A novel human protein serine/threonine phosphatase, PP5, and a structurally related phosphatase in Saccharomyces cerevisiae, PPT1, have been identified from their cDNA and gene respectively. Their predicted molecular mass is 58 kDa and they comprise a C‐terminal phosphatase catalytic domain and an N‐terminal domain, which has four repeats of 34 amino acids, three of which are tandemly arranged. The phosphatase domain possesses all the invariant motifs of the PP1/PP2A/PP2B gene family, but is not closely related to any other known member (< or = 40% identity). Thus PP5 and PPT1 comprise a new subfamily. The repeats in the N‐terminal domain are similar to the tetratricopeptide repeat (TPR) motifs which have been found in several proteins that are required for mitosis, transcription and RNA splicing. Bacterially expressed PP5 is able to dephosphorylate serine residues in proteins and is more sensitive than PP1 to the tumour promoter okadaic acid. A 2.3 kb mRNA encoding PP5 is present in all human tissues examined. Investigation of the intracellular distribution of PP5 by immunofluorescence, using two different antibodies raised against the TPR and phosphatase domains, localizes PP5 predominantly to the nucleus. This suggests that, like other nuclear TPR‐containing proteins, it may play a role in the regulation of RNA biogenesis and/or mitosis.
Protein phosphatase 5 (PP5) exhibits very low phosphatase activity, which can be stimulated > 25-fold by proteolysis. Since proteolysis cleaves the N-tenninal tetratricopeptide repeat (TPR) domain from the catalytic domain, these results indicate that the TPR domain shields the active site. Polyunsaturated fatty acids, such as arachidonic acid, and lipids containing polyunsaturated fatty acids, such as phosphatidylinositol, stimulate both bacterially expressed human and native rabbit PP5 activity > 25-fold towards casein and myelin basic protein. Phosphatidylinositol binds to the TPR domain, and not to the catalytic domain, indicating that activation by polyunsaturated fatty acids is allosteric and that it may occur by movement of the TPR domain to allow substrate access.
The specificity of the catalytic subunit of protein phosphatase-1 (PP1,) is modified by regulatory subunits that target it to particular subcellular locations. Here, we identify PP1,-binding domains on G, and G,, the subunits that target PPI, to hepatic and muscle glycogen, respectively, and on MI,", the subunit that targets PPI, to smooth muscle myosin. G,-(G63 -T93) interacted with PP1, and prevented G, from suppressing the dephosphorylation of glycogen phosphorylase, but it did not dissociate G, from PPI, or affect other characteristic properties of the PPlG, complex. These results indicate that G, contains two PP1,-binding sites, the region which suppresses the dephosphorylation of glycogen phosphorylase being distinct from that which enhances the dephosphorylation of glycogen synthase. At higher concentrations, G,-(G63 -N7.5) had the same effect as GM-(G63-T93), but not if Ser67 was phosphorylated by cyclic-AMP-dependent protein kinase. Thus, phosphorylation of Ser67 dissociates G, from PP1 because phosphate is inserted into the PP1,-binding domain of G,. M,,<)-(Ml -E309) and Ml,,)-(MI -F38), but not Ml,,,-(D39-E309), mimicked the M,,, subunit in stimulating dephosphorylation of the smooth muscle myosin P-light chain and heavy meromyosin in vitro. However, in contrast to the M,,,, subunit and Ml,~l-(MI -E309), neither Ml,,,-(MI -F38) nor M1,,-(D39-E309) suppressed the PPI,-catalysed dephosphorylation of glycogen phosphorylase. These observations suggest that the region which stimulates the dephosphorylation of myosin is situated within the N-terminal 38 residues of the M,,, subunit, while the region which suppresses the dephosphorylation of glycogen phosphorylase requires the presence of at least part of the region 39-309 which contains seven ankyrin repeats. M,,,-(MI -F38) displaced G, from PPI,, while GM-(G63-T93) displaced M,,,, from PPlC in vitro. These observations indicate that the region(s) of PP1, that interact with G,/G, and MI,, overlap, explaining why different forms of PPI, contain just a single targetting subunit.Keywords: protein phosphatase-1 ; myosin ; smooth muscle ; glycogen metabolism.Protein phosphatase-1 (PPl), one of the major protein serine/ threonine phosphatases of eukaryotic cells, participates in the control of a variety of cellular functions that include glycogen metabolism, muscle contraction, the exit from mitosis (reviewed in [I, 21) and the splicing of mRNA 131. However, evidence has been accumulating that different processes are regulated by ~ Correspondence to P. Cohen, MRC Protein Phosphorylation Unit,
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