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 phosphatase-1 (PP1), is dominantly expressed in smooth muscle, and the inhibitory activity is potentiated by protein kinase C and its related enzymes [Eto, M. et al. (1997) FEBS Lett. 410, 356-360]. In order to identify its physiological target in smooth muscle, the myofibrillar extract from porcine aorta media was analyzed by affinity chromatography on CPI17-conjugated Sepharose. The binding of phosphatases to the resin depended on thiophosphorylation of CPI17, and about 90% of the phosphatase activities toward phosphorylated myosin (p-myosin) and phosphorylase-a were bound to the resin and could be eluted with 0.5 M NaCl. The IC50 values of thiophosphorylated CPI17 toward phosphatases bound to the resin were in the range of 0.5-3 nM, as expected for the PP1 holoenzymes sensitive to CPI17. The CPI17-sensitive fraction was further separated into several peaks of phosphatase activity by column chromatography on Mono Q, which suggested multiple functions of CPI17 as a mediator of the protein kinase C-related signal transduction pathway in aorta smooth muscle. The major activity toward p-myosin was identified as the myofibril-bound PP1 (PP1M), and its subunit composition (140, 37, and 20 kDa) was consistent with that of PP1M from chicken gizzard and porcine bladder. The purified PP1M was completely inhibited by phosphorylated and thiophosphorylated CPI17. Kinetic analysis showed mixed inhibition of PP1M by CPI17 (Ki = 1.9 nM and Ki' = 5.1 nM). The concentration of CPI17 in aorta smooth muscle cells was estimated to be at least 0. 3 microM from the result of Western 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 of CPI17 in vascular smooth muscle.
Senba
S
S
Division of Chemistry, Graduate School of Science, Hokkaido University, Sapporo, Hokkaido, 060-0810, Japan.
Eto
M
M
Yazawa
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001011:Aorta / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000378:metabolism*; D020035:Holoenzymes / Q000378:metabolism; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000378:metabolism*; D009131:Muscle, Smooth, Vascular / Q000201:enzymology / Q000378:metabolism*; D043263:Myosin-Light-Chain Phosphatase; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors* / Q000302:isolation & purification / Q000378:metabolism; D010750:Phosphoproteins; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D054645:Protein Phosphatase 1; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D013552:Swine