MYOSIN PHOSPHORYLATION BY MYOSIN LIGHT CHAIN KINASE

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This project is aimed at understanding the molecular mechanisms by which calmodulin (CaM) modulates the enzymatic activity of the myosin light chain kinase (MLCK) and leads to the calcium dependent phosphorylation/activation of smooth muscle myosin. Specifically, this project is focused on the following problems: (1) The conformational transitions in the catalytic/regulatory domain of rabbit smooth muscle MLCK induced by its interaction with CaM and with the myosin regulatory light chain (RLC). The hypothesis of the intrasteric inhibitory mechanism will be tested be measuring the distance between resonance energy transfer chromophores placed at key positions in the native MLCK and in specifically designed mutants. Distances between landmark sites in the binary and ternary complexes of MLCK, CaM and RLC will be measured. The effects of nucleotides and nucleotide analogues on such complexes will be evaluated. (2) The functional role of MLCK segments outside the catalytic core. Sites interacting with actin and myosin heavy chain will be identified. The structural and functional properties of the N-terminal repetitive segment specific for the mammalian smooth muscle MLCK will be characterized. (3) In situ localization of MLCK in the smooth muscle cell. Distribution of MLCK in relation to other protein components under various contractile conditions will be determined by immunoelectron microscopy using antibodies specific to various segments of MLCK.