Regions of the 110-kDa regulatory subunit M110 required for regulation of myosin-light-chain-phosphatase activity in smooth muscle.

To characterize the in situ interactions between the subunits (regulatory 110 kDa, M110; 21-kDa, M21 and catalytic, 37-kDa, PP1c) of smooth muscle myosin phosphatase (SMPP-1M), we determined, in Triton-X-100-permeabilized rabbit portal vein contracted with microcystin-LR, the ability of the following fragments of M110 to regulate relaxation induced by exogenous PP1c: (a) M110 purified from pig bladder; (b) the 72.5-kDa N-terminal fragment expressed from rat kidney cDNA [glutathione-S-transferase-M110-(11-612)-peptide]; (c) a 58-kDa fragment, the N-terminal degradation product of M110 (M58); (d) two fragments expressed from rat aorta cDNA [M110-(1-309)-peptide and M110-(39-309)-peptide]; a synthetic fragment of M110 [M110-(1-38)-peptide]. The M110/M21 complex accelerated approximately 1.6-fold the rate of dephosphorylation of the myosin P-light chain and also relaxation induced by PP1c. The glutathione-S-transferase-M110-(11-612)-peptide and the M58 fragments, as well as the M110-(1-309)-peptide and, at higher concentration, M110-(1-38)-peptide, had similar effects that did not require the M21 subunit. Arachidonic acid, known to dissociate PP1c from the native holoenzyme and inhibit SMPP-1M activity, inhibited the regulatory action of the M110/M21 complex on PP1c activity and, to a lesser extent that of the glutathione-S-transferase-M110-(11-612)-peptide, but not that of the M58 fragment or of the shorter peptides. We conclude that, consistent with in vitro studies [8], the N-terminal sequence (1-309) of the M110 subunit is also sufficient to enhance the activity of PP1c for myosin in muscle. However, its C-terminal half (downstream from the M58 fragment) is required for inhibition by arachidonic acid. In contrast to the effect of the M110 subunit and its fragments, a peptide, corresponding to part of the PP1c-binding site of the regulatory glycogen-binding subunit from skeletal muscle GM [GM-(63-93)-peptide], specifically slowed the relaxation, induced by flash photolysis of diazo-2, of Triton X-100-permeabilized femoral artery strips, and inhibited the holoenzyme-induced relaxation in the portal vein, suggesting that the GM subunit can compete with the regulatory effect of M110 on PP1c in smooth muscle.

Duke Scholars

Author Timothy Arthur James Haystead Pharmacology & Cancer Biology