Identification of trimeric myosin phosphatase (PP1M) as a target for a novel PKC-potentiated protein phosphatase-1 inhibitory protein (CPI17) in porcine aorta smooth muscle

CPI17, a phosphorylation-dependent inhibitory protein of protein phosphatas e-1 (PP1), is dominantly expressed in smooth muscle, and the inhibitory act ivity is potentiated by protein kinase C and its related enzymes [Eto, M, e t at. (1997) FEES Lett, 410, 356-360], In order to identify its physiologic al target in smooth muscle, the myofibrillar extract from porcine aorta med ia was analyzed by affinity chromatography on CPI17-conjugated Sepharose. T he binding of phosphatases to the resin depended on thiophosphorylation of CPI17, and about 90% of the phosphatase activities toward phosphorylated my osin (p-myosin) and phosphorylase-a were bound to the resin and could be el uted with 0.5 M NaCl, The IC,, values of thiophosphorylated CPI17 toward ph osphatases bound to the resin were in the range of 0.5-3 nM, as expected fo r the PPI holoenzymes sensitive to CPI17, The CPI17-sensitive fraction was further separated into several peaks of phosphatase activity by column chro matography on Mono Q, which suggested multiple functions of CPI17 as a medi ator of the protein kinase C-related signal transduction pathway in aorta s mooth muscle. The major activity toward p-myosin was identified as the myof ibril-bound PP1 (PP1M), and its subunit composition (140, 37, and 20 kDa) w as consistent with that of PP1M from chicken gizzard and porcine bladder. T he purified PP1M was completely inhibited by phosphorylated and thiophospho rylated CPI17, Kinetic analysis showed mixed inhibition of PP1M by CPI17 (K -i = 1.9 nM and K-i'= 5.1 nM), The concentration of CPI17 in aorta smooth m uscle cells was estimated to be at least 0.3 mu M from the result of Wester n analysis. This concentration appears to be sufficient to suppress the in situ PP1M in aorta smooth muscle, and PP1M is thus identified as a target o f CPI17 in vascular smooth muscle.