Sites phosphorylated in myosin light chain in contracting smooth muscle

Purified smooth muscle myosin light chain can be phosphorylated at multiple sites by myosin light chain kinase and protein kinase C. We have determined the sites phosphorylated on myosin light chain in intact bovine tracheal smooth muscle. Stimulation with 10 microM carbachol resulted in 66 +/- 5% monophosphorylated and 11 +/- 2% diphosphorylated myosin light chain after 1 min, and 47 +/- 4% monophosphorylated and 5 +/- 2% diphosphorylated myosin light chain after 30 min. Myosin heavy chain contained 0.06 +/- 0.01 mol of phosphate/mol of protein which did not change with carbachol. At both 1 and 30 min the monophosphorylated myosin light chain contained only phosphoserine whereas the diphosphorylated myosin light chain contained both phosphoserine and phosphothreonine. Two-dimensional peptide mapping of tryptic digests of monophosphorylated and diphosphorylated myosin light chain obtained from carbachol-stimulated tissue was similar to the peptide maps of purified light chain monophosphorylated and diphosphorylated, respectively, by myosin light chain kinase; these maps were distinct from the map obtained with tracheal light chain phosphorylated by protein kinase C. Phosphorylation of tracheal smooth muscle myosin light chain by myosin light chain kinase yields the tryptic phosphopeptide ATSNVFAMFDQSQIQEFK with S the phosphoserine in the monophosphorylated myosin light chain and TS the phosphotreonine and phosphoserine in the diphosphorylated myosin light chain. Thus, stimulation of tracheal smooth muscle with a high concentration of carbachol results in formation of both monophosphorylated and diphosphorylated myosin light chain although the amount of diphosphorylated light chain is substantially less than monophosphorylated light chain. In the intact muscle, myosin light chain is phosphorylated at sites corresponding to myosin light chain kinase phosphorylation.     

Linear relationship between diphosphorylation of 20 kDa light chain of gizzard myosin and the actin-activated myosin ATPase activity

The addition of large amounts of myosin light chain kinase to the reconstituted gizzard actomyosin shows diphosphorylation of 20 kDa myosin light chain. Accompanying diphosphorylation, the actin-activated myosin ATPase activity was also enhanced. The extent of diphosphorylation and the myosin ATPase activity were clearly demonstrated to be in a linear relationship. From the time course experiment, the conversion of monophosphorylated light chain into one which was diphosphorylated seemed to be a sequential process. Moreover, analyzing phospho-amino acid by using a two-dimensional electrophoresis technique revealed that monophosphorylated light chain contained phosphoserine and diphosphorylated one contained phosphothreonine in addition to phosphoserine.     

Diphosphorylation of platelet myosin by myosin light chain kinase.

Recently, one of the authors (K.I.) and other investigators reported that myosin light chain (MLC) of smooth muscle (gizzard, arterial and tracheal) was diphosphorylated by myosin light chain kinase (MLCK) and that diphosphorylated myosin showed a marked increase in the actin-activated myosin ATPase activity in vitro and ex vivo. In this study, we prepared myosin, actin, tropomyosin (human platelet), MLCK (chicken gizzard) and calmodulin (bovine brain) and demonstrated diphosphorylation of MLC of platelet by MLCK in vitro. Our results are as follows. (1) Platelet MLC was diphosphorylated by a relatively high concentration (greater than 20 micrograms/ml) of MLCK in vitro. As a result of diphosphorylation, the actin-activated myosin ATPase activity was increased 3 to 4-fold as compared to the monophosphorylation. (2) Both di- and monophosphorylation reactions showed similar Ca2+, KCl, MgCl2-dependence. Maximal reaction was seen at [Ca2+] greater than 10(-6) M, 60 mM KCl and 2 mM MgCl2. This condition was physiological in activated platelets. (3) Di- and monophosphorylated myosin showed similar Ca2+, KCl-dependence of ATPase activity but distinct MgCl2-dependence. Diphosphorylated myosin showed maximal ATPase activity at 2 mM MgCl2 and monophosphorylated myosin showed a maximum at 10 mM MgCl2. (4) The addition of tropomyosin stimulated actin-activated ATPase activity in both di- and monophosphorylated myosin to the same degree. (5) ML-9, a relatively specific inhibitor of MLCK, inhibited the aggregation of human platelets induced by thrombin ex vivo in a dose-dependent manner. Moreover, this drug also partially inhibited both di- and monophosphorylation reactions and actin-activated ATPase activity. On the other hand, H-7, a synthetic inhibitor of protein kinase C, had little effect on the aggregation of human platelets induced by thrombin ex vivo. From these results, we conclude that diphosphorylation of platelet myosin by MLCK may play an important role in activated platelets in vivo.     

Study of the phosphorylatable light chains of skeletal and gizzard myosins by nuclear magnetic resonance spectroscopy.

31P and 1H n.m.r. studies of the phosphorylatable light chains from rabbit fast skeletal and chicken gizzard muscles in the isolated state and in the intact myosin molecule indicate that the N-terminal region of the light chain containing the sites of phosphorylation has independent segmental flexibility. The ionization behaviour of serine phosphate in both rabbit skeletal and chicken gizzard P light chains exhibits cooperativity and is compatible with the phosphate group being influenced by neighbouring positively charged side-chains. No marked difference in phosphate ionization behaviour was apparent between the monophosphorylated P light chains of rabbit skeletal and chicken gizzard myosins. From 1H and 31P n.m.r. studies of the overall conformation, side-chain ionization properties and the spectral effects of titration with an anionic paramagnetic reagent bound at the basic N-terminal region, it is concluded that Thr-18 and Ser-19 are phosphorylated in the bisphosphorylated P light chain of gizzard myosin, the latter residue being the site of monophosphorylation. In the presence of F-actin the mobility of the serine phosphate of the P light chain of intact gizzard myosin was reduced. No interaction between the isolated P light chain and F-actin was however detected. These results are discussed with reference to the observed conformational features of the P light chain.     

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.     

Ricin B chain converts a non-cytotoxic antibody-ricin A chain conjugate into a potent and specific cytotoxic agent

A method is described for the preparation of partially and fully phosphorylated chicken gizzard myosin. When fully phosphorylated it possessed an actin-activated Mg2+-ATPase of similar specific activity to that of mammalian skeletal muscle myosin. The Mg2+-ATPase activity of these preparations was related in a non-linear fashion to increasing phosphorylation of the P light chain. When P light chain phosphorylation occurred during enzymic assay the Mg2+-ATPase activity remained constant. Fully phosphorylated preparations of gizzard myosin possessed an actin-activated Mg2+-ATPase that was not Ca2+-sensitive, whereas the Mg2+-ATPase of partially phosphorylated myosin preparations was Ca2+-sensitive.     

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.     

Embryonic chicken gizzard: expression of the smooth muscle regulatory proteins caldesmon and myosin light chain kinase

The patterns of expression of the smooth muscle regulatory proteins caldesmon and myosin light chain kinase were investigated in the developing chicken gizzard. Immunofluorescent studies revealed that both proteins were expressed as early as E5 throughout the mesodermal gizzard anlage, together with actin, -actinin and a small amount of nonmuscle myosin. These proteins appear to form the scaffold for smooth muscle development, defined by the onset of smooth muscle myosin expression. During E6, a period of extensive cell division, smooth muscle myosin begins to appear in the musculi laterales close to the serosal border and, later, also in the musculi intermedii. Until about E10, myosin reactivity expands into the pre-existing thin filament scaffold. Later in development, the contractile and regulatory proteins co-localize and show a regular uniform staining pattern comparable to that seen in adult tissue. By using immunoblotting techniques, the low-molecular mass form of caldesmon and myosin light chain kinase were detected as early as E5. During further development, the expression of caldesmon switched from the low-molecular mass to the high-molecular mass form; in neonatal and adult tissue, high-molecular mass caldesmon was the only isoform expressed. The level of expression of myosin light chain kinase increased continously during embryonic development, but no embryospecific isoform with a different molecular mass was detected.     

Myosin light chain kinase expression during smooth muscle development

The expression of smooth muscle myosin light chain kinase (MLCK) was investigated during chicken gizzard development. The molecular weight and the antigenic properties of MLCK did not change during development. The use of anion exchange high performance liquid chromatography (HPLC) enabled us to distinguish between MLCKs from post-hatched and adult chickens. A partial amino acid sequence determination of 4-day-old gizzard MLCK failed to disclose differences in the primary sequences of the two proteins. The results suggest that MLCK has the same primary sequence in all sequences of the two proteins. The results suggest that MLCK has the same primary sequence in all stages of gizzard development, although charge variants due to post-translational modifications may exist.     

Identification of the native form of chicken gizzard myosin light chain kinase with the aid of monoclonal antibodies

Examination, by immunoblotting, of myosin light chain kinase-containing fractions obtained during purification of the enzyme from chicken gizzard has shown that a single species (Mr = 136,000) exists in the muscle and that this enzyme is degraded, primarily to a 130,000-dalton fragment, during purification. These conclusions were confirmed by phosphorylation of the different species of myosin light chain kinase by the isolated catalytic subunit of cyclic AMP-dependent protein kinase.     

Heterogeneity of smooth muscle myosin light chain kinase is revealed by anion-exchange high-performance liquid chromatography

When smooth muscle myosin light chain kinase, purified by standard procedures from chicken gizzard smooth muscle, was applied to an anion-exchange high-performance liquid chromatographic column, three well resolved peaks were obtained. Each peak contained a single protein whose electrophoretic mobility corresponded to that of MLCK. However each enzyme was characterized by a different specific activity. Peptide mapping experiments were unable to demonstrate different proteolytic patterns for the three proteins. Treatment of myosin light chain kinase with alkaline phosphatase, prior to ion chromatography, resulted in a change of elution profile. These experiments suggest that myosin light chain kinase could exist in three forms characterized by a different degree of phosphorylation.     

Isolation of cardiac myofibrils and myosin light chains with in vivo levels of light chain phosphorylation

Conditions are described for the preparation of functional myofibrils and myosin light chains from freeze-clamped beating hearts with the state of light chain phosphorylation chemically ‘frozen’ during the extraction procedure. Myofibrils were shown to be functionally intact by measurement of Ca²⁺ binding and ATPase activity. Highly purified cardiac myosin light chains could be routinely isolated from myofibrillar preparations using ethanol fractionation together with ion-exchange chromotography. Analysis of light chains for covalent phosphate indicated that basal levels of phosphorylation of the 18?20 000 dalton light chain of myosin in rabbit hearts beating in situ or in a perfusion apparatus were 0.3–0.4 mol/mol. Covalent phosphate content of the light chain fraction did not change during perfusion of hearts with 10 μM epinephrine.     

Spatial localization of mono-and diphosphorylated myosin II regulatory light chain at the leading edge of motile HeLa cells

Nonmuscle myosin II activity is regulated by phosphorylation of the myosin II regulatory light chain (MRLC) at Ser19 or at both Thr18 and Ser19, and the phosphorylation of MRLC promotes the contractility and stability of actomyosin. To analyze the states of MRLC phosphorylation at the leading edge in the motile HeLa cells, we have examined the subcellular distribution of monophosphorylated or diphosphorylated form of MRLC using a confocal microscope. The cross-sectional imaging revealed that monophosphorylated MRLC distributed throughout the cortical region and the leading edge, but its fluorescent signal was much stronger at the leading edge. This distribution pattern of monophosphorylated MRLC was almost identical to those of myosin II and F-actin. On the other hand, diphosphorylated MRLC is localized at the base of leading edge, spatially very close to the substrate, and colocalized with F-actin in part at the base of filopodia. Diphosphorylated MRLC was hardly detectable at the tip of filopodia and the cell cortical region, where monophosphorylated MRLC was clearly detected. These localization patterns suggest that myosin II is activated at the leading edge, especially at the base but not the tip of filopodia in motile cells. Next, we analyzed the cells expressing GFP-tagged recombinant MRLCs. Expression of GFP-tagged diphosphorylatable and monophosphorylatable MRLCs led to a significant increase in the filopodial number, compared with the cells expressing nonphosphorylatable MRLC. This result indicated that expression of phosphorylatable MRLC enhances the formation of filopodia at the wound edge.     

Purification and characterization of a phosphoprotein phosphatase that dephosphorylates myosin and the isolated light chain from chicken gizzard smooth muscle

A phosphatase that dephosphorylates myosin and the isolated light chain has been purified to near homogeneity from chicken gizzard smooth muscle. The molecular weight of the enzyme was estimated to be 100,000 and 35,000 under native and denatured conditions, respectively. It requires Mg2+ or Mn2+. The activity was measured quantitatively with a coupled enzyme system with the aid of myosin light chain kinase. The Vm and Km were determined to be 23.4 mumol/mg/min and 4.2 microM, respectively, with the isolated light chain as substrate under the optimal conditions (5 mM Mg2+ at pH 8.45). The specific activity with myosin as substrate at a concentration of 0.9 microM was found to be 1.25 mumol/mg/min, which was about one-fifth of the activity for the isolated light chain under the same conditions. The phosphatase seems to be specific to gizzard myosin. It may play an important role in the regulation of the myosin-actin interaction in smooth muscle.     

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.     

Phosphorylation of smooth muscle myosin heavy and light chains. Effects of phorbol dibutyrate and agonists

A number of different protein kinases phosphorylate purified heavy chains or the 20-kDa light chain of smooth muscle myosin. The physiological significance of these phosphorylation reactions has been examined in intact smooth muscle. Myosin heavy chain was slightly phosphorylated (0.08 mol of phosphate/mol) under control conditions in bovine tracheal tissue. Treatment with carbachol, isoproterenol, or phorbol 12,13-dibutyrate resulted in no significant change. In contrast, heavy chain was phosphorylated to 0.30 mol of phosphate/mol of heavy chain in tracheal smooth muscle cells in culture. This value increased significantly with ionomycin treatment. In control tissues, 9% of the light chain was monophosphorylated with 32P in the serine site phosphorylated by myosin light chain kinase. Carbachol (0.1 microM) alone resulted in contraction and 42% monophosphorylated light chain with 32P only in the serine site phosphorylated by myosin light chain kinase. Similarly, stimulation with histamine, 5-hydroxytryptamine, or KCl resulted in 32P incorporation into only the myosin light chain kinase serine site. Phorbol 12,13-dibutyrate (1 microM) alone resulted in 22% monophosphorylated light chain. However, only 25% of the 32P was in the myosin light chain kinase serine site, whereas 75% was in a serine site phosphorylated by protein kinase C. Phorbol 12,13-dibutyrate plus carbachol resulted in 27% monophosphorylated light chain; 75% of the 32P was in the myosin light chain kinase serine site, with the remainder in the protein kinase C serine site. These results indicate that phorbol esters act to increase phosphorylation of myosin light chain by protein kinase C. However, receptor-mediated stimulation or depolarization leading to tracheal smooth muscle contraction results in phosphorylation of myosin light chain by myosin light chain kinase alone.     

Isoforms of the small non-catalytic subunit of smooth muscle myosin light chain phosphatase.

Chicken gizzard smooth muscle myosin light chain phosphatase is composed of a approximately 37 kDa catalytic subunit, a approximately 110 kDa myosin binding or targeting subunit and a approximately 20 kDa subunit (MPs) whose function is as yet undefined. It was reported previously that a cloned chicken gizzard MPs cDNA encodes a protein of 186 amino acids (aa) [Y.H. Chen, M.X. Chen, D.R. Alessi, D.G. Gampbell, C. Shanahan, P. Cohen, P.T.W. Cohen, FEBS Lett. 356 (1994) 51-55]. More recently, we obtained by PCR amplification another MPs cDNA that encodes a protein of only 161 aa [Y. Zhang, K. Mabuchi, T. Tao, Biochim. Biophys. Acta 1343 (1997) 51-58]. In this work we obtained cDNAs corresponding to both sequences using a different set of PCR primers, indicating that the two sequences correspond to isoforms that most likely arose from alternative splicing of the same gene. Using two polyclonal antibodies, one raised against the recombinant 161 aa isoform of chicken gizzard MPs and the other against a C-terminal polypeptide that is present only in the 186 aa isoform, we found that while the 161 aa isoform is the predominant one in chicken gizzard, in chicken aorta it is the 186 aa one; in chicken stomach both isoforms are present, and in mammalian tissues such as ferret and rat only the 186 aa isoform is detected. Furthermore, we purified the MPs associated with the chicken gizzard myosin light chain phosphatase holoenzyme and determined its molecular weight, amino acid composition and six residues of its C-terminal sequence. The results from these analyses showed conclusively that the predominant isoform in chicken gizzard is the 161 aa one.     

Smooth muscle myosin light chain kinase efficiently phosphorylates serine 15 of cardiac myosin regulatory light chain

Specific phosphorylation of the human ventricular cardiac myosin regulatory light chain (MYL2) modifies the protein at S15. This modification affects MYL2 secondary structure and modulates the Ca(2+) sensitivity of contraction in cardiac tissue. Smooth muscle myosin light chain kinase (smMLCK) is a ubiquitous kinase prevalent in uterus and present in other contracting tissues including cardiac muscle. The recombinant 130 kDa (short) smMLCK phosphorylated S15 in MYL2 in vitro. Specific modification of S15 was verified using the direct detection of the phospho group on S15 with mass spectrometry. SmMLCK also specifically phosphorylated myosin regulatory light chain S15 in porcine ventricular myosin and chicken gizzard smooth muscle myosin (S20 in smooth muscle) but failed to phosphorylate the myosin regulatory light chain in rabbit skeletal myosin. Phosphorylation kinetics, measured using a novel fluorescence method eliminating the use of radioactive isotopes, indicates similar Michaelis-Menten V(max) and K(M) for regulatory light chain S15 phosphorylation rates in MYL2, porcine ventricular myosin, and chicken gizzard myosin. These data demonstrate that smMLCK is a specific and efficient kinase for the in vitro phosphorylation of MYL2, cardiac, and smooth muscle myosin. Whether smMLCK plays a role in cardiac muscle regulation or response to a disease causing stimulus is unclear but it should be considered a potentially significant kinase in cardiac tissue on the basis of its specificity, kinetics, and tissue expression.     

In vitro movement of actin filaments on gizzard smooth muscle myosin: requirement of phosphorylation of myosin light chain and effects of tropomyosin and caldesmon

ATP-dependent movement of actin filaments on smooth muscle myosin was investigated by using the in vitro motility assay method in which myosin was fixed on the surface of a coverslip in a phosphorylated or an unphosphorylated state. Actin filaments slid on gizzard myosin phosphorylated with myosin light chain kinase (MLCK) at a rate of 0.35 micron/s, but did not slide at all on unphosphorylated myosin. The movement of actin filaments on phosphorylated myosin was stopped by perfusion of phosphatase. Subsequent perfusion with a solution containing MLCK, calmodulin, and Ca2+ enabled actin filaments to move again. The sliding velocities on monophosphorylated and diphosphorylated myosin by MLCK were not different. Actin filaments did not move on myosin phosphorylated with protein kinase C (PKC). The sliding velocity on myosin phosphorylated with both MLCK and PKC was identical to that on myosin phosphorylated only with MLCK. Gizzard tropomyosin enhanced the sliding velocity to 0.76 micron/s. Gizzard caldesmon decreased the sliding velocity with increase in its concentration. At a 5-fold molar ratio of caldesmon to actin, the movement stopped completely. This inhibitory effect of caldesmon was relieved upon addition of excess calmodulin and Ca2+.