Regulatory and structural motifs of chicken gizzard myosin light chain kinase.

Abstract

The amino acid sequence for chicken smooth muscle myosin light chain kinase (smMLCK) was deduced from a full-length cDNA. This has allowed dermition ofboth the complete sequence of the inactive 64-kDa proteolytic fragment, which contains the pseudosubstrate autoregulatory sequence, and of the active 61-kDa Ca2+/calmodulin-independent fragment, which lacks the autoregulatory domain. Comparison of the two sequences shows that the autoregulatory domain extends from Asn-780 to Arg-808. The peptide Leu-774 to Ser-787 does not inhibit smMLCK, whereas peptides of similar or shorter length from the pseudosubstrate region (Ser-787 to Val-807) are potent inhibitors. These data define the autoregulatory region as being contained within and probably identical to the pseudosubstrate domain. The catalytic and regulatory regions are flanked by several copies of 100-amino acid segments containing one of two consensus motifs. These motifs are absent from mammalian skeletal muscle MLCK or from Dictyostelium discoideum MLCK but are present in the Caenorhabditis elegans unc-22 gene product and the titin molecule of skeletal muscle myofibrils. These results indicate that the amino acid sequence of smMLCK encodes multiple functional motifs in addition to the catalytic domain.Myosin light chain kinases (MLCKs)

Intermolecular tuning of calmodulin by target peptides and proteins: Differential effects on Ca2+ binding and implications for kinase activation

“…Many CaM regulated kinases, including skMLCK, smMLCK, and MKII, are activated by the removal of an autoinhibitory sequence from the active site of the enzyme, an event that can be triggered either by the binding of CaM to a nearby sequence or by phosphorylation of key residues (Zurini et al, 1984;Edelman et al, 1985;Pearson et al, 1988;Olson et al, 1990;Gallagher et al, 1993;Brickey et al, 1994;Krueger et al, 1995;Goldberg et al, 1996). For these proteins, the smaller Ca2+ affinity enhancement observed for the full-size enzyme relative to the isolated target peptide is likely to stem, at least in part, from the energetic cost of removing autoinhibition.…”

Primary structure required for the inhibition of smooth muscle myosin light chain kinase

“…In the central portion of the molecule, the sequences commonly observed in various protein kinases [3][4][5] are found and, therefore, it is assigned that the catalytic domain exists at the central part of the molecule. Calmodulin binding peptide was isolated by Lukas et al [6] and the sequence correlates with residues 797-816 of chicken gizzard MLCK sequence [3,4] which lies towards the C-terminal side of the catalytic domain. Kemp et al [7] found that residues 788-811 showed significant homology to the N-terminal sequence of the regulatory light chain of myosin and that a synthetic peptide analog of this region inhibited Ca2÷/calmodulindependent MLCK activity.…”

“…Phosphorylation of the regulatory light chain of myosin is catalyzed by a Ca~-*/ calmodulin-dependent myosin light chain kinase (MLCK). The structure-function relationships of smooth muscle MLCK are greatly assisted by the determination of the complete amino acid sequence [3][4][5]. In the central portion of the molecule, the sequences commonly observed in various protein kinases [3][4][5] are found and, therefore, it is assigned that the catalytic domain exists at the central part of the molecule.…”

“…The structure-function relationships of smooth muscle MLCK are greatly assisted by the determination of the complete amino acid sequence [3][4][5]. In the central portion of the molecule, the sequences commonly observed in various protein kinases [3][4][5] are found and, therefore, it is assigned that the catalytic domain exists at the central part of the molecule. Calmodulin binding peptide was isolated by Lukas et al [6] and the sequence correlates with residues 797-816 of chicken gizzard MLCK sequence [3,4] which lies towards the C-terminal side of the catalytic domain.…”

Ontogenesis of myosin light chain kinase mRNA and protein content in guinea pig tracheal smooth muscle

“…12 It is generally accepted that this initial phase is dependent on phosphorylation of the 20-kDa myosin light chain (MLC 20 ) and the consequent activation of the acto-myosin ATPase, with recruitment of fast-cycling cross-bridges. The regulation of MLC 20 phosphorylation is therefore a key limiting event of shortening velocity. Net MLC 20 phosphorylation reflects the opposing enzyme activity of myosin light chain kinase (MLCK) and myosin light chain phosphatase, which phosphorylate and dephosphorylate MLC 20 , respectively.…”