Phosphorylation of smooth muscle myosin light chain kinase by the catalytic subunit of adenosine 3': 5'-monophosphate-dependent protein kinase.

Turkey gizzard smooth muscle light chain kinase was purified by affinity chromatography on calcium dependent regulator weight of 125,000 +/- 5,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. When myosin light chain kinase is incubated with the catalytic subunit of cyclic AMP-dependent protein kinase, 1 mol of phosphate is incorporated per mol of myosin kinase. Brief tryptic digestion of the 32P-labeled myosin kinase liberates a single radioactive peptide with a molecular weight of approximately 22,000. Phosphorylation of myosin kinase results in a 2-fold decrease in the rate at which the enzyme phosphorylates the 20,000-dalton light chain of smooth muscle myosin. These results suggest that cyclic AMP has a direct effect on actin-myosin interaction in smooth muscle.     

Phosphorylation of smooth muscle actin by the catalytic subunit of the cAMP-dependent protein kinase

Partially purified smooth muscle (chicken gizzard) actomyosin contains two major substrates of cAMP-dependent protein kinase: a protein of M_r = 130,000, identified as the calmodulin-dependent myosin light chain kinase, and a protein of M_r = 42,000. This latter protein was shown by a variety of electrophoretic procedures to be actin. Purified smooth muscle actin also was phosphorylated by the catalytic subunit of cAMP-dependent protein kinase. The rate of phosphorylation of smooth muscle actin was significantly enhanced by depolyjerization of actin. A maximum of 2.0 mol phosphate could be incorporated per mol G-actin. Skeletal muscle F-actin was not significantly phosphorylated by protein kinase; however, skeletal G-actin is a substrate for the protein kinase although its rate of phosphorylation was significantly slower than that of smooth muscle G-actin.     

Phosphorylation of skeletal muscle myosin light chain kinase by the catalytic subunit of cAMP-dependent protein kinase

Phosphatidylethanolamine (PE) was isolated from membranes of Bacillus megaterium. The organism was grown at 20°C and 55°C. The phase equilibria in PE/water systems were studied by ²H and ³¹P nuclear magnetic resonance, and by polarized light microscopy. PE isolated from B. megaterium grown at 20°C forms a lamellar liquid crystalline phase at the growth temperature, and at low water contents a cubic liquid crystalline phase at 58°C. The ratio iso/ante-iso acyl chains was 0.3 in this lipid. PE isolated from this organism grown at 55°C forms only a lamellar liquid crystalline phase up to at least 65°C. In this lipid the ratio iso/ante-iso acyl chains was 3.2.     

A cAMP-dependent regulatory protein for RLC-a myosin kinase catalyzing the phosphorylation of scallop smooth muscle myosin light chain

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.     

Functional interactions between smooth muscle myosin light chain kinase and calmodulin

Calmodulin (CaM) binding by turkey gizzard myosin light chain kinase (MLCK) causes subtle changes in the fluorescence emission and polarization excitation spectra of the enzyme. Fluorescence experiments using 9-anthroyl-choline (9AC), which competes with ATP in binding, demonstrate mutually stabilizing interactions between the CaM and ATP binding sites corresponding to delta G = -0.6 to -0.7 kcal/mol. Fluorescence titrations in the presence of 9AC or 5,5'-bis[8-(phenylamino)-1-naphthalenesulfonate] confirm the stoichiometry of 1 mol of CaM/MLCK. Phosphorylation of MLCK has no effect on either the protein fluorescence or the binding of ATP and 9AC. The dissociation constant for the MLCL-CaM complex is increased approximately 500-fold on phosphorylation. Values of Kd for the phosphorylated enzyme range from 0.5 to 1.1 microM in 0.2 N KCl, pH 7.3, 25 degrees C. We showed competition between MLCK and other CaM binding proteins and peptides by using both fluorescence and catalytic activity measurements. Competition for CaM occurs with ACTH, beta-endorphin, substance P, glucagon, poly(L-arginine), myelin basic protein, troponin I, and histone H2A. Phosphorylation of the last three proteins by the adenosine cyclic 3',5'-phosphate dependent protein kinase diminishes their ability to compete. Phosphorylation of MLCK by the protein kinase gives 0.95 +/- 0.04 and 2.2 +/- 0.4 mol of incorporated 32P in the presence and absence of CaM, respectively. These stoichiometries agree with those recently reported [Conti, M. A. & Adelstein, R. S. (1981) J. Biol. Chem. 256, 3178].     

Purification and characterization of catalytic subunit of skeletal muscle adenosine 3':5'-monophosphate-dependent protein kinase.

The catalytic subunit of rabbit skeletal muscle cyclic adenosine 3':5'-monophosphate-dependent protein kinase has been isolated in pure form. It has a molecular weight of 41,300, as determined by sedimentation equilibrium, which is in good agreement with the value of 41,000 determined by electrophoresis in the presence of sodium dodecyl sulfate. Sedimentation velocity determinations indicate that the subunit has an S20,w value of 3.12 which is essentially independent of protein concentration. These experiments are interpreted as indicating that the catalytic subunit dissociated from the holoenzyme exists as a monomer in solution. The least abundant amino acid is half-cystine, which was calculated to be present at 2.8 mol/mol of protein. The sulfhydryl reagents, N-ethylmaleimide, p-chloromercuribenzoic acid, and 5,5'-dithiobis(2-nitrobenzoic acid) inhibit the enzymatic activity of the subunit; inhibition by the two latter compounds can be reversed by 2-mercaptoethanol. Binding of 1 mol of N-ethylmaleimide/mol of protein results in almost complete inhibition. The isolated catalytic subunit contains 2.2 mol of tightly bound phosphate/mol of protein. Identification of either O-phosphoserine or O-phosphothreonine after partial acid hydrolysis indicates that at least part of the endogeneous phosphate exists as the phospho ester of one of these amino acids. Two peaks of catalytic activity corresponding to isoelectric points of pH 7.4 and 8.5 were identified by isoelectric focusing. Both forms utilize the same substrates and have similar sedimentation constants.     

Phosphorylation of skeletal and cardiac muscle C-proteins by the catalytic subunit of cAMP-dependent protein kinase

Catecholamines are known to influence the contractility of cardiac and skeletal muscles, presumably via cAMP-dependent phosphorylation of specific proteins. We have investigated the in vitro phosphorylation of myofibrillar proteins by the catalytic subunit of cAMP-dependent protein kinase of fast- and slow-twitch skeletal muscles and cardiac muscle with a view to gaining a better understanding of the biochemical basis of catecholamine effects on striated muscles. Incubation of canine red skeletal myofibrils with the isolated catalytic subunit of cAMP-dependent protein kinase and Mg-[gamma-32P]ATP led to the rapid incorporation of [32P]phosphate into five major protein substrates of subunit molecular weights (MWs) 143,000, 60,000, 42,000, 33,000, and 11,000. The 143,000 MW substrate was identified as C-protein; the 42,000 MW substrate is probably actin; the 33,000 MW substrate was shown not to be a subunit of tropomyosin and, like the 60,000 and 11,000 MW substrates, is an unidentified myofibrillar protein. Isolated canine red skeletal muscle C-protein as phosphorylated to the extent of approximately 0.5 mol Pi/mol C-protein. Rabbit white skeletal muscle and bovine cardiac muscle C-proteins were also phosphorylated by the catalytic subunit of cAMP-dependent protein kinase, both in myofibrils and in the isolated state. Cardiac C-protein was phosphorylated to the extent of 5-6 mol Pi/mol C-protein, whereas rabbit white skeletal muscle C-protein was phosphorylated at the level of approximately 0.5 mol Pi/mol C-protein. As demonstrated earlier by others, C-protein of skeletal and cardiac muscles inhibited the actin-activated myosin Mg2+-ATPase activity at low ionic strength in a system reconstituted from the purified skeletal muscle contractile proteins (actin and myosin).(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential binding of cAMP-dependent protein kinase regulatory subunit isoforms Ialpha and IIbeta to the catalytic subunit

Limited trypsin digestion of type I cAMP-dependent protein kinase holoenzyme results in a proteolytic-resistant Delta(1-72) regulatory subunit core, indicating that interaction between the regulatory and catalytic subunits extends beyond the autoinhibitory site in the R subunit at the NH(2) terminus. Sequence alignment of the two R subunit isoforms, RI and RII, reveals a significantly sequence diversity at this specific region. To determine whether this sequence diversity is functionally important for interaction with the catalytic subunit, specific mutations, R133A and D328A, are introduced into sites adjacent to the active site cleft in the catalytic subunit. While replacing Arg(133) with Ala decreases binding affinity for RII, interaction between the catalytic subunit and RI is not affected. In contrast, mutant C(D328A) showed a decrease in affinity for binding RI while maintaining similar affinities for RII as compared with the wild-type catalytic subunit. These results suggest that sequence immediately NH(2)-terminal to the consensus inhibition site in RI and RII interacts with different sites at the proximal region of the active site cleft in the catalytic subunit. These isoform-specific differences would dictate a significantly different domain organization in the type I and type II holoenzymes.     

Inhibition of the catalytic subunit of cAMP-dependent protein kinase by dicyclohexylcarbodiimide

The hydrophobic carbodiimide dicyclohexylcarbodiimide (DCCD) has been shown to inhibit the catalytic (C) subunit of adenosine cyclic 3',5'-phosphate dependent protein kinase (EC 2.7.1.3) in a time-dependent, irreversible manner. The rate of inactivation was first order and showed saturation kinetics with an apparent Ki of 60 microM. Magnesium adenosine 5'-triphosphate (MgATP) was capable of protecting against this inhibition, whereas neither a synthetic peptide substrate nor histone afforded protection. Mg alone afforded some protection. When the catalytic subunit was aggregated with the regulatory subunit in the holoenzyme complex, no inhibition was observed. The inhibition was enhanced at low pH, suggesting that a carboxylic acid group was the target for interaction with DCCD. On the basis of the protection studies, it is most likely that this carboxylic acid group is associated with the MgATP binding site, perhaps serving as a ligand for the metal. Efforts to identify the site that was modified by DCCD included (1) modification with [14C]DCCD, (2) modification by DCCD in the presence of [3H]aniline, and (3) modification with DCCD and [14C]glycine ethyl ester. In no case was radioactivity incorporated into the protein, suggesting that the irreversible inhibition was due to an intramolecular cross-link between a reactive carboxylic acid group and a nearby amino group. Differential peptide mapping identified a single peptide that was consistently lost as a consequence of DCCD inhibition. This peptide (residues 166-189) contained four carboxylic acid residues as well as an internal Lys.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of smooth muscle myosin conformation on myosin light chain kinase binding and on phosphorylation

Conventional smooth muscle myosin preparations contain a tightly bound myosin light chain kinase activity, which is incompletely removed by gel filtration at high ionic strength. We show here that by contrast, this kinase activity is released, together with calmodulin, under conditions in which myosin is in the folded configuration. The conformation-related release of kinase occurred for dephosphorylated myosin in both the presence and absence of ATP and Ca2+. Binding of kinase to extended phosphorylated myosin was relatively weaker than to dephosphorylated myosin, but was nonetheless detected. The kinetic consequences of this binding behaviour were determined by measuring initial myosin phosphorylation rates as a function of KCl concentration. Rate optima occurred at 60 mM KCl and 300 mM KCl, conditions favouring respectively stable filaments and stable extended monomers. Phosphorylation of the folded monomer was uniformly slow at low KCl concentrations. The folded myosin monomer is thus a relatively poor substrate for the kinase, and is therefore unlikely to represent an analog of the relaxed crossbridge configuration in myosin filaments.     

Mechanism of calmodulin inhibition of cAMP-dependent protein kinase activation of phosphorylation kinase

The activation of phosphorylase kinase (EC 2.7.1.38; ATP:phosphorylase b phosphotransferase) by the catalytic subunit of cAMP-dependent protein kinase (EC 2.7.1.37; ATP:protein phosphotransferase) is inhibited by calmodulin. The mechanism of that inhibition has been studied by kinetic measurements of the interactions of the three proteins. The binding constant for calmodulin with phosphorylase kinase was found to be 90 nM when measured by fluorescence polarization spectroscopy. Glycerol gradient centrifugation studies indicated that 1 mol of calmodulin was bound to each phosphorylase kinase. Phosphorylation of the phosphorylase kinase did not reduce the amount of calmodulin bound. Kinetic studies of the activity of the catalytic subunit of cAMP-dependent protein kinase on phosphorylase kinase as a function of phosphorylase kinase and calmodulin concentrations were performed. The results of those studies were compared with mathematical models of four different modes of inhibition: competitive, noncompetitive, substrate depletion, and inhibition by a complex between phosphorylase kinase and calmodulin. The data conform best to the model in which the inhibitory species is a complex of phosphorylase kinase and calmodulin. The complex apparently competes with the substrate, phosphorylase kinase, which does not have exogenous calmodulin bound to it. In contrast, the phosphorylation of the synthetic phosphate acceptor peptide, Kemptide, is not inhibited by calmodulin.     

Some properties of the catalytic subunit of adenosine 3':5'-monophosphate-dependent protein kinase from pigeon breast muscle]

A method for the preparation of a homogenous catalytic subunit of adenosine 3':5'-monophosphate-dependent protein kinase from pigeon breast muscle was developed. The molecular weight of the enzyme as determined by electrophoresis in the presence of sodium dodecyl sulfate was found to be 42000. The pH optimum of the catalytic subunit was around 8.0. The active site of the catalytic subunit was studied using some derivatives of ATP, containing different reactive groups in the triphosphate chain of the molecule. It may be assumed that the pH optimum of the enzyme inactivation by adenosine 5'-chloromethylpyrophosphonate and the protective effect of ATP suggest covalent binding of the imidazole ring in the enzyme active site. The kinetic mechanism of the protein kinase reaction was studied using the initial rate experiments and reaction product inhibition. The results obtained were consistent with a random Bi-Bi kinetic mechanism.     

Autophosphorylation of the catalytic subunit of cAMP-dependent protein kinase.

The catalytic subunit of cAMP-dependent protein kinase contains two stable phosphorylation sites, Thr-197 and Ser-338 (Shoji, S., Titani, K., Demaille, J. G., and Fischer, E. H. (1979) J. Biol. Chem. 254, 6211-6214). Thr-197 is very resistant to dephosphorylation and thus cannot typically be autophosphorylated in vitro once the stable subunit is formed. Ser-338 is slowly dephosphorylated and can be rephosphorylated autocatalytically. In addition to these two stable phosphorylation sites, a new site of autophosphorylation, Ser-10, was identified. Phosphorylation at Ser-10 does not have a major effect on activity, and phosphates from Ser-10 or Ser-338 are not transferred to physiological substrates such as the type II regulatory subunit. Autophosphorylation at Ser-10 is associated with one of the two major isoelectric variants of the catalytic subunit. The form having the more acidic pI can be autophosphorylated at Ser-10 while the more basic form of the catalytic subunit cannot. Phosphorylation at Ser-10 does not account for the two isoenzyme forms. Since the reason for two isoelectric variants of the catalytic subunit is still unknown, it is not possible to provide a structural basis for the difference in accessibility of Ser-10 to phosphorylation. Either Ser-10 is not accessible in the more basic form of the catalytic subunit or some other type of post- or cotranslational modification causes Ser-10 to be a poor substrate. Whether the myristoyl group at the amino-terminal Gly is important for Ser-10 autophosphorylation remains to be established. The isoenzyme forms of the catalytic subunit do not correspond to the gene products coded for by the C alpha and C beta genes.     

The calmodulin binding domain of chicken smooth muscle myosin light chain kinase contains a pseudosubstrate sequence

Smooth muscle myosin light chain kinase contains a 64 residue sequence that binds calmodulin in a Ca2+-dependent manner (Guerriero, V., Jr., Russo, M. A., and Means, A. R. (1987) Biochemistry, in press). Within this region is a sequence with homology to the corresponding sequence reported for the calmodulin binding region of skeletal muscle myosin light chain kinase (Blumenthal, D. K., Takio, K., Edelman, A. M., Charbonneau, H., Titani, L., Walsh, K. A., and Krebs, E. G. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 3187-3191). Inspection of these sequences reveals that they both share a similar number and spatial arrangement of basic residues with those present in the myosin light chain substrate. We have synthesized a 22-residue peptide corresponding to residues 480-501 (determined from the cDNA) of the smooth muscle myosin light chain kinase. This peptide, Ala-Lys-Lys-Leu-Ser-Lys-Asp-Arg-Met-Lys-Lys-Tyr-Met-Ala-Arg-Arg-Lys-Trp- Gln-Lys-Thr-Gly, inhibited calmodulin-dependent activation of the smooth muscle myosin light chain kinase with an IC50 of 46 nM. At saturating concentrations of calmodulin, the 22-residue peptide inhibited myosin light chain and synthetic peptide substrate phosphorylation competitively with IC50 values of 2.7 and 0.9 microM, respectively. An 11-residue synthetic peptide analog, corresponding to part of the calmodulin-binding sequence in skeletal muscle myosin light chain kinase, Lys-Arg-Arg-Trp-Lys-Lys-Asn-Phe-Ile-Ala-Val, also competitively inhibited synthetic peptide substrate phosphorylation with a Ki of 1 microM. The competitive inhibitory activity of the calmodulin binding regions is similar to the apparent Km of 2.7 microM for phosphorylation of the 23-residue peptide analog of the smooth muscle myosin light chain and raises the possibility that the calmodulin binding region of the myosin light chain kinase may act as a pseudosubstrate inhibitor of the enzyme.     

Phosphorylation of type II regulatory subunit of cAMP-dependent protein kinase in intact smooth muscle

A monoclonal antibody was used to quantitate changes in the extent of phosphorylation of the type II regulatory subunit of cAMP-dependent protein kinase in intact bovine tracheal smooth muscle. The autophosphorylated and nonphosphorylated forms of the regulatory subunit (RII) were separated in sodium dodecyl sulfate-polyacrylamide gels and identified by immunoblot analysis. Addition of cAMP to tissue extracts resulted in rapid dephosphorylation of RII (t 1/2 = 20s at 4 degrees C) while addition of MgATP caused complete conversion to the phosphorylated form. Under basal conditions, 56% of RII in intact muscle was phosphorylated when the tissue was homogenized under conditions which fully inhibit protein kinase and phosphatase activities. Incubation with isoproterenol caused a dose-dependent decrease in the phosphorylation state of RII (EC50 = 5 X 10(-8) M). Incubation with high concentrations of isoproterenol, 1-methyl-3-isobutylxanthine, or forskolin caused maximal decreases in the phosphorylated form to 12-18% of the total RII. The effect of isoproterenol was rapid (t 1/2 = 15 s at 37 degrees C), reversible, and could be blocked with the antagonist propranolol. Contraction of the smooth muscle with K+ or low (less than 1 microM) concentrations of carbachol had no effect on the phosphorylation level. A decrease in the basal phosphorylation level to 41% was observed with 10 microM carbachol which was additive with the dephosphorylation produced by isoproterenol. The time course of isoproterenol-induced dephosphorylation of RII paralleled that of muscle relaxation, consistent with a role of cAMP-dependent protein kinase activation in relaxation of smooth muscle.     

cAMP mediated proteolysis of the catalytic subunit of cAMP-dependent protein kinase

The cAMP-dependent protein kinase from LLC-PK1 cells can be activated in vivo by calcitonin and vasopressin, or forskolin. Continuous treatment of cells with these agents results in a decrease of total cAMP-PK activity. The loss of kinase activity was enhanced when either of these three agents was incubated in the presence of isobutylmethylxanthine. Results obtained using affinity purified antibodies to the catalytic subunit show that the loss of kinase was due to specific proteolysis of this subunit.     

Interaction of calmodulin with myosin light chain kinase and cAMP-dependent protein kinase in bovine brain

Myosin light chain kinase and a fraction of type II cAMP-dependent protein kinase have been partially purified from bovine brain by affinity chromatography on calmodulin-Sepharose. The myosin kinase was purified approximately 3700-fold and has an estimated molecular weight of 130,000 +/- 10,000 by sodium dodecyl sulfate gel electrophoresis. A fraction of soluble cAMP-dependent protein kinase also bound to calmodulin-Sepharose and was purified 2300-fold. A fraction of this cAMP-dependent protein kinase after purification by glycerol gradient centrifugation was shown to contain the two subunits of calcineurin, a major calmodulin-binding protein in brain, and the two subunits of type II cAMP-dependent protein kinase in a ratio of 1:1:2:2. Its sedimentation coefficient was 8.1 S and 9.0 S when centrifuged in the absence or presence of calmodulin, suggesting the formation of a complex between calmodulin and protein kinase. Our results suggest the possibility that calcineurin may be involved in the interaction between the protein kinase and calmodulin. Furthermore, our studies imply that the regulatory subunit of the cAMP-dependent protein kinase, but not the catalytic subunit, is the site of interaction with calmodulin since the catalytic subunit of protein kinase was partially resolved from the complex by cAMP.     

Adenosine 3':5'-monophosphate-dependent protein kinase from human placenta: characterization of the catalytic subunit.

The catalytic subunit of cAMP-dependent protein kinase (EC 2.7.1.37) was purified for the first time from human placenta by DEAE-cellulose and HTP chromatography. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis showed a single band of average molecular weight of 42 kDa (SEM = 0.52). Kinetic experiments showed a Km for ATP of 12.6 +/- 1.2 mumol/l, for histone II-AS of 1.3 +/- 0.05 mg.ml-1, for kemptide of 11.4 +/- 4.4 mumol/l. The synthetic inhibitor IP20-amide showed a competitive mechanism of inhibition with a Ki of 5.0 nmol/l. The protein kinase inhibitors H7 and H9 showed an apparent Ki of 8.3 and 4.9 mumol/l respectively. Preparative isoelectric focusing revealed the presence of 5 different isoforms with an average pI of 6.17, 6.70, 7.15, 7.67, 8.9.