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

Abstract

To characterize the in situ interactions between the subunits (regulatory 110kDa, 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 SMPP-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, 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 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 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 G_M subunit can compete with the regulatory effect of M₁₁₀ on PP1_C in smooth muscle.