Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.17 (CaMKII)
 4,029 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Calmodulin, the ubiquitous and multifunctional Ca(2+)-binding protein, mediates many of the regulatory effects of Ca2+, including the contractile state of smooth muscle. The principal function of calmodulin in smooth muscle is to activate crossbridge cycling and the development of force in response to a [Ca2+]i transient via the activation of myosin light-chain kinase and phosphorylation of myosin. A distinct calmodulin-dependent kinase, Ca2+/calmodulin-dependent protein kinase II, has been implicated in modulation of smooth-muscle contraction. This kinase phosphorylates myosin light-chain kinase, resulting in an increase in the calmodulin concentration required for half-maximal activation of myosin light-chain kinase, and may account for desensitization of the contractile response to Ca2+. In addition, the thin filament-associated proteins, caldesmon and calponin, which inhibit the actin-activated MgATPase activity of smooth-muscle myosin (the cross-bridge cycling rate), appear to be regulated by calmodulin, either by the direct binding of Ca2+/calmodulin or indirectly by phosphorylation catalysed by Ca2+/calmodulin-dependent protein kinase II. Another level at which calmodulin can regulate smooth-muscle contraction involves proteins which control the movement of Ca2+ across the sarcolemmal and sarcoplasmic reticulum membranes and which are regulated by Ca2+/calmodulin, e.g. the sarcolemmal Ca2+ pump and the ryanodine receptor/Ca2+ release channel, and other proteins which indirectly regulate [Ca2+]i via cyclic nucleotide synthesis and breakdown, e.g. NO synthase and cyclic nucleotide phosphodiesterase. The interplay of such regulatory mechanisms provides the flexibility and adaptability required for the normal functioning of smooth-muscle tissues.
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PMID:Calmodulin and the regulation of smooth muscle contraction. 781 54

We investigated the presence of and the endogenous substrates for Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) in cultured bovine adrenal medullary cells. By a series of chromatographic steps using DEAE-cellulose, calmodulin affinity, and Sephacryl S-300 columns, we partially purified two CaM kinases (peaks I and III) and one calmodulin-binding protein (peak II). Both of the kinases (peaks I and III) showed broad substrate specificities. Peak I, but not peak III, was immunoprecipitated with an antibody against rat brain CaM kinase II, suggesting that peak I is CaM kinase II or a closely associated CaM kinase. Although the anticaldesmon antibody recognized a 77-kDa protein (low molecular mass caldesmon) in crude preparations from the cells, the protein in peak II was not immunoblotted with the antibody. The peak II protein was phosphorylated by the CaM kinase in peak I but not by the CaM kinase in peak III. Peak I kinase also phosphorylated purified tyrosine hydroxylase and several proteins from chromaffin granule membranes. Stimulation of cultured bovine adrenal medullary cells with 56 mM K+ evoked rapid increases in 45Ca2+ influx and autonomous CaM kinase II activity, both of which were attenuated by the addition of 20 mM MgSO4, an inhibitor of voltage-dependent Ca2+ channels. These results suggest that an isozyme of CaM kinase II exists in adrenal medullary cells and is activated by cell depolarization. Furthermore, the peak II protein is apparently a novel endogenous substrate for CaM kinase II.
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PMID:Occurrence and activation of Ca2+/calmodulin-dependent protein kinase II and its endogenous substrates in bovine adrenal medullary cells. 793 21

Endogenous caldesmon kinase activity in sheep aorta smooth muscle was purified and characterized. The enzyme was identified as a proteolytic fragment of protein kinase C by cross-reactivity with anti-protein kinase C antibodies, autophosphorylation, substrate specificity and the primary structure of the sites of phosphorylation on caldesmon. The enzyme phosphorylated aorta caldesmon both in native thin filaments and in the isolated state. Up to 2.9 mols of phosphate per mol of caldesmon were transferred. Prolonged incubation of caldesmon with the kinase resulted in phosphorylation of Ser-127, Ser-587, Ser-600, Ser-657, Ser-686, and Ser-726 (numbering corresponds to chicken gizzard caldesmon sequence). Ser-600 and Ser-587 were the major sites of phosphorylation containing more than 30% of phosphate transferred. Phosphorylation did not significantly affect the interaction of caldesmon with Ca(2+)-calmodulin. However, phosphorylation of both intact caldesmon and of its C-terminal fragment (658C), containing residues 658-756, significantly decreased their ability to inhibit acto-heavy meromyosin ATPase. This seems to be partially due to a decrease in the binding of caldesmon and 658C to actin-tropomyosin and partly due to an uncoupling of the binding-inhibition relationship.
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PMID:Phosphorylation of aorta caldesmon by endogenous proteolytic fragments of protein kinase C. 818 8

A caldesmon kinase activity was detected in an ATP extract of the myofibril-like pellet from sheep aorta. The enzyme was purified 745-fold and was identified as casein kinase II on the basis of molecular size, substrate specificity, and high sensitivity to heparin inhibition. Casein kinase II phosphorylated isolated caldesmon and caldesmon incorporated into native thin filaments, and transferred about 1 mol of phosphate per mol of caldesmon-h. Ser-73 was the main site phosphorylated by casein kinase II in chicken gizzard caldesmon. Phosphorylation of caldesmon reduced its affinity for smooth muscle myosin but had no effect upon the ability of caldesmon to inhibit the ATPase activity of actomyosin.
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PMID:Identification of casein kinase II as a major endogeneous caldesmon kinase in sheep aorta smooth muscle. 822 19

Rat vascular smooth muscle cells were synchronized to the quiescent state (G0) by serum deprivation and then stimulated to enter the cell cycle by serum refeeding. At various times of the cell cycle, cells were analyzed for the expression of p34cdc2 and mitogen-activated protein kinase homologues by immunoblotting and for kinase activity toward histone H1, myelin basic protein, and caldesmon. A small amount of p34cdc2 was expressed in the G0/G1 phase (0 to 8 hours). At the G1/S transition (12 hours), the level of p34cdc2 started to accumulate and increased by 60-fold at G2/M (18 hours), accompanied by a more slowly migrating band. Histone H1 kinase activity was undetectable in anti-p34cdc2 immunoprecipitates in the G0/G1 cells but appeared around the G1/S boundary and peaked at G2/M (18 hours). The caldesmon kinase activity exhibited two distinct phases: the first appeared at G0/G1 (0 to 8 hours), and the second appeared at G1/S and continued through G2/M. Two mitogen-activated protein kinase isoforms were expressed throughout the cell cycle. Anti-mitogen-activated protein kinase immunoprecipitates possessed kinase activities toward myelin basic protein and caldesmon, which were activated within 15 minutes after serum stimulation and declined within a few hours. These findings suggest that p34cdc2 and mitogen-activated protein kinase homologues may play significant roles in regulating the progression of the cell cycle of smooth muscle cells, the former at the G2/M transition and the latter at the G0/G1 transition.
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PMID:Smooth muscle cell proliferation. Expression and kinase activities of p34cdc2 and mitogen-activated protein kinase homologues. 838 75

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

h-Caldesmon in vascular smooth muscle is phosphorylated in response to pharmacologic stimulation. Although many kinases phosphorylate h-caldesmon, in vitro, the responsible kinase in intact tissue is unknown. The sites of phosphorylation in caldesmon from intact canine aortas have recently been identified and are consensus sequences for a proline-directed protein kinase. In this study, we investigated the phosphorylation of h-caldesmon by mitogen-activated protein kinase (MAPK). Purified, recombinant MAPK phosphorylated porcine stomach h-caldesmon to a stoichiometry approaching 2 mol phosphate/mol protein. Phosphorylated h-caldesmon was subjected to proteolysis and the phosphopeptides were purified by high performance liquid chromatography. Two major phosphopeptides were identified and sequenced. These two peptides, VTS*PTKV and S*PAPK, were identical to the sequences of the sites phosphorylated in intact tissue. Antibodies to several enzymes implicated in the cascade of activation of MAPK were used to evaluate vascular smooth muscle by Western blotting. All components were found to be present. These data suggest that MAPK can function as a 'caldesmon kinase' in vascular smooth muscle.
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PMID:Identification of mitogen-activated protein kinase phosphorylation sequences in mammalian h-Caldesmon. 848 68

1. Phosphorylation of caldesmon was assayed in canine colonic circular smooth muscle strips labelled with 32P and stimulated with 10 microM acetylcholine. Caldesmon was isolated by two-dimensional non-equilibrium pH gel electrophoresis. Stimulation with acetylcholine increased caldesmon phosphorylation significantly from a basal level of 0.6 +/- 0.07 to 1.1 +/- 0.15 mol P1 (mol caldesmon)-1 after 2 min. 2. MAP kinase activities were measured in SDS extracts of muscle by a gel reconstitution method using myelin basic protein. Myelin basic protein kinase activities were observed at 38, 44, 50 and 57 kDa by the gel reconstitution method. Endogenous caldesmon kinase activities were also identified by the gel reconstitution method at 38, 44 and 50 kDa. The 38 and 44 kDa kinases comigrated with proteins labelled by anti-ERK1 MAP kinase antibodies on Western blots. Both 38 and 44 kDa MBP kinase activities increased significantly during contractions induced by 10 microM acetylcholine, 0.1 microM neurokinin A and 70 mM potassium. 3. Phorbol dibutyrate (0.1 microM) potentiated activation of MAP kinases and contraction of depolarized muscles while producing a decrease in fura-2 fluorescence ratio. This suggests that protein kinase C activation is coupled to MAP kinase activity in colonic smooth muscle. 4. MAP kinases isolated form muscle homogenates by Mono Q chromatography were assayed using the specific MAP kinase substrate peptide APRTPGGRR. Stimulation of muscles for 2 min with 10 microM acetylcholine activated both ERK1 and ERK2 MAP kinase activities 2-fold. 5. To determine the effects of caldesmon phosphorylation by MAP kinase on the cross-bridge cycle, actin sliding velocity was measured with an in vitro motility assay. Unphosphorylated turkey gizzard caldesmon (3 microM) significantly reduced mean sliding velocity. Phosphorylation of caldesmon with sea star ERK1 MAP kinase reversed the inhibitory effect of caldesmon on sliding velocity. The results are consistent with a protein kinase cascade being activated by contractile agonists in gastrointestinal smooth muscle which activates ERK MAP kinases leading to phosphorylation of caldesmon. Phosphorylation of caldesmon in vivo may reverse inhibitory influences of caldesmon on cross-bridge cycling.
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PMID:Activation of MAP kinases and phosphorylation of caldesmon in canine colonic smooth muscle. 888 69

Caldesmon inhibits myosin ATPase activity; phosphorylation of caldesmon reverses the inhibition. The caldesmon kinase is believed to be mitogen-activated protein (MAP) kinase. MAP kinases are activated during vascular stimulation, but a cause-and-effect relationship between kinase activity and contraction has not been established. We examined the role of MAP kinase in contraction using PD-098059, an inhibitor of MAP kinase kinase (MEK). MAP kinase activity was assessed using an anti-active MAP kinase antibody and direct measurement of MAP kinase catalyzed phosphorylation of myelin basic protein, MBP-(95-98). MAP kinase phosphorylation, stimulated by histamine (50 microM) or phorbol 12,13-dibutyrate (PDBu, 0.1 microM), was inhibited by PD-098059 (100 microM). PD-098059 did not alter the sensitivity or the maximal level of force in smooth muscle stimulated by histamine or PDBu, nor did PD-098059 affect contraction of beta-escin-permeabilized tissue. Our data suggest that p44 and p42 MAP kinases are not involved in regulation of vascular smooth muscle contraction. These results do not, however, preclude a role for other isoforms of the MAP kinase family.
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PMID:Inhibition of p42 and p44 MAP kinase does not alter smooth muscle contraction in swine carotid artery. 968 5

The effect of direct phosphorylation by recombinant p44erk1 mitogen-activated protein kinase on the inhibitory activity of caldesmon and its C-terminal fragment H1 was studied in vitro. Neither inhibition of actin-tropomyosin activated ATPase of heavy meromyosin by caldesmon or H1, nor inhibition of the actin-tropomyosin motility over heavy meromyosin by H1 was significantly affected by the phosphorylation while only a moderate effect on the actin-activated component of heavy meromyosin ATPase inhibition was observed. Phosphopeptide mapping of caldesmon immunoprecipitated from [32P]PO4-labelled intact gizzard strips revealed that it is predominantly phosphorylated at mitogen-activated protein kinase sites in unstimulated tissue and that it is stimulated for 1 h with phorbol 12,13-dibutyrate. We find that phorbol 12,13-dibutyrate also induces a transitory phosphorylation of caldesmon peaking at 15 min after addition and this phosphorylation is not attributed to mitogen-activated protein kinase, protein kinase C, Ca2+/calmodulin-dependent kinase II or casein kinase II. We suggest that a yet unidentified kinase, rather than mitogen-activated protein kinase, may be involved in regulation of the caldesmon function in vivo.
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PMID:Evidence against the regulation of caldesmon inhibitory activity by p42/p44erk mitogen-activated protein kinase in vitro and demonstration of another caldesmon kinase in intact gizzard smooth muscle. 1038 1


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