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, PP1(C)) 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 PP1(C) (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 PP1(C). 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 PP1(C) from the native holoenzyme and inhibit SIMPP-1M activity, inhibited the regulatory action of the M₁₁₀/M₂₁ complex on PP1(C) 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, the N-terminal sequence (1-309) of the M₁₁₀ subunit is also sufficient to enhance the activity of PP1(C) 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 PP1(C)-binding site of the regulatory glycogen-binding subunit from skeletal muscle G(M) [G(M)-(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 G(M) subunit can compete with the regulatory effect of M₁₁₀ on PP1(C) in smooth muscle.