Regions of the 110‐kDa Regulatory Subunit M110 Required for Regulation of Myosin‐Light‐Chain‐Phosphatase Activity in Smooth Muscle

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Abstract
To characterize the in situ interactions between the subunits (regulatory 110kDa, 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, hut 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 tlash 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.

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