Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.1 (protein kinase)
 81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Calmodulin is the major intracellular Ca(2+)-binding protein, providing Ca(2+)-dependent regulation of numerous intracellular enzymes. The phosphorylation of calmodulin may provide an additional mechanism for modulating its function as a signal transducer. Phosphocalmodulin has been identified in tissues and cells, and calmodulin is phosphorylated both in vitro and in intact cells by various enzymes. Phosphorylation of calmodulin on serine/threonine residues by casein kinase II decreases its ability to activate both myosin-light-chain kinase and cyclic nucleotide phosphodiesterase. For myosin-light-chain kinase the primary effect is an inhibition of the Vmax. of the reaction, with no apparent change in the concentration at which half-maximal velocity is attained (K0.5) for either Ca2+ or calmodulin. In contrast, for phosphodiesterase, phosphorylation of calmodulin significantly increases the K0.5 for calmodulin without noticeably altering the Vmax. or the K0.5 for Ca2+. The higher the stoichiometry of phosphorylation of calmodulin, the greater the inhibition of calmodulin-stimulated activity for both enzymes. Therefore the phosphorylation of calmodulin by casein kinase II appears to provide a Ca(2+)-independent mechanism whereby calmodulin regulates at least two important target enzymes, myosin-light-chain kinase and cyclic nucleotide phosphodiesterase.
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PMID:Phosphorylation by casein kinase II alters the biological activity of calmodulin. 131 63

The smooth muscle myosin light chain kinase (smMLCK) catalytic core was modeled by using the crystallographic coordinates of the cyclic AMP-dependent protein kinase catalytic subunit (cAPK) and a bound pseudosubstrate inhibitor peptide, PKI(5-24). Despite only 30% identity in amino acid sequence, the MLCK sequence can be readily accommodated in this structure. With the exception of the short B-helix, all major elements of secondary structure in the core are very likely conserved. The active site of the modeled MLCK complements the known requirements for peptide substrate recognition. MLCK contains a pseudosubstrate sequence that overlaps the calmodulin binding domain and has been proposed to act as an intrasteric inhibitor and occupy the substrate binding site in the absence of Ca(2+)-calmodulin. The pseudosubstrate sequence can be modeled easily into the entire backbone of PKI(5-24). The results demonstrate that the intrasteric model for regulation of MLCK by intramolecular competitive inhibition is structurally plausible.
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PMID:Structural basis of the intrasteric regulation of myosin light chain kinases. 143 61

We have found that a fungal strain, Talaromyces wortmannin KY12420, produces a potent inhibitor of smooth muscle myosin light chain kinase (MLCK). This active product, designated as MS-54, was isolated and purified from the culture broth of the fungus and identified as wortmannin. The inhibition of MLCK by wortmannin was prevented by a high concentration of ATP. The activity of the catalytic domain, which was disclosed by partial tryptic digestion, was also inhibited by wortmannin. These results suggest that wortmannin acts at or near to the catalytic site of the enzyme. It was shown clearly by kinetic analyses, preincubation studies, and dialysis experiments that the inhibitory action of wortmannin on MLCK was irreversible. Under the condition of preincubation for 3 min, 0.3 microM wortmannin inhibited the activity of MLCK, while 10 microM wortmannin had no effect on the activities of cAMP-dependent protein kinase, cGMP-dependent protein kinase, and calmodulin-dependent protein kinase II, and had little effect on protein kinase C activity. These data expressed clearly the marked selectivity of the compound for MLCK. Furthermore, wortmannin also inhibited both the phosphorylation of myosin light chain and the contraction in rat thoracic aorta stimulated with KCl, which indicates the effectiveness of the compound in the cellular level as an MLCK inhibitor.
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PMID:Wortmannin, a microbial product inhibitor of myosin light chain kinase. 173 24

Proteolysis of the smooth muscle myosin-light-chain kinase with either thermolysin or endoproteinase Lys-C cleaves the enzyme towards the amino-terminus between the first and second unc domains, unc-II-1 and unc-II-2, and in the calmodulin-binding domain. The thermolytic fragment extends 532 residues from Ser275 to Ala806 and is resistant to further digestion. It is catalytically inactive and does not bind calmodulin. Further proteolysis of the thermolytic fragment with trypsin generates a constitutively active fragment. Digestion with endoproteinase Lys-C initially results in an inactive fragment of 516 residues, Ala287 to Lys802. Further digestion with Lys-C endoproteinase results in a constitutively active 474-residue fragment with the same amino-terminus, but a carboxyl-terminus at Lys760, near Arg762, the last conserved residue of protein kinase catalytic domains. There is no cleavage in the acidic-residue-rich connecting peptide between the amino-terminus of the catalytic domain and the unc-I domain, nor within the unc-II or unc-I domains or between the adjacent unc-II-2 and unc-I domains. The pattern of cleavages by these proteases reflects well the predicted domain structure of the myosin-light-chain kinase and further delineates the regulatory pseudosubstrate region. A synthetic peptide corresponding to the pseudosubstrate sequence, MLCK(787-807) was a more potent inhibitor by three orders of magnitude than the overlapping peptide MLCK(777-793) proposed by Ikebe et al. (1989) [Ikebe, M., Maruta, S. & Reardon, S. (1989) J. Biol. Chem. 264, 6967-6971] to be important in autoregulation of the myosin-light-chain kinase.
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PMID:Proteolytic cleavage sites in smooth muscle myosin-light-chain kinase and their relation to structural and regulatory domains. 191 44

Synthetic peptides corresponding to the autoinhibitory domains of calcium/calmodulin-dependent protein kinase II (CaMK-(281-309)), smooth muscle myosin light chain kinase (MLCK-(480-501)), and protein kinase C (PKC-(19-36)) as well as a peptide derived from the heat-stable inhibitor of cAMP-dependent protein kinase (PKI-tide) were tested for their inhibitory specificities. The inhibitory potencies of the four peptides were determined for each of the four protein kinases using both peptide substrates (at approximate Km concentrations) and protein substrates (at concentrations less than Km). In agreement with previous studies PKI-tide was a specific and potent inhibitor of only cAMP kinase, and none of the other inhibitory peptides gave significant inhibition of cAMP kinase at concentrations of less than 100 microM. With synthetic peptide substrates, PKC-(19-36) strongly inhibited native PKC (IC50 less than 1 microM) but also significantly inhibited autophosphorylated CaMK-II (IC50 = 30 microM) and proteolytically activated MLCK (IC50 = 35 microM). MLCK-(480-501) potently inhibited MLCK (IC50 = 0.25 microM) and also strongly inhibited both PKC and CaMK-II (IC50 = 1.4 and 1.7 microM, respectively). CaMK-(281-309) inhibited autophosphorylated CaMK-II, PKC, and proteolyzed MLCK almost equally (IC50 = 10, 38, and 48 microM, respectively). Qualitatively similar results were obtained with protein substrates. These studies validate the use of PKI-tide as a specific inhibitor of cAMP kinase in intact cell studies and suggest that PKC-(19-36) can also be used but only within a narrow concentration range. However, the autoinhibitory domain peptides from MLCK and CaMK-II are not sufficiently specific to be used in similar investigations.
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PMID:Specificities of autoinhibitory domain peptides for four protein kinases. Implications for intact cell studies of protein kinase function. 215 65

To evaluate the role of domain I of calmodulin (CaM) in the activation of target enzymes, a series of CaM mutants was constructed in which domain I (49 amino acids) was substantially deleted, or was exchanged with the homologous region (58 amino acids) of cardiac troponin C (cTnC). The proteins are 1) aM, a mutant CaM in which domain I has been deleted; 2) TaM, first domain of cTnC, last three domains of CaM; 3) TaM-BMI, same as TaM, except the nonfunctional first Ca2(+)-binding domain has been restored by mutagenesis; 4) CaT, first domain of CaM, last three domains of cTnC. These proteins were evaluated for Ca2+ binding properties and as activators of three CaM target enzymes, CaM-dependent phosphodiesterase (PDE), smooth muscle myosin light chain kinase (MLCK), and CaM-dependent multifunctional protein kinase (CaM kinase II). The chimeric proteins containing four domains bound Ca2+ in the manner expected from the number and nature of EF hands. In contrast, aM bound only two Ca2+, suggesting that deletion of domain I may have disrupted binding in one of the remaining three domains, and did not activate the three enzymes. The kinetics of activation of PDE by CaM, TaM, and TaM-BMI were identical. Although cTnC and CaT could maximally activate PDE, the Kact for these mutants were greater than 2000 times than for CaM. All mutated proteins except CaT were poor activators of CaM kinase II and this protein activated the kinase to 65% that of CaM, with a nearly identical Kact. CaT and TaM, were poor agonists of MLCK. Activation of Ca2(+)-binding site I in TaM (TaM-BMI), completely prevented activation of MLCK. In addition, TaM-BMI was a potent competitive inhibitor of MLCK activation by CaM (Ki = 66 nM). We conclude 1) a domain I is necessary to activate these target enzymes, and the substitution of the corresponding region of cTnC into CaM leads to differential effects; 2) an active first Ca2(+)-binding site is not essential for activation of PDE and the primary sequence of the first domain of CaM need not be highly conserved; 3) for CaM kinase II, determinants in the first domain are critical whereas more flexibility exists for the remaining three domains; 4) since TaM-BMI acts as a potent competitive inhibitor of MLCK binding of CaM to a target enzyme and activation can be dissociable events.
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PMID:Chimeric calmodulin-cardiac troponin C proteins differentially activate calmodulin target enzymes. 216 Sep 66

CaVPT, a target protein of Ca2(+)-vector from amphioxus muscle, was purified from its complex with CaVP after dissociation by 6 M urea and chromatographies on DEAE-cellulose and calmodulin-Sepharose. The amino acid sequence of CaVPT has been determined. The protein is composed of 243 residues and possesses an unblocked N terminus. Its molecular weight is 26,621, distinctly lower than the apparent molecular weight deduced from electrophoresis on sodium dodecyl sulfate-containing gels. CaVPT contains a potential Asn-linked glycosylation site, four potential protein kinase C phosphorylation sites, and two casein kinase II phosphorylation sites. From the sequence the following three particular domains can be inferred: a collagen-like N-terminal segment, rich in Pro and Ala, that resembles the N-terminal segment of skeletal muscle myosin light chain kinase; next to it (from residues 33 to 50) is located a strongly amphiphilic and basic alpha-helical segment which likely binds the calcium vector protein since a proteolytic cut after Arg50, occurring occasionally during the purification of CaVPT, impairs the binding to immobilized calmodulin. This segment is followed by two immunoglobulin folds. The two immunoglobulin folds typically belong to the C2 subclass and particularly resemble those present in the neural cell surface adhesion molecules NCAM, L1, F11, MAG, TAG-1, fasciclin II, and amalgam. Recently, the presence of immunoglobulin folds of this type has been reported in some intracellular muscular proteins, namely in smooth muscle myosin light chain kinase, striated muscle C protein and titin, as well as in the nematode 600-kDa protein twitchin. From this structural study we can formulate the working hypothesis that CaVPT acts on the structure of the thick filament in muscle or regulates, perhaps via other immunoglobulin fold-containing proteins.
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PMID:Primary structure of the target of calcium vector protein of amphioxus. 224 56

A 40-kDa fragment of chicken smooth muscle myosin light chain kinase was produced and partially purified from a bacterial expression system. This fragment exhibits calmodulin binding and substrate phosphorylation properties similar to those of the isolated chicken gizzard enzyme. A series of 3'-deletion mutants was prepared and used to produce proteins with the same NH2 terminus but with COOH termini varying over 180 amino acids. Results show that truncation of the enzyme at Ser-512 (based on the amino acid numbering system described for the partial cDNA clone by Guerriero, V., Jr., Russo, M. A., Olson, N. J., Putkey, J. A., and Means, A. R. (1986) Biochemistry 25, 8372-8381) does not alter calmodulin binding, calmodulin regulation, or enzymatic properties. Removal of an additional 5 residues from the COOH terminus completely inhibits calmodulin binding and results in an inactive kinase that can be fully activated by limited proteolysis. Site specific mutations within these 5 residues demonstrate that Gly-508 and Arg-509 are independently involved in calmodulin-dependent binding and activation of myosin light chain kinase. Truncation of the enzyme at residues within the protein kinase catalytic domain results in inactive protein that cannot be activated by proteolysis.
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PMID:Myosin light chain kinase structure function analysis using bacterial expression. 267 19

The isolation of an acidic protein, pI 4.5, that is abundant in turkey gizzard is described. Its apparent molecular weight measured by electrophoretic procedures is 24,000. This protein is phosphorylated by the catalytic subunit of the cAMP-dependent protein kinase and one phosphorylation site is indicated. From sequence determinations of tryptic peptides it is concluded that this protein is closely related to the C-terminal part of smooth muscle myosin light chain kinase. The initiation site for the protein is to the C-terminal side of the calmodulin-binding site. From the sequence data an estimated molecular weight is 18,000. This protein is expressed independently, as indicated by a blocked N terminus, and is probably the translation product of the 2.7-kilobase RNA detected previously in chicken gizzard (Guerriero, V., Jr., Russo, M. A., Olson, N. J., Putkey, J. A., and Means, A. R. (1986) Biochemistry 25, 8372-8381). Because of its putative origin as the C-terminal end of smooth muscle myosin light chain kinase, it is termed "telokin" (from a combination of kinase and the Greek telos, "end").
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PMID:Identification in turkey gizzard of an acidic protein related to the C-terminal portion of smooth muscle myosin light chain kinase. 276 53

N-(6-Aminoethyl)-5-chloro-1-naphthalenesulfonamide (A-3), which is a shorter alkyl chain derivative of the calmodulin (CaM) antagonist, W-7, was found to inhibit smooth muscle myosin light chain kinase (MLC-kinase) through a mechanism different from that related to W-7. Both the holoenzyme and the catalytic fragment, which is active without CaM, were susceptible to A-3 with a similar concentration dependency, thereby indicating that the inhibitory effect is due to the direct interaction of the compound with the enzyme molecule and not with the enzyme activator. Naphthalenesulfonamides are both CaM antagonists and direct inhibitors of MLC-kinase, and these actions depend on the length of the alkyl chain (C2-C6). Although the potencies in inhibiting CaM functions increased, the direct effects on MLC-kinase decreased with extension of the carbon chain of the derivatives. Kinetic studies indicated that A-3 inhibited MLC-kinase competitively with respect to ATP and that the Ki value was 7.4 microM. A-3 was also a competitive inhibitor of cAMP-dependent protein kinase, cGMP-dependent protein kinase, protein kinase C, casein kinase I, and casein kinase II, with respect to ATP. The Ki values of naphthalenesulfonamides for these enzymes also increased with extension of the carbon chain of the derivatives. These results suggest that naphthalenesulfonamides inhibit protein phosphorylation not only by inhibition of the enzyme-activating process but also by inhibition of the catalytic process. The mode of interaction between the derivatives and protein kinases differs from the interaction between the derivatives and CaM.
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PMID:Naphthalenesulfonamides as calmodulin antagonists and protein kinase inhibitors. 287 89


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