Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

---

Query: EC:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A Dictyostelium myosin light chain kinase has been purified approximately 15,000-fold to near homogeneity. The purified kinase is a single polypeptide of approximately 34 kDa that phosphorylates only the 18-kDa Dictyostelium myosin regulatory light chain and itself among substrates tested. The enzyme was purified largely by ammonium sulfate fractionation and hydrophobic (butyl) interaction chromatography. Analysis using polyclonal antibodies raised against the purified 34-kDa protein confirms that this protein is responsible for myosin light chain kinase activity. Protein microsequence of the 34-kDa protein reveals conserved protein kinase sequences. The purified Dictyostelium myosin light chain kinase exhibits a Km for Dictyostelium myosin of 4 microM and a Vmax of 8 nmol/min/mg. Unlike other characterized myosin light chain kinases, this enzyme is not regulated by calcium/calmodulin. Western blot analysis demonstrates that the purified kinase is not a proteolytic fragment that has lost calcium/calmodulin regulation. The Dictyostelium myosin light chain kinase activity is not directly regulated by cyclic nucleotides. However, this kinase undergoes an intramolecular autophosphorylation that activates the enzyme.
...
PMID:Dictyostelium myosin light chain kinase. Purification and characterization. 238 Jan 88

A cAMP-dependent regulatory protein which modulates the phosphorylation of scallop myosin regulatory light chain-a (RLC-a) by RLC-a myosin kinase (aMK) (Sohma, H. & Morita, F. (1986) J. Biochem. 100, 1155-1163) was purified from the scallop smooth muscle. RLC-a is abundant in the opaque portion of scallop smooth muscle, one of the catch muscles. The regulatory protein for aMK was purified by employing successively DEAE Toyopearl ion exchange chromatography, Sepharose 4B-8(6-aminohexylamino)cAMP affinity chromatography, and Sephadex G 100 gel filtration. The molecular mass of the regulatory protein was 41 kDa, based on the mobility in polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. With increasing amounts of the regulatory protein, the aMK activity decreased, and complete inhibition was observed at the concentration of twice that of aMK. The aMK activity inhibited by the regulatory protein was restored by the addition of cAMP. These results suggest that aMK is similar to a catalytic subunit of cAMP-dependent protein kinase, and the protein reported here is similar to its regulatory subunit. aMK may exist as an inactive form, as a combination with this regulatory protein, in vivo and be deinhibited by an increase in the intracellular concentration of cAMP. We discuss a possible correlation between the phosphorylation of RLC-a in myosin catalyzed by aMK and the catch state of the opaque portion of scallop smooth muscle.
...
PMID:A cAMP-dependent regulatory protein for RLC-a myosin kinase catalyzing the phosphorylation of scallop smooth muscle myosin light chain. 283 66

Synthetic peptides corresponding to the phosphorylation site in the myosin regulatory light chain from smooth muscle, Lys-Lys-Arg-Ala-Arg-Ala-Thr-Ser-Asn-Val-Phe-Ala ([Ala14,15]MLC(11-23] and containing a variety of hydroxyamino acid analogs at position 19, were tested as substrates for the smooth muscle myosin light chain kinase. Peptide analogs containing either D-serine or cis-hydroxyproline were not phosphorylated. The corresponding trans-hydroxyproline containing peptide was poorly phosphorylated with a Km of 2.3 microM and a Vmax of 3 X 10(-3) mumol.min-1.mg-1 compared to a Km of 12.5 microM and a Vmax of 1.43 mumol.min-1.mg-1 for the parent peptide. All three hydroxyamino acid analog peptides acted as relatively potent inhibitors of myosin light chain phosphorylation with Ki values in the range 7.5-10 microM, comparable to 7 microM for the parent peptide. Thus the failure of the hydroxyamino acid analog peptides to act as effective substrates was not the result of poor binding to the enzyme. In contrast, the same substitutions made in the peptide substrate for the cAMP-dependent protein kinase resulted in poor inhibitors. It is likely that the hydroxyl group of the substituting amino acids in the myosin light chain peptide analogs is not presented in the correct orientation in the active site for transfer of the phosphate group.
...
PMID:Hydroxyamino acid specificity of smooth muscle myosin light chain kinase. 334 50

Phospholamban, the cardiac sarcoplasmic reticulum proteolipid, is phosphorylated by cAMP-dependent protein kinase, by Ca2+/phospholipid-dependent protein kinase, and by an endogenous Ca2+/calmodulin-dependent protein kinase, the identity of which remains to be defined. The aim of this study was therefore to characterize the latter kinase, called phospholamban kinase. Phospholamban kinase was purified approximately 42-fold with a yield of 11%. The purified fraction exhibits a specific activity of 6.5 nmol of phosphate incorporated into exogenous phospholamban per minute per milligram of protein. Phospholamban kinase appears to be a high molecular weight enzyme and presents a broad substrate specificity, synapsin-1, glycogen synthase, and smooth muscle myosin regulatory light chain being the best substrates. Phospholamban kinase phosphorylates synapsin-1 on a Mr 30 000 peptide. The enzyme exhibits an optimum pH of 8.6, a Km for ATP of 9 microM, and a requirement for Mg2+ ions. These data suggest that phospholamban kinase might be an isoenzyme of the multifunctional Ca2+/calmodulin-dependent protein kinase. Consequently we have searched for Mr 50 000-60 000 phosphorylatable subunits among cardiac sarcoplasmic reticulum proteins. A Mr 56 000 protein was found to be phosphorylated in the presence of Ca2+/calmodulin. Such phosphorylation alters the electrophoretic migration velocity of the protein. In addition, this protein that binds calmodulin was always found to be present in fractions containing phospholamban kinase activity. This Mr 56 000 protein is therefore a good candidate for being a subunit of phospholamban kinase. However, the Mr 56 000 calmodulin-binding protein and the Mr 53 000 intrinsic glycoprotein which binds ATP are two distinct entities.
...
PMID:Characterization and partial purification of cardiac sarcoplasmic reticulum phospholamban kinase. 373 Mar 67

The level of phosphorylation of myosin regulatory light chain in BALB/c 3T3 and certain other cultured substrate-attached fibroblasts has been shown to be altered by several agents which influence cell shape, attachment and/or surface receptors. This was investigated by metabolic labelling with [32P]orthophosphate, followed by exposure of the cells to the chosen conditions, rapid freezing to 'fix' phosphorylation levels, extraction and concentration in the presence of kinase and phosphatase inhibitors, and final analysis by two-dimensional gel electrophoresis. Gel patterns were interpreted by comparison with immunoprecipitates with antiserum to mouse nonmuscle myosin. Treatment of cells either with ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) or dibutyryl-cAMP suppressed light chain phosphorylation as predicted from the control mechanisms proposed previously from in vitro studies for Ca++ calmodulin and cAMP-dependent protein kinase respectively. Other effects were less easily explained: in BALB/c 3T3 cells, contrasting with previously reported behaviour of CHO cells, the cAMP-induced decline was small and transitory; and in at least one cell line (16C) the EGTA-induced decline was preceded by a strong pulse of enhanced phosphorylation. A striking and unexpected result was that azide, almost certainly acting on mitochondrial function, caused myosin light chain phosphorylation to be maintained over a long period even in the presence of EGTA which would otherwise bring about an immediate drop. The cleavage (by trypsin) or binding (by con A) of surface receptors was also shown to trigger the biochemical modulation of cellular myosin.(ABSTRACT TRUNCATED AT 250 WORDS)
...
PMID:Myosin light chain phosphorylation in fibroblast shape change, detachment and patching. 379 39

Myosin light chain kinase was extracted from bovine aortic muscularis by a low ionic strength buffer containing 50% glycerol. It was purified 130-fold with a 10% yield by anion-exchange chromatography followed by affinity chromatography on calmodulin-Sepharose. The enzyme was 95% calcium/calmodulin-dependent and exhibited a specific activity of 2-6 mumol/min per mg. It phosphorylated the myosin regulatory light chain exclusively. The apparent Kd for calmodulin was 6.3 nM. Upon phosphorylation of the enzyme by the catalytic subunit of cyclic AMP-dependent protein kinase, its affinity for calmodulin decreased 4-fold, without alteration of the V. When examined by SDS-polyacrylamide gel electrophoresis, the purified enzyme was made up of two major peptides (Mr 142 000 and 131 000, respectively), with a minor 80 000 dalton peptide. All these peptides were 32P-labeled after incubation with [gamma-32P]ATP and the catalytic subunit of cyclic AMP-dependent protein kinase. Also, after non-denaturing polyacrylamide gel electrophoresis, they all exhibited myosin light chain kinase activity, suggesting that the 131 000 and 80 000 dalton species are proteolytic products of the native enzyme of Mr 142 000. Vascular smooth muscle myosin light chain kinase is therefore soluble, calcium/calmodulin dependent and phosphorylatable by cyclic AMP-dependent protein kinase with concomitant decrease in its affinity for calmodulin. These features account for the beta-adrenergic relaxation of vascular smooth muscle.
...
PMID:Cyclic adenosine 3',5'-monophosphate-dependent regulation of purified bovine aortic calcium/calmodulin-dependent myosin light chain kinase. 689 53

Drosophila ninaC gene encodes myosin homologous proteins which are classified as myosin III of the myosin superfamily, yet the physiological and biochemical function of myosin III has not characterized. We report here that myosin III does exhibit protein kinase activity. The kinase homologous domain (MYOIIIPK) of myosin III was expressed in the baculovirus expression system and purified to homogeneity. MYOIIIPK phosphorylated a number of proteins including myosin III p132 and smooth muscle myosin regulatory light chain (LC20), suggesting that myosin III is a multifunctional protein kinase. The phosphoamino acid analysis revealed that myosin III is a serine/threonine kinase but not a tyrosine kinase. The observation that MYOIIIPK phosphorylates myosin III suggests that the autophosphorylation might play a role for the regulation of myosin III function. This is the first direct demonstration of kinase activity for the myosin III class.
...
PMID:Identification of myosin III as a protein kinase. 875 17

Previously, we utilized small-angle X-ray scattering and neutron scattering with contrast variation to obtain the first low-resolution structure of 4Ca2+.calmodulin (CaM) complexed with a functional enzyme, an enzymatically active truncation mutant of skeletal muscle myosin light chain kinase (MLCK). These experiments showed that, upon binding to MLCK, CaM undergoes a conformational collapse identical to that observed when CaM binds to the isolated peptide corresponding to the CaM binding sequence of MLCK. CaM thereby was shown to release the inhibition of the kinase by inducing a significant movement of its CaM binding and autoinhibitory sequences away from the surface of the catalytic core [Krueger, J. K., Olah, G. A., Rokop, S. E., Zhi, G., Stull, J. T., and Trewhella, J. (1997) Biochemistry 36, 6017-6023]. We report here similar scattering experiments on the CaM.MLCK complex with the addition of substrates; a nonhydrolyzable analogue of adenosine-triphosphate, AMPPNP, and a peptide substrate for MLCK, a phosphorylation sequence from myosin regulatory light chain (pRLC). These substrates are shown to induce an overall compaction of the complex. The separation of the centers-of-mass of the CaM and MLCK components is shortened (by approximately 12 A), thus bringing CaM closer to the catalytic site compared to the complex without substrates. In addition, there appears to be a reorientation of CaM with respect to the kinase upon substrate binding that results in interactions between the N-terminal sequence of CaM and the kinase that were not observed in the complex without substrates. Finally, the kinase itself becomes more compact in the CaM.MLCK.pRLC.AMPPNP complex compared to the complex without substrates. This observed compaction of MLCK upon substrate binding is similar to that arising from the closure of the catalytic cleft in cAMP-dependent protein kinase upon binding pseudosubstrate.
...
PMID:Neutron-scattering studies reveal further details of the Ca2+/calmodulin-dependent activation mechanism of myosin light chain kinase. 976 Feb 34

Dictyostelium myosin II heavy chain kinase A (MHCK A), MHCK B, and MHCK C contain a novel type of protein kinase catalytic domain that displays no sequence identity to the catalytic domain present in conventional serine, threonine, and/or tyrosine protein kinases. Several proteins, including myelin basic protein, myosin regulatory light chain, caldesmon, and casein were phosphorylated by the bacterially expressed MHCK A, MHCK B, and MHCK C catalytic domains. Phosphoamino acid analyses of the proteins showed that 91 to 99% of the phosphate was incorporated into threonine with the remainder into serine. Acceptor amino acid specificity was further examined using a synthetic peptide library (MAXXXX(S/T)XXXXAKKK; where X is any amino acid except cysteine, tryptophan, serine, and threonine and position 7 contains serine and threonine in a 1.7:1 ratio). Phosphorylation of the peptide library with the three MHCK catalytic domains resulted in 97 to 99% of the phosphate being incorporated into threonine, while phosphorylation with a conventional serine/threonine protein kinase, the p21-activated kinase, resulted in 80% of the phosphate being incorporated into serine. The acceptor amino acid specificity of MHCK A was tested directly by substituting serine for threonine in a synthetic peptide and a glutathione S-transferase fusion peptide substrate. The serine-containing substrates were phosphorylated at a 25-fold lower rate than the threonine-containing substrates. The results indicate that the MHCKs are specific for the phosphorylation of threonine.
...
PMID:Specific phosphorylation of threonine by the Dictyostelium myosin II heavy chain kinase family. 1127 93

Phenothiazines inhibit the typical shape changes displayed by activated lymphocytes and thereby their migration through polycarbonate filters. The structure activity relationship of this effect is distinct from calmodulin inhibition. Our aim was to study this effect of phenothiazines on lymphocyte migration in an environment with living solid tissue cells. We assessed the effect of trifluoperazine and chlorpromazine (TFP and CP, two strong inhibitors of lymphocyte motility) and pimozide (PIM, a much weaker inhibitor of lymphocyte motility but a strong inhibitor of calmodulin) on invasion of human Molt-4 T-cells across precultured fibroblast monolayers. As expected invasion was inhibited by TFP and CP in the micromolar range that also inhibited motility. Surprisingly, PIM inhibited monolayer invasion at least as efficiently as TFP and CP (from 2.25 microM on). Preincubation of the monolayers or the lymphoid cells show that PIM exerted this novel invasion inhibiting effect on the monolayer. TFP and CP had a much weaker effect on the monolayer. Since these three compounds inhibit calmodulin in the same order, it is likely that this effect on the monolayer was caused by inhibition of a calmodulin-dependent pathway. KN-62, a specific inhibitor of calmodulin-dependent protein kinase II acted on the monolayer like PIM, whereas ML-7, a specific inhibitor of myosin regulatory light chain kinase, inhibited lymphoid cell motility like TFP and CP. In conclusion, invasion of T-cells across cellular monolayers is inhibited both by PIM and by phenothiazines like TFP and CP, but via distinct mechanisms: TFP and CP inhibit lymphocyte motility via a calmodulin independent pathway, whereas PIM impairs the monolayer's tolerance for invasion, most likely via a calmodulin and CamKII dependent pathway.
...
PMID:Inhibition of T-cell invasion across cultured fibroblast monolayers by phenothiazine-related calmodulin inhibitors: impairment of lymphocyte motility by trifluoperazine and chlorpromazine, and alteration of the monolayer by pimozide. 1132 35


1 2 3 Next >>

---