Inhibitor‐2 functions like a chaperone to fold three expressed isoforms of mammalian protein phosphatase‐1 into a conformation with the specificity and regulatory properties of the native enzyme

作者：Dario R. Alessi, Alasdair J. Street, Philip Cohen, Patricia T.W. Cohen · 发表于：European Journal of Biochemistry · 年份：1993 · DOI：10.1111/j.1432-1033.1993.tb17853.x · 被引用次数：186 · 研究领域：Alkaline Phosphatase Research Studies、Protein Kinase Regulation and GTPase Signaling、Mitochondrial Function and Pathology

Three isoforms of mammalian protein phosphatase-1 (PP1 alpha, PP1 beta and PP1 gamma) were expressed in Escherichia coli and purified to near homogeneity. The activities of all isoforms towards phosphorylase, phosphorylase kinase and myosin and their sensitivities to inhibitor-2 were similar to the native PP1 catalytic subunit (PP1C) isolated from vertebrate tissues. Like PP1C, they each formed a complex with the glycogen-targetting(G) subunit which directs PP1C to glycogen particles in skeletal muscle. However, other properties differed strikingly from native PP1C. The expressed isoforms were 100-600-fold less sensitive to inhibitor-1, 3-5-fold less sensitive to okadaic acid, 5-100-fold less sensitive to microcystin-LR and approximately 20-fold more active in dephosphorylating histone H1 than native PP1C. Although PP1 gamma (like PP1C) was active in the absence of Mn2+, expressed PP1 alpha and PP1 beta were completely dependent on Mn2+ for activity. PP1 beta, like PP1C, interacted with the myofibrillar-targetting(M) complexes from skeletal-muscle and smooth-muscle producing species with enhanced myosin-phosphatase activity, whereas expressed PP1 alpha and PP1 gamma did not. The expressed isoforms of PP1 combined with inhibitor-2 to form an inactive complex (PP1I) that could be reactivated by the glycogen-synthase-kinase-3(GSK3)-catalysed phosphorylation of inhibitor-2. This procedure transformed the properties of all three expressed isoforms to those of native PP1C. Their se...