Domain organization of chicken gizzard myosin light chain kinase deduced from a cloned cDNA

Abstract

Myosin light chain kinases (MLCK) are the most studied of the calmodulin-activated enzymes; however, minimal sequence information is available for the smooth muscle form of the enzyme. The production of an antibody against the enzyme and the use of expression vectors for constructing cDNA libraries have facilitated the isolation of a cDNA for this kinase. The derived amino sequence was found to contain a region of high homology (54%) to the rabbit skeletal muscle enzyme and also very significant homology (35%) to the catalytic subunit of phosphorylase b kinase and cGMP-dependent protein kinase. All of these homologies were found in the known catalytic domains of these enzyme, thus enabling us to predict the location of the catalytic domain for the chicken gizzard myosin light chain kinase. Within the catalytic domain a consensus sequence for an ATP-binding site was located. Subcloning and expression of different regions of the cDNA defined a 192 base pair fragment coding for the calmodulin-binding domain of MLCK. Both of the cAMP-dependent protein kinase phosphorylation sites were identified by sequence homology. A linear model for MLCK is presented placing the various domains in relative position. Northern blot analysis and S1 protection and mapping experiments have revealed that the mRNA for MLCK is 5.5 kilobases in length, but there also exists a second mRNA of 2.7 kilobases that shares a high degree of homology with about 520 base pairs at the 3' end of the cDNA for MLCK.

Isolation and characterization of a cDNA clone encoding avian skeletal muscle C-protein: an intracellular member of the immunoglobulin superfamily.

“…Another protein, chicken smooth muscle myosin light chain kinase (smMLCK) (38), was found in a computer search to exhibit significant similarity to the derived amino acid sequence of AC-86. Upon comparing the sequences, it was seen that smMLCK contains regions similar to the immunoglobulin-like (15-30% matching residues) and Fn-like (22-27% matching residues) domains of C-protein (Fig.…”

The actin cytoskeleton in cancer cell motility

“…Phosphorylation of the myosin II light chains (MLC) is a key mechanism for regulation of actin-myosin contractility [43]. MLC phosphorylation promotes the release of the [52,53] and MLCK [54] ( Table 1). The ability of these various kinases to phosphorylate MLC allows for multiple signalling pathways to converge on the regulation of actin-myosin contractility.…”

Myosin light chain phosphorylation regulates barrier function by remodeling tight junction structure

“…Moreover, in the event that MLCK activation alone could trigger TJ regulation, we sought to determine morphological and biochemical changes in TJ structure that accompany MLC phosphorylation-induced barrier function decreases. We developed a Caco-2 cell line stably expressing an inducible transactivating element, Tet-off (Gossen and Bujard, 1992;Yu Journal of Cell Science 119 (10) et al, 2001), that drives expression of a constitutively active truncated mutant of MLC kinase, tMLCK (Guerriero et al, 1986). By suppressing tMLCK expression until the Caco-2 cells formed fully differentiated polarized monolayers, we were able to test the role of MLCK in the acute regulation of TJ function.…”