Abstract 16572: Cardiac Mlc2 Kinase Is Localized To The Z-disc And Interacts With α-actinin2

IntroductionCardiac contractility is enhanced by phosphorylation of myosin light chain 2 (MLC2) by cardiac-specific MLC kinase (cMLCK), located at the neck region of myosin heavy chain. In normal mouse and human hearts, the level of phosphorylation is maintained relatively constant, around 30 - 40% of total MLC2, likely by well-balanced phosphorylation and phosphatase-dependent dephosphorylation. Overexpression of cMLCK promotes sarcomere organization, while the loss of cMLCK leads to cardiac atrophy in vitro and in vivo. In neonatal cardiomyocytes, cMLCK protein is localized diffusely in the cytoplasm, with occasional striated patterns that do not overlap with its substrate MLC2.HypothesiscMLCK has a novel function, in addition to phosphorylating MLC2.MethodsProtein crosslinking followed by liquid chromatography tandem mass spectrometry was utilized for screening proteins interacting with cMLCK. To detect protein-protein interactions, we utilized bio-layer interferometry, co-immunoprecipitation assays with multiple mutants and co-immunostaining.ResultsIn normal adult mouse and human hearts, cMLCK is predominantly expressed at the Z-disc with additional diffuse cytosolic expression. We found that cMLCK interacts with the Z-disc protein, α-actinin2, with a high-affinity kinetic value of 13.4 ± 0.1 nM through the N-terminus region of cMLCK, unique to cardiac-isoform. cMLCK mutant deficient for interacting with α-actinin2 did not promote sarcomeric organization, and reduced cardiomyocyte cell size. In contrast, a cMLCK kinase-deficient mutant showed effects similar to wild-type cMLCK on sarcomeric organization and cardiomyocyte cell size.ConclusioncMLCK is predominantly expressed at the Z-disc and interacts with the Z-disc protein α-actinin2. Our results suggest that cMLCK plays a role in sarcomere organization, likely distinct from its role in phosphorylating MLC2, both of which contributes to the enhancement of cardiac contractility.