The amino acid sequence of a novel mammalian protein phosphatase, termed PPX (and designated PPP4 in the human genome nomenclature), has been deduced from the cDNA and shown to be 65% identical to PP2A alpha and PP2A beta and 45% identical to PPI isoforms, the predicted molecular mass being 35 kDa. PPX was expressed in the baculovirus system. Its substrate specificity and sensitivity to the inhibitors, okadaic acid and microcystin, were similar (but not identical) to the catalytic subunit of PP2A. However, PPX did not bind the 65 kDa regulatory subunit of PP2A. The intracellular localization of PPX was investigated by immunofluorescence using two different antibodies raised against bacterially expressed PPX and a PPX‐specific peptide. These showed that although PPX was distributed throughout the cytoplasm and the nucleus, intense staining occurred at centrosomes. The centrosomal staining was apparent in interphase and at all stages of mitosis, except telophase. In contrast, antibodies directed against bacterially expressed PP2A were not specifically localized to centrosomes. The human autoantibody #5051, which stains the pericentriolar material, colocalizes with PPX antibodies, suggesting that PPX may play a role in microtubule nucleation.
Three isoforms of mammalian protein phosphatase-1 (PPla, PPlp and PPly) 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 (PPlC) isolated from vertebrate tissues. Like PPlC, they each formed a complex with the glycogen-targetting(G) subunit which directs PPlC to glycogen particles in skeletal muscle. However, other properties differed strikingly from native PP1 C. 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 PPlC. Although PPly (like PPlC) was active in the absence of Mn2+, expressed PPla and PPlp were completely dependent on Mn2+ for activity. PPlP, like PPlC, interacted with the myofibrillar-targetting(M) complexes from skeletal-muscle and smooth-muscle producing species with enhanced myosin-phosphatase activity, whereas expressed PP1 a and PP1 y did not.The expressed isoforms of PP1 combined with inhibitor-2 to form an inactive complex (PPlI) 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 PPlC. Their sensitivities to inhibitor-1 , okadaic acid and microcystin-LR were increased greatly, their histone-phosphatase activities decreased and the activities of PPla and PPlP became independent of MnZ+. Furthermore PPla and PPly now interacted with the M complexes in a similar manner to PPlp and PPlC. Conversely, incubation of native PPlC with 50 mM NaF caused conversion to a Mn"+-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 PPlC from activated PPlI, but not inactive PPlI, to form G-subunitlPP1C and M-complexPP1C heterodimeric complexes. Inhibitor-2 was also found to be essential for the reactivation of PP1 C 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 PPlC polypeptides, that its function is similar to that of a molecular chaperone and that it acts as a cytosolic reservoir of PPlC molecules which can be directed to the required subcellular locations following the synthesis of specific targetting subunits.Protein phosphatase-1 (PPl), one of the major types of protein serine/threonine phosphatase in eukaryotic cells, is characterised by its specific dephosphorylation of the P-subunit of phosphorylase kinase and sensitivity to the two small
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