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

TY - JOUR

T1 - Regions of the 110-kDa regulatory subunit M110 required for regulation of myosin-light-chain-phosphatase activity in smooth muscle

AU - Gailly, Philippe

AU - Wu, Xuqiong

AU - Haystead, Timothy A.J.

AU - Somlyo, Andrew P.

AU - Cohen, Patricia T.W.

AU - Cohen, Philip

AU - Somlyo, Avril V.

PY - 1996/7/1

Y1 - 1996/7/1

N2 - 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.

AB - 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.

KW - Arachidonic acid

KW - Calcium senstization

KW - Protein phosphatase

KW - Smooth muscle

KW - Smooth muscle myosin phosphatase

UR - https://www.scopus.com/pages/publications/0030037103

U2 - 10.1111/j.1432-1033.1996.0326u.x

DO - 10.1111/j.1432-1033.1996.0326u.x

M3 - Article

C2 - 8706736

AN - SCOPUS:0030037103

SN - 0014-2956

VL - 239

SP - 326

EP - 332

JO - European Journal of Biochemistry

JF - European Journal of Biochemistry

IS - 2

ER -