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)

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

Nonmuscle caldesmon from bovine brain bound to microtubules with a stoichiometry of five tubulin dimers to one molecule of caldesmon with values of Ka 4.5 x 10(5) M-1. The binding of caldesmon to microtubules was inhibited in the presence of Ca2+ and calmodulin. The phosphorylation of caldesmon by cdc2 kinase also eliminated the microtubule-binding activity. These results suggest that caldesmon may play a physiological role in the functions of microtubules.
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PMID:The binding of nonmuscle caldesmon from brain to microtubules. Regulations by Ca(2+)-calmodulin and cdc2 kinase. 154 74

One of the profound changes in cellular morphology during mitosis is a massive alteration in the organization of microfilament cytoskeleton. It has been recently discovered that nonmuscle caldesmon, an actin and calmodulin binding microfilament-associated protein of relative molecular mass Mr = 83,000, is dissociated from microfilaments during mitosis, apparently as a consequence of mitosis-specific phosphorylation. cdc2 kinase, which is a catalytic subunit of MPF (maturation or mitosis promoting factor), is found to be responsible for the mitosis-specific phosphorylation of caldesmon. Because caldesmon is implicated in the regulation of actin myosin interactions and/or microfilament organization, these results suggest that cdc2 kinase directly affects microfilament re-organization during mitosis.
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PMID:Mitosis-specific phosphorylation of caldesmon: possible molecular mechanism of cell rounding during mitosis. 177 11

One of the profound changes in cellular morphology which occurs during mitosis is a massive alteration in the organization of the microfilament cytoskeleton. This change, together with other mitotic events including nuclear membrane breakdown, chromosome condensation and formation of mitotic spindles, is induced by a molecular complex called maturation promoting factor. This consists of at least two subunits, a polypeptide of relative molecular mass 45,000-62,000 (Mr 45-62K) known as cyclin, and a 34K catalytic subunit which has serine/threonine kinase activity and is known as cdc2 kinase. Non-muscle caldesmon, an 83K actin- and calmodulin-binding protein, is dissociated from microfilaments during mitosis, apparently as a consequence of mitosis-specific phosphorylation. We now report that cdc2 kinase phosphorylates caldesmon in vitro principally at the same sites as those phosphorylated in vivo during mitosis, and that phosphorylation reduces the binding affinity of caldesmon for both actin and calmodulin. Because caldesmon inhibits actomyosin ATPase, our results suggest that cdc2 kinase directly causes microfilament reorganization during mitosis.
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PMID:Phosphorylation of non-muscle caldesmon by p34cdc2 kinase during mitosis. 198 9

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

Phosphorylation of rat non-muscle caldesmon by cdc2 kinase causes reduction in most of caldesmon's properties, including caldesmon's binding to actin, myosin, and calmodulin, as well as its inhibition of actomyosin ATPase. We have generated and characterized the COOH terminus of caldesmon mutants lacking mitosis-specific phosphorylation sites, because the COOH-terminal half of caldesmon contains all 7 putative Ser or Thr sites for cdc2 kinase. Codons for the 7 putative Ser or Thr residues have been mutated to Ala, and resultant mutants were bacterially expressed. Analyses of the phosphopeptide maps of these mutants have identified 6 sites, including Ser-249, Ser-462, Thr-468, Ser-491, Ser-497, and Ser-527 as the mitosis-specific phosphorylation sites, whereas the phosphorylation of the remaining site, Thr-377, is not detected by this assay method. Actin binding experiments have suggested that 5 sites including Ser-249, Ser-462, Thr-468, Ser-491, and Ser-497 are important for the phosphorylation-dependent reduction in actin binding. Characterization of a mutant lacking all 7 Ser or Thr sites (7-fold mutant) has revealed that 7-fold mutation eliminates all phosphorylation sites by cdc2 kinase. While the in vitro properties of the 7-fold mutant, including actin, myosin, and calmodulin binding and inhibition of actomyosin ATPase, are very similar to those of nonmutated protein, such properties are not affected by the treatment with cdc2 kinase in contrast to nonmutated protein. This mutant should thus be useful to explore the functions of the mitosis-specific phosphorylation of caldesmon.
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PMID:Characterization of the COOH terminus of non-muscle caldesmon mutants lacking mitosis-specific phosphorylation sites. 787 50

Mitosis-specific phosphorylation by cdc2 kinase causes nonmuscle caldesmon to dissociate from microfilaments during prometaphase. (Yamashiro, S., Y. Yamakita, R. Ishikawa, and F. Matsumura. 1990. Nature (Lond.). 344:675-678; Yamashiro, S., Y. Yamakita, H. Hosoya, and F. Matsumura. 1991. Nature (Lond.) 349:169-172). To explore the functions of caldesmon phosphorylation during cytokinesis, we have examined the relationship between the phosphorylation level, actin-binding, and in vivo localization of caldesmon in cultured cells after their release of metaphase arrest. Immunofluorescence studies have revealed that caldesmon is localized diffusely throughout cytoplasm in metaphase. During early stages of cytokinesis, caldesmon is still diffusely present and not concentrated in contractile rings, in contrast to the accumulation of actin in cleavage furrows during cytokinesis. In later stages of cytokinesis, most caldesmon is observed to be yet diffusely localized although some concentration of caldesmon is observed in cortexes as well as in cleavage furrows. When daughter cells begin to spread, caldesmon shows complete colocalization with F-actin-containing structures. These observations are consistent with changes in the levels of microfilament-associated caldesmon during synchronized cell division. Caldesmon is missing from microfilaments in prometaphase cells arrested by nocodazole treatment, as shown previously (Yamashiro, S., Y. Yamakita, R. Iskikawa, and F. Matsumura. 1990. Nature (Lond.). 344:675-678). The level of microfilament-associated caldesmon stays low (12% of that of interphase cells) when some cells start cytokinesis at 40 min after the release of metaphase arrest. When 60% of cells finish cytokinesis at 60 min, the level of microfilament-associated caldesmon is recovered to 50% of that of interphase cells. The level of microfilament-associated caldesmon is then gradually increased to 80% when cells show spreading at 120 min. Dephosphorylation appears to occur during cytokinesis. It starts when cells begin to show cytokinesis at 40 min and completes when most cells finish cytokinesis at 60 min. These results suggest that caldesmon is not associated with microfilaments of cleavage furrows at least in initial stages of cytokinesis and that dephosphorylation of caldesmon appears to couple with its reassociation with microfilaments. Because caldesmon is known to inhibit actomyosin ATPase and/or regulate actin assembly, its continued dissociation from microfilaments may be required for the assembly and/or activation of contractile rings.
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PMID:Localization of caldesmon and its dephosphorylation during cell division. 838 77

Caldesmon phosphatase was identified in chicken gizzard smooth muscle by using as substrates caldesmon phosphorylated at different sites by protein kinase C, Ca2+/calmodulin-dependent protein kinase II and cdc2 kinase. Most (approximately 90%) of the phosphatase activity was recovered in the cytosolic fraction. Gel filtration after (NH4)2SO4 fractionation of the cytosolic fraction revealed a single major peak of phosphatase activity which coeluted with calponin phosphatase [Winder, Pato and Walsh (1992) Biochem. J. 286, 197-203] and myosin LC20 phosphatase. Further purification of caldesmon phosphatase was achieved by sequential chromatography on columns of DEAE-Sephacel, omega-amino-octyl-agarose, aminopropyl-agarose and thiophosphorylated myosin LC20-Sepharose. A single peak of caldesmon phosphatase activity was detected at each step of the purification. The purified phosphatase was identified as SMP-I [Pato and Adelstein (1980) J. Biol. Chem. 255, 6535-6538] by subunit composition (three subunits, of 60, 55 and 38 kDa) and Western blotting using antibodies against the holoenzyme which recognize all three subunits and antibodies specific for the 38 kDa catalytic subunit. SMP-I is a type 2A protein phosphatase [Pato, Adelstein, Crouch, Safer, Ingebritsen and Cohen (1983) Eur. J. Biochem. 132, 283-287; Winder et al. (1992), cited above]. Consistent with the conclusion that SMP-I is the major caldesmon phosphatase of smooth muscle, purified SMP-I from turkey gizzard dephosphorylated all three phosphorylated forms of caldesmon, whereas SMP-II, -III and -IV were relatively ineffective. Kinetic analysis of dephosphorylation by chicken gizzard SMP-I of the three phosphorylated caldesmon species and calponin phosphorylated by protein kinase C indicates that calponin is a significantly better substrate of SMP-I than are any of the three phosphorylated forms of caldesmon. We therefore suggest that caldesmon phosphorylation in vivo can be maintained after kinase inactivation due to slow dephosphorylation by SMP-I, whereas calponin and myosin are rapidly dephosphorylated by SMP-I and SMP-III/SMP-IV respectively. This may have important functional consequences in terms of the contractile properties of smooth muscle.
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PMID:Smooth-muscle caldesmon phosphatase is SMP-I, a type 2A protein phosphatase. 839 39

While cyclin-dependent kinase 5 (Cdk5) is widely distributed in mammalian tissues and in cultured cell lines, Cdk5-associated kinase activity has been demonstrated only in mammalian brains. An active form of Cdk5, called neuronal cdc2-like kinase (Nclk) has been purified from mammalian brain and shown to be a heterodimer of Cdk5 and a 25 kDa protein, which is derived proteolytically from a 35 kDa brain and neuron-specific protein. The protein is essential for the kinase activity of Cdk5 and is therefore designated neuronal Cdk5 activator, p25/35Nck5a. Nclk appears to have important neuronal functions. The changes in Cdk5 and Nck5a expression appear to correlate with the terminal differentiation of neurons of the mouse embryonic brain. Transfection of cultured cortical neurons with dominant negative cdk5 mutants or Nck5a antisense DNA may reduce neurite growth, suggesting that Nclk plays an active role in neuron differentiation. A number of cytoskeletal proteins including neurofilament proteins, the neuron-specific microtubule associated protein tau, and the actin binding protein caldesmon are in vitro substrates of Nclk. Although Nck5a has cyclin-like activity, it shows minimal amino acid sequence identity to members of cyclin family proteins. The mechanism of activation of Cdk5 by Nck5a differs from that of cyclin activation of Cdks in that full Cdk5 kinase activity can be achieved in the absence of phosphorylation of Cdk5. An isoform of Nck5a, a 39 kDa protein has been cloned and shown to share extensive amino acid identity and the mechanism of Cdk5 activation with Nck5a. These proteins may represent a subfamily of Cdk activators distinct from cyclins.
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PMID:Cyclin-dependent kinase 5 (Cdk5) and neuron-specific Cdk5 activators. 955 97

Human fascin is an actin-bundling protein and is thought to play a role in the formation of microfilament bundles of microspikes and stress fibers in cultured cells. To explore the regulation of fascin-actin interaction, we have examined the effects of culture cell caldesmon and tropomyosin (TM) on actin binding activity of human fascin. Caldesmon alone or TM alone has little or no effect on the actin binding of fascin. However, caldesmon together with TM completely inhibits actin binding of human fascin. When calmodulin is added, the inhibition of fascin-actin interaction by caldesmon and TM becomes Ca2+ dependent because Ca2+/calmodulin blocks actin binding of caldesmon. Furthermore, as phosphorylation of caldesmon by cdc2 kinase inhibits actin binding of caldesmon, phosphorylation can also control actin binding of fascin in the presence of TM. As expected by the inhibition of fascin-actin binding, caldesmon coupled with TM also inhibits actin bundling activity of fascin. Whereas smooth muscle caldesmon alone or TM alone shows no effect, caldesmon together with TM completely inhibits actin bundling activity of fascin. This inhibition is again Ca2+ dependent when calmodulin is added to the system. These results suggest important roles for caldesmon and TM in the regulation of the function of human fascin.
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PMID:Regulation of actin binding and actin bundling activities of fascin by caldesmon coupled with tropomyosin. 975 49


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