Investigation of the Myosin Light Chain Kinase Mechanism of Phosphorylation using Reconstituted Smooth Muscle Myosin Filaments

Abstract

We are investigating the mechanism by which the Myosin Light Chain Kinase-Calmodulin (MLCK-CaM) complex phosphorylates Smooth Muscle Myosin (SMM). MLCK-CaM phosphorylation of SMM is the primary regulator of smooth muscle contraction, and is required for the catalytic activity of the myosin motor. We have previously shown that MLCK-CaM-SMM forms a tightly-associated complex and that MLCK-CaM co-purifies with SMM. This co-purified MLCK-CaM can phosphorylate SMM which was shown by actin filament motility in an in vitro motility assay. In addition, using total internal reflectance fluorescence (TIRF) microscopy, we were able to characterize dynamic interactions of MLCK with monomeric SMM using Quantum Dots (QD) to label MLCK.The goal of our current study is to determine how a small amount of MLCK is capable of phosphorylating a much larger amount of SMM. The ratio of MLCK to SMM in smooth muscle cells is approximately 1:70, yet the MLCK is able to produce contractions in the cell within seconds. To answer this question, we are investigating the interaction of MLCK-QD with reconstituted SMM filaments. This approach is moving more towards the in vivo conditions, and will help us answer the long term question of how MLCK is phosphorylating SMM within smooth muscle cells.We have to reconstituted SMM filaments which are identical in structure to native SMM filaments, and are capable of being phosphorylated by MLCK. Our reconstituted filaments are fluorescently labeled and are lightly cross-linked, which allows them to be stable in the presence of ATP and/or high ionic strength. We have shown that QD-MLCK co-localizes with our reconstituted SMM filaments. Our data proposes a mechanism of SMM phosphorylation by the physical movement of MLCK along a contractile fiber within the smooth muscle cell.