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

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Abstract
Three isoforms of mammalian protein phosphatase-1 (PP1α, PP1β and PP1γ) 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 PP1γ (like PP1C) was active in the absence of Mn2+, expressed PP1α and PP1β were completely dependent on Mn2+ for activity. PP1β, like PP1C, interacted with the myofibrillar-targetting(M) complexes from skeletal-muscle and smooth-muscle producing species with enhanced myosin-phosphatase activity, whereas expressed PP1α and PP1γ 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 sensitivities to inhibitor-1, okadaic acid and microcystin-LR were increased greatly, their histone-phosphatase activities decreased and the activities of PP1α and PP1β became independent of Mn2+. Furthermore PP1α and PP1γ now interacted with the M complexes in a similar manner to PP1β and PP1C. Conversely, incubation of native PP1C with 50 mM NaF caused conversion to a Mn2+-dependent form with properties similar to those of the expressed isozymes. The G subunit from skeletal muscle or the M complex from smooth muscle could displace PP1C from activated PP1I, but not inactive PP1I, to form G-subunit/PP1C and M-complex/PP1C heterodimeric complexes. Inhibitor-2 was also found to be essential for the reactivation of PP1C from 6 M guanidinium chloride in the absence of Mn2+. Taken together, the results suggest that inhibitor-2 is critical for the correct folding of nascent PP1C polypeptides, that its function is similar to that of a molecular chaperone and that it acts as a cytosolic reservoir of PP1C molecules which can be directed to the required subcellular locations following the synthesis of specific targetting subunits.