Regulatory Mechanisms of Smooth Muscle Contraction

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The regulatory mechanisms of myosin phosphatase (MP), the domain structure of the large subunit of MP (MYPT1) and human isoform of MYPT1 have been investigated.1.The activity of myosin phosphatase is inhibited by the phosphorylation of the large regulatory subunit (MYPT1). The kinases involved are the endogenous kinase and Pho-kinase. Introduciton of Rho-kinase into the permeabilized smooth muscle provoked a contraction. These results suggest that Rho-Kinase modulates smooth muscle contraction through the regulation of MP.2.MYPT1 binds to PP1c and also to myosin and thereby effects an activation of phosphatase activity. To investigate the sequence involved in these interaction various mutants of MYPT1 were expressed and assayd. Two regions of MYPT1 are important for binding of PP1c, i.e.the ankyrin repeats and the N-terminal sequence 1 to 38. The C-terminal ankyrin repeats appear dominant in binding to PP1c but there is an interaction that N-terminal repeats may also be involved. The N-terminal peptide has two apparent functions ; the binding to PP1c via the consensus binding sequence and activation of PP1c by the sequence to 16.3.Two cDNA clones encoding the human large subunit of myosin phosphatase were isolated and termed MYPT1 and MYPT2. Sequence analysis revealed that MYPT1 and MYPT2 contained 1030 and 982 amino acids, respectively. The amino acid sequence of MYPT1 was more than 89% identical to that of the rat MYPT1, and MYPT2 contained 61% identity with human MYPT1. Conserved sequences for the two isoforms were in the ankyrin repeats, the leucine zipper motif, and the central 50 residues containing the putative regulatory phosphorylation site. MYPT1 was present mainly in smooth muscle, while MYPT2 is dominant in heart. FISH analysis placed human MYPT1 and MYPT2 genes over chromosome 12q5-21.2 and 1q32-1, respectively.