Senba S, Eto M, Yazawa M
Division of Chemistry, Graduate School of Science, Hokkaido University, Sapporo, Hokkaido, 060-0810, Japan.
J Biochem. 1999 Feb;125(2):354-62. doi: 10.1093/oxfordjournals.jbchem.a022294.
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.
CPI17是蛋白磷酸酶-1（PP1）的一种磷酸化依赖性抑制蛋白，在平滑肌中大量表达，其抑制活性可被蛋白激酶C及其相关酶增强[Eto，M.等人（1997年）《欧洲生物化学学会联合会快报》410，356 - 360]。为了确定其在平滑肌中的生理靶点，通过在CPI17偶联的琼脂糖凝胶上进行亲和层析，对猪主动脉中膜的肌原纤维提取物进行了分析。磷酸酶与树脂的结合取决于CPI17的硫代磷酸化，并且大约90%针对磷酸化肌球蛋白（p - 肌球蛋白）和磷酸化酶 - a的磷酸酶活性与树脂结合，并且可以用0.5 M NaCl洗脱。硫代磷酸化的CPI17对与树脂结合的磷酸酶的IC50值在0.5 - 3 nM范围内，这对于对CPI17敏感的PP1全酶来说是预期的。通过在Mono Q柱上进行层析，对CPI17敏感的部分进一步分离为几个磷酸酶活性峰，这表明CPI17作为主动脉平滑肌中蛋白激酶C相关信号转导途径的介质具有多种功能。针对p - 肌球蛋白的主要活性被鉴定为与肌原纤维结合的PP1（PP1M），其亚基组成（140、37和20 kDa）与来自鸡砂囊和猪膀胱的PP1M一致。纯化的PP1M被磷酸化和硫代磷酸化的CPI17完全抑制。动力学分析表明CPI17对PP1M有混合抑制作用（Ki = 1.9 nM和Ki' = 5.1 nM）。根据蛋白质印迹分析结果，估计主动脉平滑肌细胞中CPI17的浓度至少为0.3 microM。该浓度似乎足以抑制主动脉平滑肌中的原位PP1M，因此PP1M被确定为CPI17在血管平滑肌中的一个靶点。
