INHIBITOR-2 FUNCTIONS LIKE A CHAPERONE TO FOLD 3 EXPRESSED ISOFORMS OF MAMMALIAN PROTEIN PHOSPHATASE-1 INTO A CONFORMATION WITH THE SPECIFICITY AND REGULATORY PROPERTIES OF THE NATIVE ENZYME

Three isoforms of mammalian protein phosphatase-1 (PP1alpha, PP1beta a nd PP1gamma) were expressed in Escherichia coli and purified to near h omogeneity. The activities of all isoforms towards phosphorylase, phos phorylase kinase and myosin and their sensitivities to inhibitor-2 wer e similar to the native PP1 catalytic subunit (PP1C) isolated from ver tebrate tissues. Like PP1C, they each formed a complex with the glycog en-targetting(G) subunit which directs PP1C to glycogen particles in s keletal muscle. However, other properties differed strikingly from nat ive PP1C. The expressed isoforms were 100-600-fold less sensitive to i nhibitor-1, 3-5-fold less sensitive to okadaic acid, 5-100-fold less s ensitive to microcystin-LR and approximately 20-fold more active in de phosphorylating histone H1 than native PP1C. Although PP1gamma (like P P1C) was active in the absence of Mn2+, expressed PP1alpha and PP1beta were completely dependent on Mn2+ for activity. PP1beta, like PP1C, i nteracted with the myofibrillar-targetting(M) complexes from skeletal- muscle and smooth-muscle producing species with enhanced myosin-phosph atase activity, whereas expressed PP1alpha PP1gamma did not. The expre ssed isoforms of PP1 combined with inhibitor-2 to form an inactive com plex (PP1I) that could be reactivated by the glycogen-synthase-kinase- 3(GSK3)-catalysed phosphorylation of inhibitor-2. This procedure trans formed the properties of all three expressed isoforms to those of nati ve PP1C. Their sensitivities to inhibitor-1, okadaic acid and microcys tin-LR were increased greatly, their histone-phosphatase activities de creased and the activities of PP1alpha PP1beta became independent of M n2+. Furthermore PP1alpha PP1gamma now interacted with the M complexes in a similar manner to PP1beta and PP1C. Conversely, incubation of na tive PP1C with 50 mM NaF caused conversion to a Mn2+-dependent form wi th properties similar to those of the expressed isozymes. The G subuni t from skeletal muscle or the M complex from smooth muscle could displ ace 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 guanidini um chloride in the absence of Mn2+. Taken together, the results sugges t that inhibitor-2 is critical for the correct folding of nascent PP1C polypeptides, that its function is similar to that of a molecular cha perone and that it acts as a cytosolic reservoir of PP1C molecules whi ch can be directed to the required subcellular locations following the synthesis of specific targetting subunits.