REGIONS OF THE 110-KDA REGULATORY SUBUNIT M(110) REQUIRED FOR REGULATION OF MYOSIN-LIGHT-CHAIN-PHOSPHATASE ACTIVITY IN SMOOTH-MUSCLE

To characterize the in situ interactions between the subunits (regulat ory 110 kDa, M(110); 21-kDa, M(21) and catalytic, 37-kDa, PP1(c)) of s mooth muscle myosin phosphatase (SMPP-1M), we determined, in Triton-x- 100-permeabilized rabbit portal vein contracted with microcystin-LR, t he ability of the following fragments of M(110) to regulate relaxation induced by exogenous PP1(c): (a) M(110) purified from pig bladder; (b ) the 72.5-kDa N-terminal fragment expressed from rat kidney cDNA luta thione-S-transferase-M(110)-(11-612)-peptide]; (c) a 58-kDa fragment, the N-terminal degradation product of M(110) (M(58)); (d) two fragment s expressed from rat aorta cDNA [M(110)-(1-309)-peptide and M(110)-(39 -309)-peptide]; a synthetic fragment of M(110) [M(110)-(1-38)-peptide] . The M(110)/M(21) 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-M(110)-(11-612)-pepti de and the M(58) fragments, as well as the M(110)-(1-309)-peptide and, at higher concentration, M(110)-(1-38)-peptide, had similar effects t hat did not require the M(21) subunit. Arachidonic acid, known to diss ociate PP1(c) from the native holoenzyme and inhibit SMPP-1M activity, inhibited the regulatory action of the M(110)/M(21), complex on PP1(c ) activity and, to a lesser extent that, of the glutathione-S-transfer ase-M(110)-(11-612)-peptide, but not that of the M(58) fragment or of the shorter peptides. We conclude that, consistent with in vitro studi es [8], the N-terminal sequence (1-309) of the M(110) subunit is also sufficient to enhance the activity of PP1(c) for myosin in muscle. How ever, its C-terminal half (downstream from the M(58) fragment) is requ ired for inhibition by arachidonic acid. In contrast to the effect of the M(110), subunit and its fragments, a peptide, corresponding to par t of the PP1(c)-binding site of the regulatory glycogen-binding subuni t from skeletal muscle G(M) [G(M)-(63 -93)-peptide], specifically slow ed 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) subuni t can compete with the regulatory effect of M(110) on PP1(c) in smooth muscle.