Autoinhibition of Myosin Light Chain Kinase

Abstract

Smooth muscle myosin light chain kinase (MLCK) is a Ca2+/calmodulinactivated enzyme that regulates contraction by phosphorylation of the regulatory light chains of myosin. Inactivity of the enzyme in the absence of Ca2+/calmodulin is thought to result from interaction of the catalytic core with another region of the enzyme known as the inhibitory region. This inhibitory region resembles the light chain substrate of the kinase, and therefore is called the pseudosubstrate. A partial cDNA for chicken gizzard MLCK was expressed in bacteria and used to make truncation and point mutations to test if the pseudosubstrate sequence was the inhibitory sequence. Truncation at A796 removes most of the pseudosubstrate sequence, but the mutant was inactive. Truncation at K793 resulted in a constitutively active mutant, indicating that only three amino acids of the pseudosubstrate sequence are needed for inhibition. These results required reevaluation of the pseudosubstrate hypothesis. Based on a model showing the interaction of the pseudosubstrate sequence with the catalytic core, Y794 interacts with a hydrophobic pocket in the catalytic core. We propose that this interaction is enough to inhibit the enzyme.