Phosphorylation of synthetic peptides by skeletal muscle myosin light chain kinases

Substrate determinants for rabbit and chicken skeletal muscle myosin light chain kinases were examined with synthetic peptides. Both skeletal muscle myosin light chain kinases had similar phosphorylation kinetics with synthetic peptide substrates. Average kinetic constants for skeletal muscle myosin light chain heptadecapeptide, (formula; see text) where S(P) is phosphoserine, were Km, 2.3 microM and Vmax, 0.9 mumol/min/mg of enzyme. Km values were 122 and 162 microM for skeletal muscle peptides containing A-A for basic residues at positions 2-3 and 6-7, respectively. Average kinetic constants for smooth muscle myosin light chain peptide, (formula; see text), were Km, 1.4 microM and Vmax 27 mumol/min/mg of enzyme. Average Km values for the smooth muscle peptide, residues 11-23, were 10 microM which increased 6- and 11-fold with substitutions of alanine at residues 12 and 13, respectively. Vmax values decreased and Km values increased markedly by substitution of residue 16 with glutamate in the 11-23 smooth muscle tridecapeptide. Basic residues located 3 and 6-7 residues toward the NH2 terminus from phosphoserine in smooth muscle myosin light chain and 6-8 and 10-11 residues toward the NH2 terminus from phosphoserine in skeletal muscle myosin light chain appear to be important substrate determinants for skeletal muscle myosin light chain kinases. These properties are different from myosin light chain kinase from smooth muscle.     

Substrate specificity of a multifunctional calmodulin-dependent protein kinase

The substrate specificity of the multifunctional calmodulin-dependent protein kinase from skeletal muscle has been studied using a series of synthetic peptide analogs. The enzyme phosphorylated a synthetic peptide corresponding to the NH2-terminal 10 residues of glycogen synthase, Pro-Leu-Ser-Arg-Thr-Leu-Ser-Val-Ser-Ser-NH2, stoichiometrically at Ser-7, the same residue phosphorylated in the parent protein. The synthetic peptide was phosphorylated with a Vmax of 12.5 mumol X min-1 X mg-1 and an apparent Km of 7.5 microM compared to values of 1.2 mumol X min-1 X mg-1 and 3.1 microM, respectively, for glycogen synthase. Similarly, a synthetic peptide corresponding to the NH2-terminal 23 residues of smooth muscle myosin light chain was readily phosphorylated on Ser-19 with a Km of 4 microM and a Vmax of 5.4 mumol X min-1 X mg-1. The importance of the arginine 3 residues NH2-terminal to the phosphorylated serine in each of these peptides was evident from experiments in which this arginine was substituted by either leucine or alanine, as well as from experiments in which its position in the myosin light chain sequence was varied. Positioning arginine 16 at residues 14 or 17 abolished phosphorylation, while location at residue 15 not only decreased Vmax 14-fold but switched the major site of phosphorylation from Ser-19 to Thr-18. It is concluded that the sequence Arg-X-Y-Ser(Thr) represents the minimum specificity determinant for the multifunctional calmodulin-dependent protein kinases. Studies with various synthetic peptide substrates and their analogs revealed that the specificity determinants of the multifunctional calmodulin-dependent protein kinase were distinct from several other "arginine-requiring" protein kinases.     

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.     

Amino acid sequence of the phosphorylation site of bovine cardiac myosin light chain

Amino acid sequences of peptides containing the phosphorylation site of bovine cardiac myosin light chain (L2) were determined. The site was localized to a serine residue in the tentative amino terminus of the light chain and is homologous to phosphorylation sites in other myosin light chains. Phosphorylation of bovine cardiac light chain by chicken gizzard myosin light chain kinase was Ca2+-calmodulin dependent. Kinetic data gave a Km of 107; microM and a Vmax of 23.6 mumol min-1 mg-1. In contrast to what has been observed with smooth muscle light chains, neither the phosphorylation site fragment of the cardiac light chain nor a synthetic tetradecapeptide containing the phosphorylation site were effectively phosphorylated by the chicken gizzard kinase. Phosphorylation of cardiac myosin light chains by chicken gizzard myosin light chain kinase, therefore, requires other regions of the light chain in addition to a phosphate acceptor site.     

Characterization of the phosphotyrosyl protein phosphatase activity of calmodulin-dependent protein phosphatase

Calmodulin-dependent protein phosphatase from bovine brain and heart was assayed for phosphotyrosine and phosphoserine phosphatase activity using several substrates: 1) smooth muscle myosin light chain (LC20) phosphorylated on tyrosine or serine residues, 2) angiotensin I phosphorylated on tyrosine, and 3) synthetic phosphotyrosine- or phosphoserine-containing peptides with amino acid sequences patterned after the autophosphorylation site in Type II regulatory subunit of the cAMP-dependent protein kinase. The phosphatase was activated by Ni2+ and Mn2+, and stimulated further by calmodulin. In the presence of Ni2+ and calmodulin, it exhibited similar kinetic constants for the dephosphorylation of phosphotyrosyl LC20 (Km = 0.9 microM, and Vmax = 350 nmol/min/mg) and phosphoseryl LC20 (Km = 2.6 microM, Vmax = 690 nmol/min/mg). Dephosphorylation of phosphotyrosyl LC20 was inhibited by phosphoseryl LC20 with an apparent Ki of 2 microM. Compared to the reactions with phosphotyrosyl LC20 as the substrate, reactions with phosphotyrosine-containing oligopeptides exhibited slightly higher Km and lower Vmax values. The reaction with the phosphoseryl peptide based on the Type II regulatory subunit sequence exhibited a slightly higher Km (23 microM), but a much higher Vmax (4400 nmol/min/mg) than that with its phosphotyrosine-containing counterpart. Micromolar concentrations of Zn2+ inhibited the phosphatase activity; vanadate was less potent, and 25 mM NaF was ineffective. The study provides quantitative data to serve as a basis for comparing the ability of the calmodulin-dependent protein phosphatase to act on phosphotyrosine- and phosphoserine-containing substrates.     

Characterization of chicken skeletal muscle myosin light chain kinase. Evidence for muscle-specific isozymes

Myosin light chain kinase purified from chicken white skeletal muscle (Mr = 150,000) was significantly larger than both rabbit skeletal (Mr = 87,000) and chicken gizzard smooth (Mr = 130,000) muscle myosin light chain kinases, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Km and Vmax values with rabbit or chicken skeletal, bovine cardiac, and chicken gizzard smooth muscle myosin P-light chains were very similar for the chicken and rabbit skeletal muscle myosin light chain kinases. In contrast, comparable Km and Vmax data for the chicken gizzard smooth muscle myosin light chain kinase showed that this enzyme was catalytically very different from the two skeletal muscle kinases. Affinity-purified antibodies to rabbit skeletal muscle myosin light chain kinase cross-reacted with chicken skeletal muscle myosin light chain kinase, but the titer of cross-reacting antibodies was approximately 20-fold less than the anti-rabbit skeletal muscle myosin light chain kinase titer. There was no detectable antibody cross-reactivity against chicken gizzard myosin light chain kinase. Proteolytic digestion followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis or high performance liquid chromatography showed that these enzymes are structurally very different with few, if any, overlapping peptides. These data suggest that, although chicken skeletal muscle myosin light chain kinase is catalytically very similar to rabbit skeletal muscle myosin light chain kinase, the two enzymes have different primary sequences. The two skeletal muscle myosin light chain kinases appear to be more similar to each other than either is to chicken gizzard smooth muscle myosin light chain kinase.     

Identification, phosphorylation, and dephosphorylation of a second site for myosin light chain kinase on the 20,000-dalton light chain of smooth muscle myosin

At relatively high concentrations of myosin light chain kinase, a second site on the 20,000-dalton light chain of smooth muscle myosin is phosphorylated (Ikebe, M., and Hartshorne, D. J. (1985) J. Biol. Chem. 260, 10027-10031). In this communication the site is identified and kinetics associated with its phosphorylation and dephosphorylation are described. The doubly phosphorylated 20,000-dalton light chain from turkey gizzard myosin was hydrolyzed with alpha-chymotrypsin and the phosphorylated peptide was isolated by reverse phase chromatography. Following amino acid analyses and partial sequence determinations the second site of phosphorylation is shown to be threonine 18. This site is distinct from the threonine residue phosphorylated by protein kinase C. The time courses of phosphorylation of serine 19 and threonine 18 in isolated light chains follow a single exponential indicating a random process, although the phosphorylation rates differ considerably. The values of kcat/Km for serine 19 and threonine 18 for isolated light chains are 550 and 0.2 min-1 microM-1, respectively. With intact myosin, phosphorylation of serine 19 is biphasic; kcat/Km values are 22.5 and 7.5 min-1 microM-1 for the fast and slow phases, respectively. In contrast, phosphorylation of threonine 18 in intact myosin is a random, but markedly slower process, kcat/Km = 0.44 min-1 microM-1. Dephosphorylation of doubly phosphorylated myosin (approximately 4 mol of phosphate/mol of myosin) and isolated light chains (approximately 2 mol of phosphate/mol of light chain) follows a random process and dephosphorylation of the serine 19 and threonine 18 sites occurs at similar rates.     

Substrate based inhibitors of smooth muscle myosin light chain kinase.

Activation of myosin light chain kinase is a prerequisite for smooth muscle activation. In this study, short peptide analogs of the phosphorylation site of the myosin light chain were studied for their effects on several contractile protein systems. The peptides inhibited phosphorylation of isolated ventricular and smooth muscle myosin light chains by smooth muscle myosin light chain kinase, but they were only weak inhibitors of phosphorylation of intact myosin and actomyosin. The peptides were also unable to block force development or myosin light chain phosphorylation in glycerol permeabilized fibers of swine carotid media. Apparently, the association of the myosin light chain with myosin changes its conformation such that substrate analogs which are potent inhibitors of the phosphorylation of isolated myosin light chains by myosin light chain kinase are ineffective at blocking phosphorylation of the intact molecule.     

Biochemical properties of chimeric skeletal and smooth muscle myosin light chain kinases.

The molecular and biochemical properties of myosin light chain kinases from chicken skeletal and smooth muscle were investigated by recombinant DNA techniques. Deletion of the amino-terminal region of either the smooth or skeletal muscle myosin light chain kinase resulted in a decrease in Vmax with no significant change in Km values for light chain substrates. Skeletal/smooth muscle chimeric kinases were inactive when a 65-residue region amino-terminal of the catalytic core was exchanged between the two forms. Changing alanine 494 to glutamic acid within this region in the chicken skeletal muscle myosin light chain kinase increased the Km values for light chains 10-fold. These results are consistent with the hypothesis that the region amino-terminal of the catalytic core in myosin light chain kinases is involved in light chain recognition. A skeletal muscle kinase which contained the smooth muscle calmodulin binding domain remained regulated by Ca2+/calmodulin. Thus, the calmodulin binding domains of smooth and skeletal muscle myosin light chain kinases share structural elements necessary for regulation.     

N-(6-phenylhexyl)-5-chloro-1-naphthalenesulfonamide, a novel activator of protein kinase C

Naphthalenesulfonamide derivatives were used to study the mechanism of regulation of Ca2+-dependent smooth muscle myosin light chain phosphorylation catalyzed by Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C) and myosin light chain kinase. Derivatives such as N-(6-phenylhexyl)-5-chloro-1-naphthalenesulfonamide (SC-9), with a hydrophobic residue at the end of a hydrocarbon chain, stimulated Ca2+-activated, phospholipid-dependent myosin light chain phosphorylation in a Ca2+-dependent fashion. There was no significant effect of these compounds on Ca2+-calmodulin (CaM) dependent myosin light chain phosphorylation. On the other hand, derivatives with the guanidino or amino residue at the same position had an inhibitory effect on both Ca2+-phospholipid- and Ca2+-CaM-dependent myosin light chain phosphorylation. These observations suggest that activation of Ca2+-activated, phospholipid-dependent myosin light chain phosphorylation by naphthalenesulfonamide derivatives depends on the chemical structure at the end of hydrocarbon chain of each compound. SC-9 was similar to phosphatidylserine with regard to activation, and the apparent Km values for Ca2+ of the enzyme with this compound and phosphatidylserine were 40 microM and 80 microM, respectively. Kinetic analysis indicated that 12-O-tetradecanoylphorbol 13-acetate increased the affinity of the enzyme with SC-9 for calcium ion. However, kinetic constants revealed that the Km value of protein kinase C activated by SC-9 for substrate myosin light chain was 5.8 microM, that is, about 10 times lower than that of the enzyme with phosphatidylserine, and that the Vmax value with SC-9 was 0.13 nmol X min-1, that is, 3-fold smaller than that seen with phosphatidylserine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Substrate specificity of myosin light chain kinases.

Skeletal muscle myosin light chain kinase can phosphorylate myosin light chains isolated from skeletal or smooth muscle. In contrast, smooth muscle myosin light chain kinase specifically phosphorylates light chains isolated from smooth muscle. In this study, we have identified residues within the rabbit smooth and skeletal muscle myosin light chain kinases which may interact with the basic residues that are important substrate determinants in the light chains. Mutation of aspartic acid 270 amino-terminal of the catalytic core of the skeletal muscle myosin light chain kinase increased the Km value for both smooth and skeletal muscle light chains. Although deletions of the analogous region of the smooth muscle myosin light chain kinase (residues 663-678) markedly increased the Km value for light chain, mutation of any single acidic residue within this region did not have a similar effect. Mutation of single residues within the catalytic core of the skeletal muscle (E377 and E421) and smooth muscle (E777 and E821) myosin light chain kinases increased Km values for the smooth muscle light chain at least 35- and 100-fold, respectively. It is proposed that these residues may form ionic interactions with the arginine that is 3 residues amino-terminal of the phosphorylatable serine in the smooth muscle light chain.     

Spatial requirements for location of basic residues in peptide substrates for smooth muscle myosin light chain kinase

The requirement of basic residues as substrate specificity determinants for the chicken gizzard myosin light chain kinase has been studied using synthetic peptide analogs of the local phosphorylation site sequence in the myosin light chains, Lys-Lys-Arg13-Pro-Gln-Arg16-Ala-Thr-Ser19-Asn-Val-Phe- Ala. The basic residue, Arg-16, was found to have a strong influence on the kinetics of phosphorylation similar to that reported previously for the three adjacent residues, Lys-11, Lys-12, and Lys-13 (Kemp, B. E., Pearson, R. B., and House, C. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 7471-7475). The location of Arg-16 in relation to Ser-19 as well as the distance between Arg-13 and Arg-16 had a profound effect on both the kinetics and the site specificity of phosphorylation. Placement of Arg-16 at position 15 resulted in a complete switch in phosphorylation site specificity from Ser-19 to Thr-18. Increasing the number of alanine residues between Arg-13 and Arg-16 in the model peptide, Lys-Lys-Arg-(Ala)n-Arg-Ala-Thr-Ser-Asn-Val-Phe-Ala, also influenced the kinetics and site specificity of peptide phosphorylation. With two or three alanines (n = 2 or 3), the apparent Km was 7.5 and 10 microM, respectively, and 97% of the phosphate was esterified to Ser-19. Increasing or decreasing the number of alanines (n = O to n = 4) was accompanied by an increase in the apparent Km and phosphorylation of both Thr-18 and Ser-19. These results support the concept that both the presence and location of basic residues play an essential role in the substrate specificity of the smooth muscle myosin light chain kinase.     

Nonmuscle myosin phosphorylation sites for calcium-dependent and calcium-independent protein kinases

Thymus myosin, light chains and a synthetic peptide (S-S-K-R-A-K-A-K-T-T-K-K-R-P-Q-R-A-T-S-N-V-F-S) corresponding to the N-terminal sequence of smooth muscle myosin light chains were compared as substrates for calcium/calmodulin-dependent protein kinase (MLCK), calcium/phospholipid-dependent protein kinase (PKC), and a MgATP-activated protein kinase (H4PK) from lymphoid cells. All protein kinases catalyzed phosphorylation of the substrates although H4PK showed higher affinity for isolated light chains and the peptide. Phosphoamino acid analysis and analysis of thermolysin peptides established that PKC catalyzed phosphorylation of threonine-9 or 10. In addition, PKC and H4PK catalyzed phosphorylation at serine-19, the MLCK site. Collectively the data support the hypothesis that myosin filament assembly in nonmuscle cells may be regulated by a variety of calcium-dependent and calcium-independent protein kinases.     

Calmodulin activates bovine-cardiac myosin light-chain kinase by increasing the affinity for myosin light-chain 2

The basic mechanism by which calmodulin activates bovine-cardiac muscle myosin light-chain kinase was investigated using highly purified preparations of mixed bovine-cardiac myosin light chains or isolated myosin light chain 2. The apparent contamination of these substrate proteins by calmodulin, as detected by activation of calmodulin-sensitive phosphodiesterase, was less than 4 parts/million and was undetectable by antibodies against calmodulin. The apparent KA for calmodulin was 2 nM and 20 nM in the presence of isolated myosin light-chain 2 and mixed myosin light chains, respectively. Purified bovine cardiac troponin C activated myosin light-chain kinase by about 10% at a concentration of 2 microM. Mixed myosin light chains were phosphorylated in the absence and presence of calmodulin and in the presence of calcium with a V of 11.1 and 11.0 mumol phosphate transferred min-1 (mg enzyme)-1, respectively. The apparent Km values for mixed myosin light chains were 8.0 and 0.35 mg/ml in the absence and presence of calmodulin, respectively. Similarly calmodulin lowered the Km value for isolated myosin light-chain 2 over 20-fold and increased the V value only about 1.5-fold. Activity observed in the absence of calmodulin was dependent on the presence of calcium and was suppressed by chelating free calcium either before or during a phosphorylation reaction. The apparent KA for calcium was 1.2 microM and 0.4 microM in the absence and presence of calmodulin. Activity in the absence of calmodulin was inhibited at very high concentrations of the 'specific' calmodulin antagonists W-7, trifluoperazine and R24571 with apparent IC50 values of 0.3 mM, 0.2 mM and 0.02 mM. Antibiotics raised against calmodulin suppressed completely the kinase activity in the presence of calmodulin but had no effect on the activity measured in its absence. These results suggest that calmodulin stimulates the activity of bovine-cardiac myosin light-chain kinase by increasing over 20-fold the affinity for its substrate myosin light-chain 2.     

Phosphorylation kinetics of skeletal muscle myosin and the effect of phosphorylation on actomyosin adenosinetriphosphatase activity

Purified rabbit skeletal muscle myosin is phosphorylated on one type of light-chain subunit (P-light chain) by calmodulin-dependent myosin light chain kinase and dephosphorylated by phosphoprotein phosphatase C. Analyses of the time courses of both phosphorylation and dephosphorylation of skeletal muscle myosin indicated that both reactions, involving at least 90% of the P-light chain, were kinetically homogeneous. These results suggest that phosphorylation and dephosphorylation of rabbit skeletal muscle myosin heads are simple random processes in contrast to the sequential phosphorylation mechanism proposed for myosin from gizzard smooth muscle. We also examined the effect of phosphorylation of rabbit skeletal muscle myosin on the actin-activated ATPase activity. We observed an apparent 2-fold decrease in the Km for actin, from about 6 microM to about 2.5 microM, with no significant effect on the Vmax (1.8s-1) in response to P-light-chain phosphorylation. There was no significant effect of phosphorylation on the ATPase activity of myosin alone (0.045 s-1). ATPase activation could be fully reversed by addition of phosphatase catalytic subunit. The relationship between the extents of P-light-chain phosphorylation and ATPase activation (at 3.5 microM actin and 0.6 microM myosin) was essentially linear. Thus, in contrast to results obtained with myosin from gizzard smooth muscle, these results suggest that cooperative interactions between the myosin heads do not play an important role in the activation process in skeletal muscle. Since the effect of P-light-chain phosphorylation is upon the Km for actin, it would appear to be associated with a significant activation of ATPase activity only at appropriate concentrations of actin and salt.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetic parameters of the protein tyrosine kinase activity of EGF-receptor mutants with individually altered autophosphorylation sites.

Epidermal growth factor (EGF)-receptor mutants in which individual autophosphorylation sites (Tyr1068, Tyr1148 or Tyr1173) have been replaced by phenylalanine residues were expressed in NIH-3T3 cells lacking endogenous EGF-receptors. Kinetic parameters of the kinase of wild-type and mutant receptors were compared. Both wild-type and mutant EGF-receptors had a Km(ATP) 1-3 microM for the autophosphorylation reaction, and a Km(ATP) of 3-7 microM for the phosphorylation of a peptide substrate. These are similar to the Km(ATP) values reported for EGF-receptor of A431 cells. A synthetic peptide representing the major in vitro autophosphorylation site Tyr1173 of the EGF-receptor (KGSTAENAEYLRV) was phosphorylated by wild-type receptor with a Km of 110-130 microM, and the peptide inhibited autophosphorylation with a Ki of 150 microM. Mutant EGF-receptors phosphorylated the peptide substrate with a Km of 70-100 microM. A similar decrease of Km (substrate) was obtained when the phosphorylation experiments were performed with the commonly applied substrates angiotensin II and a peptide derived from c-src. The Km of angiotensin II phosphorylation was reduced from 1100 microM for wild-type receptor to 890 microM for mutant receptor and for c-src peptide from 1010 microM to 770 microM respectively. The Vmax of the kinase was dependent on receptor concentration, but was not significantly affected by the mutation. Analogs of the Tyr1173 peptide in which the tyrosine residue was replaced by either a phenylalanine or an alanine residue also inhibited autophosphorylation with Ki of 650-750 microM. These analyses show that alterations of individual autophosphorylation sites do not have a major effect on kinase activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phosphorylation of vertebrate nonmuscle and smooth muscle myosin heavy chains and light chains

In this article we review the various amino acids present in vertebrate nonmuscle and smooth muscle myosin that can undergo phosphorylation. The sites for phosphorylation in the 20 kD myosin light chain include serine-19 and threonine-18 which are substrates for myosin light chain kinase and serine-1 and/or-2 and threonine-9 which are substrates for protein kinase C. The sites in vertebrate smooth muscle and nonmuscle myosin heavy chains that can be phosphorylated by protein kinase C and casein kinase II are also summarized.Original data indicating that treatment of human T-lymphocytes (Jurkat cell line) with phorbol 12-myristate 13-acetate results in phosphorylation of both the 20 kD myosin light chain as well as the 200 kD myosin heavy chain is presented. We identified the amino acids phosphorylated in the human T-lymphocytes myosin light chains as serine-1 or serine-2 and in the myosin heavy chains as serine-1917 by 1-dimensional isoelectric focusing of tryptic phosphopeptides. Untreated T-lymphocytes contain phosphate in the serine-19 residue of teh myosin light chain and in a residue tentatively identified as serine-1944 in the myosin heavy chain.Abbreviations MLC myosin light chain - MHC myosin heavy chain - Tris tris(hydroxymethyl)aminomethane - EGTA [ethylenebis(oxyethylenenitrilo)]tetraacetic acid - EDTA ethylenediaminetetraacetate - TPCK N-tosyl-L-phenylalanine chloromethyl ketone - PMA phorbol 12-myristate 13-acetate     

Properties of a monoclonal antibody directed to the calmodulin-binding domain of rabbit skeletal muscle myosin light chain kinase

A synthetic peptide representing the calmodulin-binding domain of rabbit skeletal muscle myosin light chain kinase (K-R-R-W-K-K-N-F-I-A-V-S-A-A-N-R-F-K-K-I-S-S-S-G-A-L) was used as an antigen to produce a monoclonal antibody. The antibody (designated MAb RSkCBP1, of the IgM class) reacted with similar affinity (KD approximately 20 nM) by competitive enzyme-linked immunoassay (ELISA) with the antigen peptide and intact rabbit skeletal muscle myosin light chain kinase. MAb RSkCBP1 inhibited rabbit skeletal muscle myosin light chain kinase activity competitively with respect to calmodulin (Ki = 20 nM). The antibody also inhibited myosin light chain kinase activity in extracts of skeletal muscle from several mammalian species (rabbit, sheep, and bovine) and an avian species (chicken). The concentration of MAb RSKCBP1 required for 50% inhibition of enzyme activity was similar for the mammalian species (80 nM) but was significantly higher for the avian species (1.2 microM). A competitive ELISA protocol was used to analyze weak cross-reactivity to other calmodulin-binding peptides and proteins. This assay demonstrated no cross-reactivity with the venom peptides melittin or mastoparan; smooth muscle myosin light chain kinases from hog carotid, bovine trachea, or chicken gizzard; bovine brain calmodulin-dependent calcineurin; or rabbit skeletal muscle troponin I. These data support the contention that the synthetic peptide used as the antigen represents the calmodulin-binding domain of rabbit skeletal muscle myosin light chain kinase and that the calmodulin-binding domains of different calmodulin-regulated proteins may have distinct primary and/or higher order structures.     

Purification of myosin light chain kinase from Limulus muscle

It has previously been shown that the regulatory light chains of myosin from Limulus, the horseshoe crab, can be phosphorylated either by purified turkey gizzard smooth muscle myosin light chain (MLC) kinase or by a crude kinase fraction prepared from Limulus muscle [Sellers, J. R. (1981) J. Biol. Chem. 256, 9274-9278]. This phosphorylation was shown to be associated with a 20-fold increase in the actin-activated MgATPase activity of the myosin. We have now purified the Ca2+-calmodulin-dependent MLC kinase from Limulus muscle to near homogeneity by using a combination of low ionic strength extraction, ammonium sulfate fractionation, and chromatography on Sephacryl S-300 and DEAE-Sephacel. The final purification was achieved by affinity chromatography on a calmodulin-Sepharose 4B column. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis showed 95% of the protein to be comprised of a doublet with Mr = 39000 and 37000. Electrophoresis of the kinase fraction under nondenaturing conditions resulted in a partial separation of the two major bands and demonstrated that each had catalytic activity. An SDS-polyacrylamide gel overlayed with 125I-calmodulin demonstrated that both the Mr 39K and the Mr 37K proteins bind calmodulin. Neither of the bands could be phosphorylated by the catalytic subunit of cAMP-dependent protein kinase. With Limulus myosin light chains as a substrate, the Vmax was 15.4 mumol min-1 mg-1, and the Km was 15.6 microM. The KD for calmodulin was determined to be 6 nM. The enzyme did not phosphorylate histones, casein, actin, or tropomyosin.     

Comparison of the substrate specificity of adenosine 3':5'-monophosphate- and guanosine 3':5'-monophosphate-dependent protein kinases. Kinetic studies using synthetic peptides corresponding to phosphorylation sites in histone H2B.

The substrate specificities of cyclic GMP-dependent and cyclic AMP-dependent protein kinases have been compared by kinetic analysis using synthetic peptides as substrates. Both enzymes catalyzed the transfer of phosphate from ATP to calf thymus histone H2B, as well as to two synthetic peptides, Arg-Lys-Arg-Ser32-Arg-Lys-Glu and Arg-Lys-Glu-Ser36-Tyr-Ser-Val, corresponding to the amino acid sequences around serine 32 and serine 36 in histone H2B. Serine 38 in the latter peptide was not phosphorylated by either enzyme. Cyclic GMP-dependent kinase and cyclic AMP-dependent kinase catalyzed the incorporation of 1.1 and 2.0 mol of phosphate/mol of histone H2B, respectively. The phosphorylation of histone H2B, respectively. The phosphorylation of histone H2B by cyclic GMP-dependent kinase showed two distinct optima as the magnesium concentration was increased. However, the phosphorylation of either synthetic peptide by this enzyme was depressed at high magnesium concentrations. As the pH of reaction mixtures was elevated from pH 6 to pH 9, the rate of phosphorylation of Arg-Lys-Arg-Ser32-Arg-Lys-Glu by cyclic GMP-dependent kinase continually increased. Acetylation of the NH2 terminus of the peptide did not qualitatively affect this pH profile, but did increase the Vmax value of the enzyme 3-fold. The apparent Km and Vmax values for the phosphorylation of Arg-Lys-Arg-Ser32-Arg-Lys-Glu by cyclic GMP-dependent kinase were 21 microM and 4.4 mumol/min/mg, respectively. The synthetic peptide Arg-Lys-Glu-Ser36-Tyr-Ser-Val was a relatively poor substrate for cyclic GMP-dependent kinase, exhibiting a Km value of 732 microM, although the Vmax was 12 micromol/min/mg. With histone H2B as substrate for the cyclic GMP-dependent kinase, two different Km values were apparent. The Km values for cyclic AMP-dependent kinase for either synthetic peptide were approximately 100 microM, but the Vmax for Arg-Lys-Arg-Ser32-Arg-Lys-Glu was 1.1 mumol/min/mg, while the Vmax for Arg-Lys-Glu-Ser36-Tyr-Ser-Val was 16.5 mumol/min/mg. These data suggest that although the two cyclic nucleotide-dependent protein kinases have similar substrate specificities, the determinants dictated by the primary sequence around the two phosphorylation sites in histone H2B are different for the two enzymes.