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.     

Neural and smooth muscle development in the chicken gizzard

The development of the autonomic ganglia of Auerbach's plexus and gizzard smooth muscle was studied in chicken embryos. Nervous system and smooth-muscle-specific antibodies were employed in immunofluorescence stainings on tissue sections to investigate the temporal and spatial frame of neural and muscular differentiation in relation to each other. Subserosal clusters of neural cells were clearly demonstrable at embryonic day 5 (ED5), the earliest stage analysed, with the monoclonal antibody El (SGIII-1). Fine nerve fibres (ED6) and, later, large axon bundles projecting from subserosal neuron clusters towards the lumen were followed and found to reach the luminal border by ED11. Already in early development the area of the future laminar tendons on the ventral and dorsal surface of the gizzard was devoid of neuroblasts, and nerve fibres were not extending to the muscle-tendon borderline until ED16. Double stainings with antibodies to smooth muscle myosin (SMM) and El revealed that SMM expression, taken as an indicator for muscle differentiation, followed neural growth. It was first detectable in close apposition to the differentiating neuroblasts in the caudal and cranial portion of the gizzard at ED6. With further development, myosin expression proceeded inward towards the lumen in a wave which followed the ingrowth of E1-positive nerve fibres from the prospective Auerbach plexus. Neuromuscular differentiation deviated from this pattern in the lateral tendon area where nerve growth was delayed and myosin expression preceeded the arrival of E1-positive nerve fibres. The findings suggest that the gizzard could serve as a model system for the analysis of potential early nervous system imprints on smooth premuscle mesenchyme differentiation.     

Vascular smooth muscle caldesmon

Caldesmon, a major actin- and calmodulin-binding protein, has been identified in diverse bovine tissues, including smooth and striated muscles and various nonmuscle tissues, by denaturing polyacrylamide gel electrophoresis of tissue homogenates and immunoblotting using rabbit anti-chicken gizzard caldesmon. Caldesmon was purified from vascular smooth muscle (bovine aorta) by heat treatment of a tissue homogenate, ion-exchange chromatography, and affinity chromatography on a column of immobilized calmodulin. The isolated protein shared many properties in common with chicken gizzard caldesmon: immunological cross-reactivity, Ca2+-dependent interaction with calmodulin, Ca2+-independent interaction with F-actin, competition between actin and calmodulin for caldesmon binding only in the presence of Ca2+, and inhibition of the actin-activated Mg2+-ATPase activity of smooth muscle myosin without affecting the phosphorylation state of myosin. Maximal binding of aorta caldesmon to actin occurred at 1 mol of caldesmon: 9-10 mol of actin, and binding was unaffected by tropomyosin. Half-maximal inhibition of the actin-activated myosin Mg2+-ATPase occurred at approximately 1 mol of caldesmon: 12 mol of actin. This inhibition was also unaffected by tropomyosin. Caldesmon had no effect on the Mg2+-ATPase activity of smooth muscle myosin in the absence of actin. Bovine aorta and chicken gizzard caldesmons differed in several respects: Mr (149,000 for bovine aorta caldesmon and 141,000 for chicken gizzard caldesmon), extinction coefficient (E1%280nm = 19.5 and 5.0 for bovine aorta and chicken gizzard caldesmon, respectively), amino acid composition, and one-dimensional peptide maps obtained by limited chymotryptic and Staphylococcus aureus V8 protease digestion. In a competitive enzyme-linked immunosorbent assay, using anti-chicken gizzard caldesmon, a 174-fold molar excess of bovine aorta caldesmon relative to chicken gizzard caldesmon was required for half-maximal inhibition. These studies establish the widespread tissue and species distribution of caldesmon and indicate that vascular smooth muscle caldesmon exhibits physicochemical differences yet structural and functional similarities to caldesmon isolated from chicken gizzard.     

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.     

Complete amino-acid sequence of subfragment-2 in adult chicken skeletal muscle myosin

Although the complete amino-acid sequence of the short subfragment-2 (short S-2) and the partial sequence of the hinge region derived from adult chicken skeletal muscle myosin have been reported previously, the sequence of the N-terminal portion of subfragment-2 (S-2) and the connective portion between the above two regions could not be determined. In this study, the amino-acid sequence of these undetermined portions were completely sequenced. Furthermore, overlaps of cyanogen bromide (CNBr) peptides in the hinge region were also isolated and sequenced. Peptides obtained by hydrolysis with dilute formic acid and by digestion with lysyl endopeptidase of S-2 were purified and sequenced. These results established the complete amino-acid sequence of S-2 composed of 429 amino-acid residues. This sequence of adult chicken skeletal muscle myosin was compared with that of chicken embryonic skeletal muscle, chicken gizzard muscle and rabbit cardiac muscle myosin (alpha-myosin heavy chain) and shows degrees of 96%, 38% and 84% sequence identities, respectively. The frequency with which hydrophobic residues are present at position "a" in seven-residues repeats of the hinge region was markedly reduced when compared to the short S-2 sequence of the chicken skeletal muscle myosin.     

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.     

A smooth muscle-specific monoclonal antibody recognizes smooth muscle actin isozymes

Injection of chicken gizzard actin into BALB/c mice resulted in the isolation of a smooth muscle-specific monoclonal antibody designated CGA7. When assayed on methanol-Carnoy's fixed, paraffin-embedded tissue, it bound to smooth muscle cells and myoepithelial cells, but failed to decorate striated muscle, endothelium, connective tissue, epithelium, or nerve. CGA7 recognized microfilament bundles in early passage cultures of rat aortic smooth muscle cells and human leiomyosarcoma cells but did not react with human fibroblasts. In Western blot experiments, CGA7 detected actin from chicken gizzard and monkey ileum, but not skeletal muscle or fibroblast actin. Immunoblots performed on two-dimensional gels demonstrated that CGA7 recognizes gamma-actin from chicken gizzard and alpha- and gamma-actin from rat colon muscularis. This antibody was an excellent tissue-specific smooth muscle marker.     

Phosphorylation of chicken gizzard myosin: Myosin filament hypothesis of calcium regulation

Myosin from chicken gizzard smooth muscle was found to be characteristically different from rabbit skeletal striated myosin: i) ATP induced a profound change in the conformation of gizzard myosin molecules. ii) ATP also induced disassembling of gizzard myosin filaments. iii) Enzymic phosphorylation of gizzard myosin light chains rendered both the myosin conformation and the myosin filaments resistant to the actions of ATP. iv) Very high concentrations of magnesium were required for formation of the ATP-resistant filaments as well as for superprecipitation (a model contraction) of actomyosin suspensions. v) ITP was a very poor substrate for MLCK, and was accordingly incapable of inducing “Ca-tension” in glycerinated fibers of gizzard muscle, but it did induce “Mg-tension.” Primarily from these findings, it was proposed that tje mechanism of gizzard muscle contraction involves ATP-induced changes in the morphology of myosin filaments which are reversibly altered by enzymic phosphorylation and dephosphorylation of myosin light chains in the presence of relatively high concentrations of magnesium.     

Embryonic chicken gizzard: smooth muscle and non-muscle myosin isoforms

Antibodies to smooth muscle and non-muscle myosin allow the development of smooth muscle and its capillary system in the embryonic chicken gizzard to be followed by immunofluorescent techniques. Although smooth muscle development proceeds in a serosal to luminal direction, angiogenetic cell clusters develop independently at the luminal side close to the epithelial layer, and the presumptive capillaries invade the developing muscle in a luminal to serosal direction. The smooth muscle and non-muscle myosin heavy chains in this avian system cannot be separated by SDS polyacrylamide gel electrophoresis and do not show isoform specificity in immunoblotting, unlike the system found in mammals. Only two myosin heavy chains with M_r of 200 and 196 kDa were separable and considerable immunological cross-reactivity was found between the denatured myosin isoform heavy chains.     

Expression of immunoreactive myosin and myoglobin in the developing chicken gizzard

Summary Antibodies to adult-type myosin and myoglobin from chicken gizzard were used to study the expression of these proteins during chicken embryogenesis. Using the indirect immunofluorescent technique, myosin was detected as discrete fluorescent foci in the central part of the presumptive chicken gizzard as early as day 5 of development. During the following days, immunoreactive myosin extends both craniocaudally as well as laterally and reaches the serosal and luminal borders by day 13/14. On day 16, the adult fascicular pattern is achieved. As judged by enzymelinked immunoassay and spectroscopic methods, myoglobin did not appear until day 18.Dedicated to Mrs. C.F. Schoenberg, Department of Anatomy, Cambridge, Great Britain     

Type VI collagen represents a major fraction of connective tissue collagens

A new method for the isolation of type VI collagen from peptic tissue digests is presented which gives tenfold higher yields than methods previously reported. From the amounts of purified protein obtained from human placenta, bovine uterus, chicken gizzard and entire mouse bodies we conclude that type VI collagen represents a major fraction of connective tissue collagens.     

Changes in myosin isozymes during development of chicken gizzard muscle

The distribution of myosin isozymes in embryonic and adult chicken gizzard muscle were examined by electrophoresis in a non-denaturing gel system (pyrophosphate acrylamide gel electrophoresis), and both light and heavy chains of embryonic and adult myosin isozymes were compared. In pyrophosphate acrylamide gel electrophoresis, there were three isozyme components in embryonic gizzard myosin, but only one isozyme in adult gizzard myosin. The mobility of the fastest migrating embryonic isozyme was similar to that of the adult isozyme. The three embryonic isozymes differ from each other in the light chain distribution. Two of them contain an embryo-specific myosin light chain, which is characterized by its molecular weight and isoelectric point, whereas the other embryonic myosin isozyme contained the same light chains as the adult myosin. The pattern of peptide fragments of embryonic heavy chain produced by digestion with alpha-chymotrypsin in the presence of SDS was not distinguishable from that of adult myosin heavy chain. Thus there are myosin isozymes specific to embryonic gizzard muscle which exhibit embryo-specific light chain compositions, but are similar to adult gizzard myosin in their heavy chain structure.     

Comparison of the effects of smooth and skeletal muscle actins on smooth muscle actomyosin Mg2+-ATPase

Actin has been purified from smooth muscle (chicken gizzard) by two different procedures and its activation of smooth muscle myosin Mg2+-ATPase activity compared with that achieved with rabbit skeletal muscle actin. The procedure of Pardee and Spudich (Methods Enzymol. (1982) 85, 164-181) for the purification of rabbit skeletal muscle actin is readily applicable to the isolation of chicken gizzard actin, enabling large quantities to be purified in two days. Smooth muscle actin could be successfully stored as F-actin at -80 degrees C and survived freezing and thawing at least twice. Smooth muscle actin activated myosin Mg2+-ATPase to a higher level than its skeletal muscle counterpart (77.9 nmol Pi/min/mg myosin vs 48.1 nmol Pi/min/mg myosin).     

Tissue-specific and developmentally regulated alternative splicing of a visceral isoform of smooth muscle myosin heavy chain.

Previous work demonstrated that the rabbit smooth muscle myosin heavy chain gene showed sequence divergence at the 25kDa/50kDa junction of the S1 subfragment when compared to chicken gizzard and chicken epithelial nonmuscle myosin. RNase protection analysis with a probe spanning this region detected two partially protected fragments which were not present in RNA from vascular tissue and only found in RNA from visceral tissue. The polymerase chain reaction was used to amplify a 162bp product from primers spanning the putative region of divergence and DNA sequence analysis revealed a seven amino acid insertion not previously detected in other characterised cDNA clones. RNase protection analysis using the PCR product as probe showed that the inserted sequence was expressed exclusively in RNA from visceral tissue. Similar RNA analysis showed that the visceral isoform was not expressed in 20 day fetal rabbit smooth muscle tissues. These results indicated that the new visceral isoform was expressed in a tissue-specific and developmentally regulated manner. Genomic DNA sequencing and mapping of the exon-intron boundaries showed that the visceral isoform was the product of cassette-type alternative splicing. The inclusion of a visceral-specific sequence near the Mg-ATPase domain and at the 25kDa/50kDa junction suggests that the visceral isoform may be important for myosin function in smooth muscle cells.     

Potentiation of actomyosin ATPase activity by filamin

It was found that thin filaments from chicken gizzard muscle activate skeletal muscle myosin Mg2+-ATPase to a greater extent than does the complex of chicken gizzard actin and tropomyosin. The protein factor responsible for this additional activation has been now identified as the high Mr actin binding protein, filamin.     

Chicken gizzard filamin, retina filamin and cgABP260 are respectively, smooth muscle-, non-muscle- and pan-muscle-type isoforms: distribution and localization in muscles

We determined the full cDNA sequences of chicken gizzard filamin and cgABP260 (chicken gizzard actin-binding protein 260). The primary and secondary structures predicted by these sequences were similar to those of chicken retina filamin and human filamins. Like mammals, chickens have 3 filamin isoforms. Comparison of their amino acid sequences indicated that gizzard filamin, retina filamin, and cgABP260 were the counterparts of human FLNa (filamin a), b, and c, respectively. Antibodies against the actin-binding domain (ABD) of these 3 filamin isoforms were raised in rabbits. Using immunoabsorption and affinity chromatography, we prepared the monospecific antibody against the ABD of each filamin. In immunoblotting, the antibody against the gizzard filamin ABD detected a single band in gizzard, but not in striated muscles or brain. In brain, only the antibody against the retina filamin ABD produced a strong single band. The antibody against the cgABP260 ABD detected a single peptide band in smooth, skeletal, and cardiac muscle. In immunofluorescence microscopy of muscular tissues using these antibodies, the antibody against the gizzard filamin ABD only stained smooth muscle cells, and the antibody against the retina filamin ABD strongly stained endothelial cells of blood vessels and weakly stained cells in connective tissue. The antibody against the cgABP260 ABD stained the Z-lines and myotendinous junctions of breast muscle, the Z-lines and intercalated disks of cardiac muscle, and dense plaques of smooth muscle. These findings indicate that chicken gizzard filamin, retina filamin, and cgABP260 are, respectively, smooth muscle-type, non-muscle-type, and pan-muscle-type filamin isoforms.     

Calcium sensitivity of contractile proteins from chicken gizzard muscle.

Actin, myosin, and "native" tropomyosin (NTM) were separately isolated from chicken gizzard muscle and rabbit skeletal muscle. With various combinations of the isolated contractile proteins, Mg-ATPase activity and superprecipitation activity were measured. It was thus found that gizzard myosin and gizzard NTM behaved differently from skeletal myosin and skeletal NTM, whereas gizzard actin functioned in the same wasy as skeletal actin. It was also found that gizzard myosin preparations were often Ca-sensitive, that is, that the two activities of gizzard myosin plus actin without NTM were activated by low concentrations of Ca2+. The Mg-ATPase activity of a Ca-insensitive preparation of gizzard myosin was not activated by actin even in the presence of Ca2+. When Ca-sensitive gizzard myosin was incubated with ATP (and Mg2+) in the presence of Ca2+, a light-chain component of gizzard myosin was phosphorylated. The light-chain phosphorylation also occurred when Ca-insensitive myosin was incubated with gizzard NTM and ATP (plus Mg2+) in the presence of Ca2+. In either case, the light-chain phosphorylation required Ca2+. Phosphorylated gizzard myosin in combination with actin was able to exhibit superprecipitation, and Mg-ATPase of the phosphorylated gizzard myosin was activated by actin; the actin activation and superprecipitation were found to occur even in the absence of Ca2+ and NTM or tropomyosin. The phosphorylated light-chain component was found to be dephosphorylated by a partially purified preparation of gizzard myosin light-chain phosphatase. Gizzard myosin thus dephosphorylated behaved exactly like untreated Ca-insensitive gizzard myosin; in combination with actin, it did not superprecipitate either in the presence of Ca2+ or in its absence, but did superprecipitated in the presence of NTM and Ca2+. Ca-activated hydrolysis of ATP catalyzed by gizzard myosin B proceeded at a reduced rate after removal of Ca2+ (by adding EGTA), whereas that catalyzed by a combination of actin, gizzard myosin, and gizzard NTM proceeded at the same rate even after removal of Ca2+. However, addition of a partially purified preparation of gizzard myosin light-chain phosphatase was found to make the recombined system behave like myosin B. Based on these findings, it appears that myosin light-chain kinase and myosin light-chain phosphatase can function as regulatory proteins for contraction and relaxation, respectively, of gizzard muscle.     

Phosphorylation of regulatory light chain a (RLC-a) in smooth muscle myosin of scallop, Patinopecten yessoensis

One of the two regulatory light chains, RLC-a, of scallop smooth muscle myosin was fully phosphorylated by myosin light chain kinase of chicken gizzard muscle. The residue phosphorylated was Ser. It may be the Ser at number 11 from the N-terminal. The sequence of 9 residues around the Ser-11, QRATSNVFA, is identical with that around the phosphorylatable Ser of LC20 of chicken gizzard myosin. RLC-a was also phosphorylated slowly by cAMP-dependent protein kinase. The phosphorylation of RLC-a may be involved in the regulatory system for the catch contraction of scallop muscle.     

Molecular characterization of a mammalian smooth muscle myosin light chain kinase.

A 5.6-kilobase cDNA clone has been isolated which includes the entire coding region for the myosin light chain kinase from rabbit uterine tissue. This cDNA, expressed in COS cells, encodes a Ca2+/calmodulin-dependent protein kinase with catalytic properties similar to other purified smooth muscle myosin light chain kinases. A module (TLKPVGNIKPAE), repeated sequentially 15 times, has been identified near the N terminus of this smooth muscle kinase. It is not present in chicken gizzard or rabbit skeletal muscle myosin light chain kinases. This repeat module and a subrepeat (K P A/V) are similar in amino acid content to repeated motifs present in other proteins, some of which have been shown to associate with chromatin structures. Immunoblot analysis after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, used to compare myosin light chain kinase present in rabbit, bovine, and chicken smooth and nonmuscle tissues, showed that within each species both tissue types have myosin light chain kinases with indistinguishable molecular masses. These data suggest that myosin light chain kinases present in smooth and nonmuscle tissues are the same protein.