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

Abstract

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

Microarray profiling of skeletal muscle tissues from equally obese, non-diabetic insulin-sensitive and insulin-resistant Pima Indians

“…The other transcripts encode PPP1R11 and PPP1R2, regulatory subunits of protein phosphatase-1 (PP1), a key enzyme that regulates the activity of glycogen synthase. PPP1R2 has been proposed to act as a molecular "chaperone" that aids the folding of newly synthesized PP1 into a biologically active conformation [31,32] and it has been shown to translocate to the nucleus during the S-phase of cell cycle [33]. There were no polymorphisms in the exons and exon-intron splice junctions of the PPP1R2 gene in selected Pima Indian subjects [34] but potential polymorphisms in the regulatory region of the gene might account for the differential gene expression.…”

Protein phosphatase 1 interacts with p53BP2, a protein which binds to the tumour suppressor p53

“…This indicates that p53BP2 is likely to have a selective effect on the substrate specificity of PP1, as has been noted for other regulatory subunits of PP1. For example, the M Ltu subunit decreases the phosphorylase phosphatase activity of PPl and enhances its myosin P-light chain phosphatase activity [22]. In contrast, the cytosolic protein, inhibitor-2, inhibits both the phosphorylase phosphatase and myosin P-light chain phosphatase activity of PP1, but has little effect on the dephosphorylation of histone H I [22].…”

“…For example, the M Ltu subunit decreases the phosphorylase phosphatase activity of PPl and enhances its myosin P-light chain phosphatase activity [22]. In contrast, the cytosolic protein, inhibitor-2, inhibits both the phosphorylase phosphatase and myosin P-light chain phosphatase activity of PP1, but has little effect on the dephosphorylation of histone H I [22]. Thus p53BP2 affects the specificity of PP1 in a different manner from other known regulatory subunits.…”

“…They were phosphorylated with ATP at the 5" ends using T4 kinase, hybridised by heating in equimolar amounts in 50 mM Tris-HCl pH 8.0 to 95°C, allowed to cool to 60°C, maintained at 60-65°C for 2 h and then allowed to cool to room temperature, PPIy: cDNA in pBS vector [21,22] was cleaved with BclI and HindIII. The fragment containing the vector and the region coding for amino acids 1-317 of PPI?…”

“…A NdeI-HindlIl fragment encoding PP1Ta-EFMPME(H)6 was cleaved from one transformant with the correct sequence and subcloned into the expression vector pCW [23] using the same restriction sites. The pCW-PP1y]-EFMPME(H)6 construct was transformed into E, coil BL21(DE3) and expressed as described for PP 1 ~1 in [22]. Purification of the His-tagged protein was by metal affinity chromotography.…”