble in growth arrested cells, but cdk4 is not phosphorylated in vivo by the cdk-activating kinase (CAK) and remains inactive. Although undetectable in lysates of cAMP-treated cells, active CAK is recovered after antibody precipitation, indicating that it is not the direct target of inhibition. Levels of the cdk inhibitor p27Klp1 increase in cAMP-treated cells, and its immunodepletion from inhibitory lysates restores CAK-mediated cdk4 activation. Kip1 does not bind to CAK, but its association with cyclin D-cdk4 prevents CAK from phosphorylating and activating the holoenzyme.
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PMID:Cyclic AMP-induced G1 phase arrest mediated by an inhibitor (p27Kip1) of cyclin-dependent kinase 4 activation. 795 14

The mechanism by which transforming growth factor beta (TGF beta) exerts growth stimulatory effects was examined in C3H/10T1/2 mouse fibroblasts by study of cell cycle regulation of the retinoblastoma gene product (p110Rb) and transcriptional regulation of the p110Rb-associated transcription factor, E2F. Northern blotting analysis shows that TGF beta and/or epidermal growth factor (EGF) stimulate by three to sixfold the level of Rb mRNA which is also reflected by the increased levels of p110Rb. p110Rb becomes phosphorylated in mid-G1 and further phosphorylated at the G1/S transition. Hyperphosphorylation of p110Rb by TGF beta can be observed when cells are in S phase. TGF beta stimulates by three to fourfold the activity of cdk2 kinase consistent with the observed phosphorylation of p110Rb and also with the possibility that the kinase is involved in phosphorylating p110Rb close to the G1/S transition. Thus, TGF beta as a growth stimulator induces, as does EGF, the phosphorylation of p110Rb during cell cycle progression. Transient transfection of E2F promoter constructs was used to analyze the effect of TGF beta on the modulation of E2F-mediated transcription. The data revealed that TGF beta can stimulate wild-type adenoviral E2 promoter activity by 12-fold. Taken together, TGF beta-induced phosphorylation of p110Rb in mouse fibroblasts appears to exert a positive regulatory function upon genes that have a pivotal role in the G1/S transition of the cell cycle.
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PMID:Transforming growth factor-beta regulation of retinoblastoma gene product and E2F transcription factor during cell cycle progression in mouse fibroblasts. 802 Dec 88

Yeasts p13suc1/p18CKS and their human homologues, p9CKShs1/p9CKShs2, strongly interact with p34cdc2 and p34cdk2. While attempting to purify the starfish oocyte p13suc1 homologue, we discovered a 15-kDa protein cross-reactive with anti-p9CKShs2/anti-p13suc1 antibodies. p15cdk-BP-Sepharose binds an anti-PSTAIRE cross-reactive protein of 33 kDa when loaded with starfish oocyte extracts. The p15cdk-BP-bound "PSTAIRE signal" is part of a 250-kDa complex distinct from p34cdc2/cyclin B. p15cdk-BP-Sepharose beads retain a kinase phosphorylating HMG I/Y, P1, and myelin basic protein (among 24 substrates tested). Major cdc2 kinase substrates are not phosphorylated by the p15cdk-BP-bound kinase. Phosphopeptide maps of P1 phosphorylated by the p15cdk-BP-bound kinase, p34cdc2/cyclin B, p 33cdk5/p25, and casein kinase 2 showed that these kinases phosphorylate P1 on different sites. Phosphopeptide maps of P1 phosphorylated by the p15cdk-BP-bound starfish kinase and p15cdk-BP-bound human p34cdk4/cyclin D are largely coincident. To investigate the nature of the p15cdk-BP-bound kinase, extracts of mammalian tissues and cells were loaded on p9CKShs1- and p15cdk-BP-Sepharose and the bound proteins were analyzed using specific anti-cdk antibodies. cdc2 and cdk2 bind to p9CKShs1-Sepharose, but not to p15cdk-BP. cdk4 and cdk5 bind to p15cdk-BP-Sepharose, but not to p9CKShs1-Sepharose. We conclude that p15cdk-BP specifically binds the cdk4/cyclin D and cdk5 kinases and, along with p13suc1 and p9CKShs, may be part of a larger family of cdk-binding proteins.
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PMID:Purification of a 15-kDa cdk4- and cdk5-binding protein. 817 58

Titin is a giant structural protein of striated muscle (M(r) approximately 3000 kDa) and single molecules span sarcomeres from the M- to Z-lines. We have cloned and sequenced the C-terminal region of the titin molecule, which is an integral part of M-lines and forms intimate contacts with the 165 and 190 kDa M-line proteins. In contrast to the regular motif patterns of the A-band portion of titin, the 5.7 kb of titin sequences from the M-line show a complex structure of immunoglobulin-C2 repeats, separated by unique interdomain insertion sequences. As a striking feature, one interdomain insertion comprises four KSP repeats analogous to the multi-phosphorylation repeats of neurofilament subunits H and M. In vitro phosphorylation assays with expressed titin KSP sequences detect high levels of titin KSP phosphorylating kinases in developing but not in differentiated muscle. Since this kinase activity can be depleted from myocyte extracts by antibodies against cdc2 kinase and p13suc1 beads, the titin KSP kinase is structurally related to cdc2 kinase. We suggest that titin C-terminal phosphorylation by SP-specific kinases is regulated during differentiation, and that this may control the assembly of M-line proteins into regular structures during myogenesis.
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PMID:Phosphorylation of KSP motifs in the C-terminal region of titin in differentiating myoblasts. 840 52

Immunocytochemistry has been used to assess the distribution of the mitosis-associated protein kinase, cdc2, in mature neurons of rat brain. A robust signal is apparent in most, but not all neurons in cerebellum, cortex and hippocampus. The signal is concentrated in neuronal nuclei with lower levels of immunoreactivity in neuronal cytoplasm. High resolution analysis indicates that the amount of cdc2 is uneven, suggesting that regulated amounts of enzyme may reflect different functional status of the neurons. In many cases, the kinase is concentrated at the nuclear periphery, implying that it may be particularly involved in phosphorylating substrates in this subnuclear compartment. These results indicate additional roles for cdc2 in terminally differentiated neurons which have undergone final mitosis.
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PMID:Demonstration of cdc2 kinase in mature neuronal nuclei. 851 45

The dynamic instability of microtubules is thought to be regulated by MAPs and phosphorylation. Here we describe the effect of the neuronal microtubule-associated protein tau by observing the dynamics of single microtubules by video microscopy. We used recombinant tau isoforms and tau mutants, and we phosphorylated tau by the neuronal kinases MARK (affecting the KXGS motifs within tau's repeat domain) and cdk5 (phosphorylating Ser-Pro motifs in the regions flanking the repeats). The variants of tau can be broadly classified into three categories, depending on their potency to affect microtubule dynamics. "Strong" tau variants have four repeats and both flanking regions. "Medium" variants have one to three repeats and both flanking regions. "Weak" variants lack one or both of the flanking regions, or have no repeats; with such constructs, microtubule dynamics is not significantly different from that of pure tubulin. N- or C-terminal tails of tau have no influence on dynamic instability. The two ends of microtubules (plus and minus) showed different activities but analogous behavior. These results are consistent with the "jaws" model of tau where the flanking regions are considered as targeting domains whereas the addition of repeats makes them catalytically active in terms of microtubule stabilization. The dominant changes in the parameters of dynamic instability induced by tau are those in the dissociation rate and in the catastrophe rate (up to 30-fold). Other rates change only moderately or not at all (association rate increased up to twofold, rates of rescue or rapid shrinkage decreased up to approximately twofold). The order of repeats has little influence on microtubule dynamics (i.e., repeats can be re-arranged or interchanged), arguing in favor of the "distributed weak binding" model proposed by Butner and Kirschner (1991); however, we confirmed the presence of a "hotspot" of binding potential involving Lys274 and Lys281 observed by Goode and Feinstein, 1994. Phosphorylation of Ser-Pro motifs by cdk5 (mainly Ser 202, 235, and 404) in the flanking regions had a moderate effect on microtubule dynamics while phosphorylation at the "Alzheimer"-site Ser262 MARK eliminated tau's interactions with microtubules. In both cases the predominant effects of phosphorylation are on the rates of tubulin dissociation and catastrophe whereas the effects on the rates of association or rescue are comparatively small.
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PMID:Domains of tau protein, differential phosphorylation, and dynamic instability of microtubules. 859 Aug 13

The adenovirus E1A protein of 243 amino acids has been shown to affect a variety of cellular functions, most notably the immortalization of primary cells and the promotion of quiescent cells into S phase. The activity of E1A is derived, in part, from its association with various cellular proteins, many of which play important roles in regulating cell cycle progression. E1A is known to have multiple sites of phosphorylation. It has been suggested that cell cycle-dependent phosphorylation may also control some of E1A's functions. We find now that immune complexes of cyclin-dependent kinases such as cdk4, cdk2, and cdc2 are all capable of phosphorylating E1A in vitro. Additionally, the sites on E1A phosphorylated by these kinases in vitro are similar to the E1A sites phosphorylated in vivo. We have also found that a phosphorylated E1A is far more efficient than an unphosphorylated E1A in associating with pRB and in disrupting E2F/DP-pRB complexes as well. On the basis of our findings and the differences in timing and expression levels of the various cyclins regulating cdks, we suggest that E1A functions at different control points in the cell cycle and that phosphorylation controls, to some extent, its biological functions.
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PMID:Cyclin-dependent kinases phosphorylate the adenovirus E1A protein, enhancing its ability to bind pRb and disrupt pRb-E2F complexes. 862 66


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