Properties of Myosin Light Chain Kinase Prepared from Rabbit Skeletal Muscle by an Improved Method1

Nagamoto, Hisashi; Yagi, Koichi

doi:10.1093/oxfordjournals.jbchem.a134700

Cited by 24 publications

(11 citation statements)

References 0 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…4). Fluorescence enhancement was maximal (=45%) at a molar ratio of 1 mol of peptide per mol of calmodulin, consistent with the stoichiometry of interaction between intact MLCK and calmodulin (8,11,14,(19)(20)(21)(22). Similar (18).…”

Section: Resultssupporting

confidence: 71%

Identification of the calmodulin-binding domain of skeletal muscle myosin light chain kinase.

Blumenthal¹,

Takio²,

Edelman³

et al. 1985

Proc. Natl. Acad. Sci. U.S.A.

305

153

View full text Add to dashboard Cite

In the course of determining the primary structure of rabbit skeletal muscle myosin light chain kinase (MLCK; ATP:protein phosphotransferase, EC 2.7.1.37) a peptide fragment was obtained that appears to represent the calmodulin-binding domain of this enzyme. Low concentrations of the peptide inhibited calmodulin activation of MLCK (Ki 1 nM). The peptide was not associated with a catalytically active, calmodulin-independent form of MLCK that was obtained by limited proteolysis. The peptide is 27 residues in length and represents the carboxyl terminus of MLCK. The sequence of the peptide shows no significant homology with any known protein sequence. The peptide contains one tryptophanyl residue and a high percentage of basic and hydrophobic residues, but no acidic or prolyl residues. Much of the sequence has a high probability of forming a helix. A chemically synthesized peptide has been prepared to study the interactions of the peptide and calmodulin in more detail. The intrinsic tryptophan fluorescence of the synthetic peptide shows a significant enhancement (w45%) in the presence of Ca2' and calmodulin; fluorescence enhancement is maximal at a peptide:calmodulin stoichiometry of 1:1. Calmodulin-Sepharose affinity chromatography in the presence of 2 M urea indicates that the interaction of peptide and calmodulin is Ca2+-dependent. The results of these studies indicate that the catalytic and calmodulin-binding domains of MLCK represent distinct and separable regions of the protein. In addition, the results provide a basis for future studies of the molecular and evolutionary details of calmodulin-dependent enzyme regulation.Calmodulin is a Ca2+-binding protein that is ubiquitously distributed and highly conserved throughout eukaryotic evolution. Although it is known to regulate many Ca2+-dependent enzymes (recently reviewed in ref. 1), little is known about the interactions of calmodulin with these proteins at the molecular level. Myosin light chain kinase (MLCK; ATP:protein phosphotransferase, EC 2.7.1.37) is one of the better characterized calmodulin-regulated enzymes (reviewed in refs. 2 and 3) and is found in both muscle and nonmuscle tissues. The enzyme occurs in tissue-specific forms, which vary widely in molecular weight, antigenic determinants, and enzymatic properties (3, 4). It catalyzes the phosphorylation of a specific class of myosin light chain subunit, termed the P-light chain (5). In smooth muscle this phosphorylation is required for initiation of contraction (reviewed in refs. 2 and 6), whereas in skeletal muscle the phosphorylation appears to modulate the degree of tension produced during isometric contraction (3, 7).A determination of the amino acid sequence of rabbit skeletal muscle MLCK was undertaken by these several laboratories as part of a long-range study of protein kinase and cal- Ca2' and 5 gM calmodulin. This calmodulin-independent form of MLCK was denoted C35. Details of the preparation and properties of these proteolyzed forms of MLCK will be described elsewhere. CNBr digesti...

show abstract

Section: Resultssupporting

confidence: 71%

Identification of the calmodulin-binding domain of skeletal muscle myosin light chain kinase.

Blumenthal¹,

Takio²,

Edelman³

et al. 1985

Proc. Natl. Acad. Sci. U.S.A.

305

153

View full text Add to dashboard Cite

show abstract

“…Nonphosphorylated and phosphorylated aorta RLC were prepared as described previously (15). Rabbit skeletal muscle myosin light chain kinase was prepared according to the method of Nagamoto and Yagi (16). Skeletal myosin was phosphorylated under the same conditions as those for the phosphorylation of smooth muscle myosin (17), except for the use of skeletal myosin light chain kinase.…”

Section: Methodsmentioning

confidence: 99%

Skeletal Muscle Myosin Monomer in Equilibrium with Filaments Forms a Folded Conformation

Katoh

Konishi

Yazawa

1998

Journal of Biological Chemistry

View full text Add to dashboard Cite

Rabbit skeletal myosin forms stable filaments under physiological conditions, and only a small amount stays as a monomer in equilibrium with filaments. The myosin monomers were observed in two conformational states, as extended and folded forms upon electron microscopy and gel filtration high performance liquid chromatography. The fraction of monomers in the folded conformation increased with a decrease in the concentration of NaCl below 0.2 M, and the conformational state was affected neither by the presence of ATP nor by the phosphorylation of regulatory light chain. In most of the folded monomers, the tail bent back toward the heads at one region, 45 nm apart from the head-tail junction, and the remaining tail portion containing the C-terminal tip appeared to interact with the head-tail junction. Only a small percentage of the folded monomers was in a more compact conformation close to the 10 S conformation of vertebrate smooth muscle and non-muscle myosins. The folded monomers, however, may not trap the products of ATP hydrolysis as assessed by single turnover experiments. The percentage of monomers in the 10 S-like conformation was increased by the exchange of a regulatory light chain with the smooth muscle light chain, indicating the participation of head-tail junction, including the regulatory light chain in the formation of folded conformation. The folded conformation may be common to various myosin IIs, suggestive of common roles for the folded monomers.

show abstract

“…A range of solutions containing different [Ca 2+ ] free were prepared by mixing pCa 9.0 and pCa 4.5 solutions. The catalytic subunit of bovine smooth muscle myosin light chain kinase (MLCK) was prepared as described previously (Nagamoto & Yagi, 1984) and stored at −80 • C until used in an experiment. The catalytic subunit of bovine protein kinase A (PKA) was purchased from Sigma (no.…”

Section: Experimental Solutionsmentioning

confidence: 99%

Differential roles of regulatory light chain and myosin binding protein-C phosphorylations in the modulation of cardiac force development

Colson

Locher

Bekyarova

et al. 2010

The Journal of Physiology

155

199

View full text Add to dashboard Cite

Phosphorylation of myosin regulatory light chain (RLC) by myosin light chain kinase (MLCK)and myosin binding protein-C (cMyBP-C) by protein kinase A (PKA) independently accelerate the kinetics of force development in ventricular myocardium. However, while MLCK treatment has been shown to increase the Ca 2+ sensitivity of force (pCa 50 ), PKA treatment has been shown to decrease pCa 50 , presumably due to cardiac troponin I phosphorylation. Further, MLCK treatment increases Ca 2+ -independent force and maximum Ca 2+ -activated force, whereas PKA treatment has no effect on either force. To investigate the structural basis underlying the kinase-specific differential effects on steady-state force, we used synchrotron low-angle X-ray diffraction to compare equatorial intensity ratios (I 1,1 /I 1,0 ) to assess the proximity of myosin cross-bridge mass relative to actin and to compare lattice spacings (d 1,0 ) to assess the inter-thick filament spacing in skinned myocardium following treatment with either MLCK or PKA. As we showed previously, PKA phosphorylation of cMyBP-C increases I 1,1 /I 1,0 and, as hypothesized, treatment with MLCK also increased I 1,1 /I 1,0 , which can explain the accelerated rates of force development during activation. Importantly, interfilament spacing was reduced by ∼2 nm ( 3.5%) with MLCK treatment, but did not change with PKA treatment. Thus, RLC or cMyBP-C phosphorylation increases the proximity of cross-bridges to actin, but only RLC phosphorylation affects lattice spacing, which suggests that RLC and cMyBP-C modulate the kinetics of force development by similar structural mechanisms; however, the effect of RLC phosphorylation to increase the Ca 2+ sensitivity of force is mediated by a distinct mechanism, most probably involving changes in interfilament spacing.

show abstract

Properties of Myosin Light Chain Kinase Prepared from Rabbit Skeletal Muscle by an Improved Method1

Cited by 24 publications

References 0 publications

Identification of the calmodulin-binding domain of skeletal muscle myosin light chain kinase.

Identification of the calmodulin-binding domain of skeletal muscle myosin light chain kinase.

Skeletal Muscle Myosin Monomer in Equilibrium with Filaments Forms a Folded Conformation

Differential roles of regulatory light chain and myosin binding protein-C phosphorylations in the modulation of cardiac force development

Contact Info

Product

Resources

About