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)

p58, also referred to as the lamin B receptor, is an intrinsic protein of the inner nuclear membrane that binds in vitro to lamin B. Previous studies have demonstrated that p58 is phosphorylated in vivo and removal of its phosphate moieties affects lamin B binding. Using affinity-purified antipeptide antibodies, we have now immunoisolated p58 from bird erythrocyte lysates under isotonic, non-denaturing conditions. Analysis of the immunopurified material shows that five distinct proteins are tightly and specifically associated with p58. Two of these polypeptides can be identified as nuclear lamins A and B. The immunoisolate also contains a kinase activity that phosphorylates p58 in vivo and in vitro, exclusively at serine residues, as indicated by phosphoamino acid analysis and two-dimensional phosphopeptide mapping. Cell fractionation experiments and in vitro phosphorylation assays demonstrate that the p58 kinase resides in the nuclear envelope and is distinct from protein kinase A and cdc2 kinase, for both of which p58 is an in vitro substrate. These data suggest that p58 is interacting in vivo with a p58 kinase and the nuclear lamins.
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PMID:The inner nuclear membrane protein p58 associates in vivo with a p58 kinase and the nuclear lamins. 132 55

The cdc25 tyrosine phosphatase is known to activate cdc2 kinase in the G2/M transition by dephosphorylation of tyrosine 15. To determine how entry into M-phase in eukaryotic cells is controlled, we have investigated the regulation of the cdc25 protein in Xenopus eggs and oocytes. Two closely related Xenopus cdc25 genes have been cloned and sequenced and specific antibodies generated. The cdc25 phosphatase activity oscillates in both meiotic and mitotic cell cycles, being low in interphase and high in M-phase. Increased activity of cdc25 at M-phase is accompanied by increased phosphorylation that retards electrophoretic mobility in gels from 76 to 92 kDa. Treatment of cdc25 with either phosphatase 1 or phosphatase 2A removes phosphate from cdc25, reverses the mobility shift, and decreases its ability to activate cdc2 kinase. Furthermore, the addition of okadaic acid to egg extracts arrested in S-phase by aphidicolin causes phosphorylation and activation of the cdc25 protein before cyclin B/cdc2 kinase activation. These results demonstrate that the activity of the cdc25 phosphatase at the G2/M transition is directly regulated through changes in its phosphorylation state.
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PMID:Periodic changes in phosphorylation of the Xenopus cdc25 phosphatase regulate its activity. 139 80

The Xenopus cdk2 gene encodes a 32-kDa protein kinase with sequence similarity to the 34-kDa product of the cdc2 gene. Previous studies have shown that the kinase activity of the protein product of the cdk2 gene oscillates in the Xenopus embryonic cell cycle with a high in M-phase and a low in interphase. In the present study cdk2 was found not to be associated with any newly synthesized proteins during the cell cycle, but the enzyme did undergo periodic changes in phosphorylation. Upon exit from metaphase, cdk2 became increasingly phosphorylated on both tyrosine and serine residues, and labeling on these residues increased progressively until entry into mitosis, when tyrosine residues were markedly dephosphorylated. Phosphopeptide mapping of cdk2 demonstrated the major sites of phosphorylation were in a phosphopeptide with a pI of 3.7 that contained both phosphoserine and phosphotyrosine. This phosphopeptide accumulated in egg extracts blocked in S-phase with aphidicolin and was not evident in cdc2 immunoprecipitated under the same conditions. Under the same conditions cdc2 was phosphorylated primarily on a phosphopeptide containing both phosphothreonine and phosphotyrosine residues, most likely threonine 14 and tyrosine 15. Affinity-purified human GST-cdc25 was able to dephosphorylate and activate cdk2 isolated from interphase cells. Phosphopeptide mapping demonstrated that the phosphate was specifically removed from the same phosphopeptide identified as the major in vivo site of phosphorylation. These results demonstrate that cdk2 is regulated in the cell cycle by phosphorylation and dephosphorylation on both serine and tyrosine residues. Moreover, the increased phosphorylation of cdk2 in aphidicolin-blocked extracts and the ability of cdc25 to mediate cdk2 dephosphorylation in vitro suggest the possibility that cdk2 is part of the mechanism ensuring mitosis is not initiated until completion of DNA replication. It also implies cdc25 may have other functions in addition to the regulation of cdc2 kinase activity.
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PMID:Cdc25 regulates the phosphorylation and activity of the Xenopus cdk2 protein kinase complex. 151 36

Mitosis-specific phosphorylation by cdc2 kinase causes nonmuscle caldesmon to dissociate from microfilaments (Yamashiro, S., Yamakita, Y., Ishikawa, R., and Matsumura, F. (1990) Nature 344, 675-678; Yamashiro, S., Yamakita, Y., Hosoya, H., and Matsumura, F. (1991) Nature 349, 169-172). To explore the function of mitosis-specific phosphorylation of caldesmon, in vivo- and in vitro-phosphorylated caldesmons have been characterized. We have found that both in vivo and in vitro phosphorylation of caldesmon causes similar changes in the properties, including reduction in actin, calmodulin, and myosin binding of caldesmon, and a decrease in the inhibition of actomyosin ATPase by caldesmon. Rat non-muscle caldesmon is phosphorylated in vitro up to a ratio of 7 mol/mol of protein. Actin-binding constants of both a high affinity (K a = 1.2 x 10(7) M-1) and a low affinity (K a = 1 x 10(6) M-1) site of unphosphorylated caldesmon are reduced to less than 10(5) M-1 with 5 mol of phosphate incorporation per mol of protein. Actin-bound caldesmon can be phosphorylated by cdc2 kinase, which results in the dissociation of caldesmon from F-actin. Caldesmon has a second myosin-binding site in the C terminus, in addition to the N terminus myosin-binding domain previously reported, because the bacterially expressed C terminus of caldesmon shows binding to myosin. Phosphorylation of the C-terminal fragments decreases their myosin-binding affinity as observed with intact caldesmon. These results suggest that caldesmon loses most of its in vitro functions during mitosis as a result of phosphorylation, which may be required for the reorganization of microfilaments during mitosis.
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PMID:Characterization of mitotically phosphorylated caldesmon. 153 4

Genetic and biochemical studies have indicated that the cdc25 protein controls the entry into mitosis by triggering tyrosine dephosphorylation of the cdc2 protein kinase. We show that the isolated cdc25 protein can catalyze dephosphorylation of several model phosphatase substrates, including p-nitrophenyl phosphate and two distinct tyrosine-phosphorylated peptides. The cdc25-dependent cleavage reaction closely resembles dephosphorylation by known tyrosine phosphatases: the reaction requires a reducing agent, shows high sensitivity to sodium vanadate, and proceeds efficiently in the presence of metal chelators. Moreover, the phosphatase activity of the cdc25 protein is eliminated by treatment with N-ethylmaleimide or by alteration of a single conserved cysteine residue by site-directed mutagenesis. These observations indicate that the cdc25 protein can function as a tyrosine phosphatase in the absence of any other protein.
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PMID:The cdc25 protein contains an intrinsic phosphatase activity. 165 74

wee1 acts antagonistically to cdc25 in the tyrosine dephosphorylation and activation of cdc2, yet biochemical evidence suggests that wee1 is not required for tyrosine phosphorylation and its role is obscure. We show here that a related 66 kd kinase, called mik1, acts redundantly with wee1 in the negative regulation of cdc2 in S. pombe. A null allele of mik1 has no discernible phenotype, but a mik1 wee1 double mutant is hypermitotically lethal: all normal M phase checkpoints are bypassed, including the requirement for initiation of cell cycle "start," completion of S phase, and function of the cdc25+ mitotic activator. In the absence of mik1 and wee1 activity, cdc2 rapidly loses phosphate on tyrosine, both in strains undergoing mitotic lethality and in those that are viable owing to a compensating mutation within cdc2. The data suggest that mik1 and wee1 act cooperatively on cdc2, either directly as the inhibitory tyrosine kinase or as essential activators of that kinase.
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PMID:mik1 and wee1 cooperate in the inhibitory tyrosine phosphorylation of cdc2. 170 23

Mammalian high-mobility group I nonhistone protein (HMG-I) is a DNA-binding chromatin protein that has been demonstrated both in vitro and in vivo to be localized to the A + T-rich sequences of DNA. Recently an unusual binding domain peptide, "the A.T-hook" motif, that mediates specific interaction of HMG-I with the minor groove of DNA in vitro has been described. Inspection of the A.T-hook region of the binding domain showed that it matches the consensus sequence for phosphorylation by cdc2 kinase. Here we demonstrate that HMG-I is a substrate for phosphorylation by purified mammalian cdc2 kinase in vitro. The site of phosphorylation by this enzyme is a threonine residue at the amino-terminal end of the principal binding-domain region of the protein. Labeling of mitotically blocked mouse cells with [32P]phosphate demonstrates that this same threonine residue in HMG-I is also preferentially phosphorylated in vivo. Competition binding studies show that cdc2 phosphorylation of a synthetic binding-domain peptide significantly weakens its interaction with A + T-rich DNA in vitro, and a similar weakening of DNA binding has been observed for intact murine HMG-I protein phosphorylated by the kinase in vitro. These findings indicate that cdc2 phosphorylation may significantly alter the DNA-binding properties of the HMG-I proteins. Because many cdc2 substrates are DNA-binding proteins, these results further suggest that alteration of the DNA-binding affinity of a variety of proteins is an important general component of the mechanism by which cdc2 kinase regulates cell cycle progression.
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PMID:Phosphorylation of the DNA-binding domain of nonhistone high-mobility group I protein by cdc2 kinase: reduction of binding affinity. 200 Mar 76

A recent report that mitosis-specific phosphorylation causes the nonmuscle caldesmon to dissociate from microfilaments (Yamashiro, S., Yamakita, Y., Ishikawa, R., and Matsumura, F. (1990) Nature 344, 675-678) suggests that this process may contribute to the major structural reorganization of the eukaryotic cell at mitosis. In this study we have demonstrated that smooth muscle caldesmon is phosphorylated in vitro by cdc2 kinase from mitotic phase HeLa cells to 1.2 mol of phosphate/mol of caldesmon. Tryptic maps showed three major phosphorylated spots and approximately equal amounts of phosphorylated Ser and Thr were identified. F-actin or calmodulin in the presence of Ca2+ blocks the phosphorylation of caldesmon. Phosphorylation of caldesmon greatly reduced its binding to F-actin. The phosphorylation sites were located in a 10,000-Da CnBr fragment at the COOH-terminal end of the caldesmon molecule known to house the binding sites for actin and calmodulin (Bartegi A., Fattoum, A., Derancourt, J., and Kassab, R. (1990) J. Biol. Chem. 265, 15231-15238). Our finding supports the model that phosphorylation of caldesmon by cdc2 kinase at mitosis may contribute to the disassembly of the microfilament bundles during prophase.
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PMID:Phosphorylation of caldesmon by cdc2 kinase. 201 82

The phosphorylation pattern of simian virus 40 (SV40) large tumor (T) antigen purified from insect cells infected with a recombinant baculovirus was compared with that reported previously for T antigen from SV40-infected monkey cells. The specific activity of metabolic phosphate labeling of baculovirus T antigen was reduced, and the phosphopeptide map of the baculovirus protein, while qualitatively similar to that of lytic T, revealed several quantitative differences. The most striking difference was the prominence in the baculovirus map of peptides containing phosphothreonine 124. These peptides are known to arise from other phosphopeptides upon dephosphorylation of neighboring serines, suggesting that baculovirus T may be underphosphorylated at these serines and perhaps other sites. Functional assays used to further investigate the phosphorylation state of the baculovirus protein included SV40 DNA binding after enzymatic dephosphorylation with alkaline phosphatase and after phosphorylation by a murine homolog of cdc2 protein kinase. The results imply that baculovirus T antigen is underphosphorylated, in particular at those serine residues whose phosphorylation is responsible for down regulation of DNA-binding activity at site II in the core origin of DNA replication. In contrast, no evidence for a functionally significant underphosphorylation at threonine 124 could be found.
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PMID:Altered phosphorylation pattern of simian virus 40 T antigen expressed in insect cells by using a baculovirus vector. 216 68

Using NMR spectroscopy to visualise tyrosine phosphorylation kinetics in real time, we have investigated the sequence-dependent determinants of the selectivity of the human insulin receptor protein-tyrosine kinase for different tyrosine residues. The peptides used encompass the multiple-tyrosine-containing autophosphorylation site sequences from the insulin receptor kinase core domain (Tyr1158, Tyr1162 and Tyr1163) and from its specific C-terminal tail domain (Tyr1328 and Tyr1334). Comparison of the phosphorylation kinetics with those found for the tyrosine residues on a peptide comprising the regulatory tyrosine phosphorylation site of cdc2 points to the role of the primary sequence context of the phosphate acceptor. The particularly deleterious influence of a basic residue immediately C-terminal to the tyrosine is discussed in relation to the autophosphorylation properties of the regulatory loop regions of the insulin and epidermal growth factor receptor kinases. The data further suggest that receptor tyrosine kinase active sites and their substrate targets act in concert to ensure that specific downstream effects are activated.
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PMID:Structural determinants of substrate selection by the human insulin-receptor protein-tyrosine kinase. 752 41


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