Regulation of rhodopsin dephosphorylation by arrestin

We have characterized the opsin phosphatase activities in extracts of rod outer segments and determined their relationship to known protein phosphatases. The opsin phosphatase activity in the extracts was not due to protein phosphatases 1, 2B, or 2C because it was neither stimulated by Mg2+ or Ca2+/calmodulin nor inhibited by protein phosphatase inhibitors-1 or -2. Opsin phosphatase activity in rod outer segment extracts was potently inhibited by okadaic acid (IC50 approximately 10 nM), a preferential inhibitor of protein phosphatase 2A. Moreover, during chromatography on DEAE-Sepharose, the opsin phosphatase activity co-eluted with three peaks of protein phosphatase 2A activity, termed protein phosphatases 2A0, 2A1, and 2A2. The opsin phosphatase activity of each peak was stimulated by polylysine, a known activator of protein phosphatase 2A. Finally, treatment of rod outer segment extracts with 80% ethanol at room temperature converted the activity from a high molecular weight form characteristic of the protein phosphatase 2A0, 2A1, and 2A2 species to a low molecular weight form characteristic of the protein phosphatase 2A catalytic subunit. We conclude that protein phosphatase 2A is likely to be the physiologically relevant rhodopsin phosphatase. The 48-kDa rod outer segment protein arrestin (S-antigen) was found to inhibit the dephosphorylation of freshly photolyzed rhodopsin by protein phosphatase 2A but did not inhibit the dephosphorylation of unbleached rhodopsin. Arrestin has no effect on the dephosphorylation of phorphorylase a, indicating that the effect was substrate-directed. It appears that dephosphorylation of the photoreceptor protein phosphorhodopsin occurs only after decay of the photoactivated protein and that this may be regulated in vivo by arrestin. The binding of arrestin to photolyzed phosphorylated rhodopsin, i.e. the binding of a regulatory protein to a protein phosphatase substrate to form a complex resistant to dephosphorylation represents a novel mechanism for the regulation of protein phosphatase 2A.     

Alpha4 protein as a common regulator of type 2A-related serine/threonine protein phosphatases

The catalytic activity of the C subunit of serine/threonine phosphatase 2A is regulated by the association with A (PR65) and B subunits. It has been reported that the alpha4 protein, a yeast homolog of the Tap42 protein, binds the C subunit of serine/threonine phosphatase 2A and protein phosphatase 2A-related protein phosphatases such as protein phosphatase 4 and protein phosphatase 6. In the present study, we showed that alpha4 binds these three phosphatases and the association of alpha4 reduces the activities of these phosphatases in vitro. In contrast, PR65 binds to the C subunit of serine/threonine phosphatase 2A but not to protein phosphatase 4 and protein phosphatase 6. These results suggest that the alpha4 protein is a common regulator of the C subunit of serine/threonine phosphatase 2A and protein phosphatase 2A-related protein phosphatases.     

Decreased protein phosphatase 2A activity in hippocampal long-term potentiation

Using autophosphorylated Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) as substrate, we now find that long-term potentian (LTP) induction and maintenance are also associated with a significant decrease in calyculin A-sensitive protein phosphatase (protein phosphatase 2A) activity, without changes in Mg2+-dependent protein phosphatase (protein phosphatase 2C) activity. This decrease in protein phosphatase 2A activity was prevented when LTP induction was inhibited by treatment with calmidazolium or D-2-amino-5-phosphonopentanoic acid. In addition, the application of high-frequency stimulation to 32P-labeled hippocampal slices resulted in increases in the phosphorylation of a 55-kDa protein immunoprecipitated with anti-phosphatase 2A antibodies. Use of a specific antibody revealed that the 55-kDa protein is the B'alpha subunit of protein phosphatase 2A. Following purification of brain protein phosphatase 2A, the B'alpha subunit was phosphorylated by CaM kinase II, an event that led to the reduction of protein phosphatase 2A activity. These results suggest that the decreased activity in protein phosphatase 2A following LTP induction contributes to the maintenance of constitutively active CaM kinase II and to the long-lasting increase in phosphorylation of synaptic components implicated in LTP.     

The catalytic subunit of phosphatase 2A dephosphorylates phosphoopsin

Rod cell outer segments were found to contain a protein phosphatase activity toward phosphoopsin with properties very similar to those of protein phosphatase 1 or 2A. The opsin phosphatase activity was stable to ethanol precipitation, had a Mr of 35,000-38,000 as determined by gel filtration, and was not dependent on divalent cations for activity. The chromatographic properties on DEAE-cellulose of the rod outer segment protein phosphatase were also similar to those reported for protein phosphatase 1 or 2A. In order to distinguish between these two protein phosphatases, we tested homogeneous preparations of protein phosphatases 1 and 2A from skeletal muscle for activity toward phosphoopsin. Protein phosphatase 2A dephosphorylated phosphoopsin at approximately 10% of its rate toward phosphorylase a, whereas protein phosphatase 1 had no activity toward phosphoopsin. We conclude that protein phosphatase 2A is present in the rod cell outer segment and that it is a likely candidate to perform the in vivo dephosphorylation of rhodopsin in the visual cycle.     

Identification of a novel protein phosphatase catalytic subunit by cDNA cloning

A cDNA encoding a novel protein phosphatase catalytic subunit (protein phosphatase X) has been isolated from a rabbit liver library. It codes for a protein having 45% and 65% amino acid sequence identity, respectively, to the catalytic subunits of protein phosphatase 1 and protein phosphatase 2A from skeletal muscle. The enzyme is neither the hepatic form of protein phosphatase 1 or 2A, nor is it protein phosphatase 2B or 2C. The possible identity of protein phosphatase X is discussed.     

mTOR-dependent suppression of protein phosphatase 2A is critical for phospholipase D survival signals in human breast cancer cells

A critical aspect of tumor progression is the generation of survival signals that overcome default apoptotic programs. Recent studies have revealed that elevated phospholipase D activity generates survival signals in breast and perhaps other human cancers. We report here that the elevated phospholipase D activity in the human breast cancer cell line MDA-MB-231 suppresses the activity of the putative tumor suppressor protein phosphatase 2A in a mammalian target of rapamycin (mTOR)-dependent manner. Increasing the phospholipase D activity in MCF7 cells also suppressed protein phosphatase 2A activity. Elevated phospholipase D activity suppressed association of protein phosphatase 2A with both ribosomal subunit S6-kinase and eukaryotic initiation factor 4E-binding protein 1. Suppression of protein phosphatase 2A by SV40 small t-antigen has been reported to be critical for the transformation of human cells with SV40 early region genes. Consistent with a critical role for protein phosphatase 2A in phospholipase D survival signals, either SV40 small t-antigen or pharmacological suppression of protein phosphatase 2A restored survival signals lost by the suppression of either phospholipase D or mTOR. Blocking phospholipase D signals also led to reduced phosphorylation of the pro-apoptotic protein BAD at the protein phosphatase 2A dephosphorylation site at Ser-112. The ability of phospholipase D to suppress protein phosphatase 2A identifies a critical target of an emerging phospholipase D/mTOR survival pathway in the transformation of human cells.     

Association of protein phosphatase 2A with its substrate vimentin intermediate filaments in 9L rat brain tumor cells

The importance of protein phosphatases in maintaining the integrity of intermediate filaments is supported by the fact that intermediate filaments would undergo a massive reorganization in cells treated with inhibitors of protein phosphatases 1 and 2A. Herein we used okadaic acid to investigate the differential roles of protein phosphatases 1 and 2A in the maintenance of intermediate filament integrity in 9L rat brain tumor cells. Protein phosphatase 2A activity was substantially inhibited after treatment with 400 nM okadaic acid for 2 h, whereas the activity of protein phosphatase 1 was only slightly affected. Furthermore, protein phosphatase 2A shows selective specificity toward phosphovimentin, which was immunologically precipitated from isotopically labeled and okadaic acid-treated cells. Further biochemical fractionation and microscopic studies revealed that vimentin intermediate filaments were colocalized with protein phosphatase 2A, but not protein phosphatase 1, in control cells. On okadaic acid treatment, vimentin filament disassembled and protein phosphatase 2A redistributed throughout the cytoplasm, suggesting that these two proteins separate from each other, whereas protein phosphatase 2A was inhibited. This working hypothesis was further supported by treatment with a low concentration (40 nM) of okadaic acid, which causes the same phenomenon. Taken together, our results showed that protein phosphatase 2A could be assigned to the intermediate filaments to serve the physiological role in maintaining the proper phosphorylation level of intermediate filaments in normal cells. This finding should pave the way for the elucidation of the regulatory mechanism of intermediate filament organization governed by protein phosphorylation.     

Saccharomyces cerevisiae protein phosphatase 2A performs an essential cellular function and is encoded by two genes.

Two genes (PPH21 and PPH22) encoding the yeast homologues of protein serine-threonine phosphatase 2A have been cloned from a Saccharomyces cerevisiae genomic library using a rabbit protein phosphatase 2A cDNA as a hybridization probe. The PPH genes are genetically linked on chromosome IV and are predicted to encode polypeptides each with 74% amino acid sequence identity to rabbit type 2A protein phosphatase, indicating once again the extraordinarily high degree of sequence conservation shown by protein-phosphatases from different species. The two PPH genes show less than 10% amino acid sequence divergence from each other and while disruption of either PPH gene alone is without any major effect, the double disruption is lethal. This indicates that protein phosphatase 2A activity is an essential cellular function in yeast. Measurement of type 2A protein phosphatase activity in yeast strains lacking one or other of the genes indicates that they account for most, if not all, protein phosphatase 2A activity in the cell.     

Specificity of the heat-stable protein inhibitor of the branched-chain alpha-keto acid dehydrogenase phosphatase

A potent, heat-stable protein inhibitor of branched-chain alpha-keto acid dehydrogenase (BCKDH) phosphatase has been identified and purified to near homogeneity from bovine kidney mitochondria (Damuni, Z., Humphreys, J. S., and Reed, L. J., Proc. Natl. Acad. Sci. U.S.A., in press). This protein is a noncompetitive inhibitor of BCKDH phosphatase, with a Ki about 0.13 nM. By contrast, this protein inhibitor did not affect the activity of the cytosolic protein phosphatase-1 and phosphatase-2A or the mitochondrial pyruvate dehydrogenase (PDH) phosphatase at concentrations up to 10 nM. The cytosolic protein phosphatase inhibitor-1 and inhibitor-2 had no effect on the activity of BCKDH phosphatase or PDH phosphatase at concentrations up to 50 and 300 nM respectively. These results, together with previous evidence, demonstrate that BCKDH phosphatase and its inhibitor protein are distinct from the cytosolic protein phosphatase-1 and phosphatase-2A and from protein phosphatase inhibitor-1 and inhibitor-2, respectively.     

Pertussis Toxin Modification of PC12 Cells Inhibits a Protein Phosphatase 2A-Like Phosphatase

Abstract: We have found that modification of rat PC12 cells with pertussis toxin resulted in an ∼50% inhibition of a protein phosphatase 2A-like phosphatase. Protein phosphatase 2A (PP2A) is a major cellular serine/threonine-specific protein phosphatase. Treatment of extracts from pertussis toxin-modified PC12 cells with either immobilized alkaline phosphatase or Ca²⁺ reversed this inhibition. Reactivation of the PP2A-like phosphatase in Ca²⁺ appears to result from the dephosphorylation of a protein by the Ca²⁺/calmodulin-dependent protein phosphatase calcineurin. The PP2A-like phosphatase in extracts from pertussis toxin-modified PC12 cells eluted from a Mono Q column at a higher ionic strength than did the PP2A-like phosphatase in extracts from control cells. After incubation in Ca²⁺, the PP2A-like phosphatase in extracts from pertussis toxin-modified cells eluted from a Mono Q column at the same ionic strength as did the PP2A-like phosphatase in extracts from control cells. These results indicate that the effect of pertussis toxin on this PP2A-like activity results from the phosphorylation of either one of the subunits of the PP2A-like phosphatase or a protein that when phosphorylated binds to and inhibits this phosphatase. Pertussis toxin modification did not result in the phosphorylation of the catalytic subunit of PP2A. Because phosphorylation regulates the activities of many enzymes and cell surface receptors, a pertussis toxin-induced decrease in PP2A activity could alter signaling pathways and other cellular processes in which G proteins are not directly involved.     

Autophosphorylation of ataxia-telangiectasia mutated is regulated by protein phosphatase 2A

Ionizing radiation induces autophosphorylation of the ataxia-telangiectasia mutated (ATM) protein kinase on serine 1981; however, the precise mechanisms that regulate ATM activation are not fully understood. Here, we show that the protein phosphatase inhibitor okadaic acid (OA) induces autophosphorylation of ATM on serine 1981 in unirradiated cells at concentrations that inhibit protein phosphatase 2A-like activity in vitro. OA did not induce gamma-H2AX foci, suggesting that it induces ATM autophosphorylation by inactivation of a protein phosphatase rather than by inducing DNA double-strand breaks. In support of this, we show that ATM interacts with the scaffolding (A) subunit of protein phosphatase 2A (PP2A), that the scaffolding and catalytic (C) subunits of PP2A interact with ATM in undamaged cells and that immunoprecipitates of ATM from undamaged cells contain PP2A-like protein phosphatase activity. Moreover, we show that IR induces phosphorylation-dependent dissociation of PP2A from ATM and loss of the associated protein phosphatase activity. We propose that PP2A plays an important role in the regulation of ATM autophosphorylation and activity in vivo.     

Expression of rat protein phosphatase 2C (IA) in Escherichia coli

A cDNA containing the entire coding sequence of rat type 2C (IA) protein phosphatase was expressed in Escherichia coli. An extract of bacterial cells harboring the recombinant plasmid contained a major (Mr = 41,000 - 43,000) and a minor (Mr = 30,000) protein band; both of these reacted with an anti-type 2C protein phosphatase serum. The size of the major protein band agrees well with that of the 2C phosphatase conceptualized from the cognate cDNA. A Mg2+-dependent protein phosphatase activity was detected in extracts containing the recombinant protein, but not in host cell extracts. Based on these results, it is concluded that the isolated cDNA clone encodes a functional type 2C protein phosphatase.     

Effects of phosphatase inhibitors and a protein phosphatase on norepinephrine secretion by permeabilized bovine chromaffin cells.

A protein phosphatase and phosphatase inhibitors were used to examine the role of protein phosphorylation in the regulation of norepinephrine secretion in digitonin-permeabilized bovine chromaffin cells. Addition of okadaic acid, a potent inhibitor of type 1 and type 2A protein phosphatases, or 1-naphthylphosphate, a more general phosphatase inhibitor, to digitonin-permeabilized chromaffin cells caused about a 100% increase in the amount of norepinephrine secreted in the absence of Ca2+ (in 5 mM EGTA) without affecting the amount of norepinephrine secreted in the presence of 10 microM free Ca2+. This stimulation of norepinephrine secretion by protein phosphatase inhibitors suggests that in the absence of Ca2+ there is a slow rate phosphorylation and that this phosphorylation triggers secretion. Addition of an exogenous type 2A protein phosphatase caused almost a 50% decrease in Ca(2+)-dependent norepinephrine secretion. Thus, the amounts of norepinephrine released both in the absence of Ca2+ and in the presence of Ca2+ appear to depend upon the level of protein phosphorylation.     

Purification and characterization of a divalent cation-independent, spermine-stimulated protein phosphatase from bovine kidney mitochondria

A divalent cation-independent and spermine-stimulated phosphatase (protein phosphatase SP) that is active toward the phosphorylated pyruvate dehydrogenase complex has been purified about 15,000-fold to near homogeneity from extracts of bovine kidney mitochondria. Half-maximal stimulation, 1.5- to 3-fold at pH 7.0-7.3, occurred at 0.5 mM spermine. Protein phosphatase SP exhibited an apparent Mr = 140,000-170,000 as estimated by gel-filtration chromatography on Sephacryl S-300. Two major subunits, with apparent Mr = 60,000 and 34,000, were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gel-permeation chromatography of protein phosphatase SP on Sephacryl S-200 in the presence of 6 M urea and 1.4 M NaCl increased its activity 3- to 6-fold and was accompanied by conversion to the catalytic subunit with an apparent Mr = approximately 34,000. Protein phosphatase SP was inactive with p-nitrophenyl phosphate and was not inhibited by protein phosphatase inhibitor 1, inhibitor 2, or the protein inhibitor of branched-chain alpha-keto acid dehydrogenase phosphatase. Protein phosphatase SP was inhibited by sheep antibody to the catalytic subunit of protein phosphatase 2A from rabbit skeletal muscle. It appears that protein phosphatase SP is related to protein phosphatase 2A.     

Antibodies directed against synthetic peptides distinguish between the catalytic subunits of protein phosphatases 1 and 2A.

Two antipeptide antibodies (designated type 1 antibody and type 2A antibody) were raised against synthetic peptides, Cys-Thr-Pro-Pro-Arg-Asn-Ser-Ala-Lys-Ala-Lys-Lys and Cys-Val-Thr-Arg-Arg-Thr-Pro-Asp-Try-Phe-Leu, corresponding to the carboxyl termini of the catalytic subunits of protein phosphatase 1 and phosphatase 2A (Cys was added for specific coupling to carrier protein). These antipeptide antibodies were highly specific and were useful in discriminating between protein phosphatase 1 and phosphatase 2A in crude extracts or purified preparations. Type 2A antibody reacted with both native and denatured protein phosphatase 2A whereas under the same condition type 1 antibody reacted only with denatured protein phosphatase 1.     

Elevated levels of protein phosphatase 1 and phosphatase 2A may contribute to cardiac dysfunction in diabetes

Although protein phosphorylation and dephosphorylation are known to regulate the activities of different enzymes, sufficient information on the role of dephosphorylation in cardiac function is not available. Since protein phosphatases mediate dephosphorylation, it is possible that cardiac dysfunction induced by diabetes may be due to alterations in the activities of these enzymes. We therefore determined cardiac protein phosphatase activity as well as protein contents of phosphatase 1 and phosphatase 2A in diabetic animals. For this purpose, rats were made diabetic by administering a single intravenous injection of streptozotocin (65 mg/kg body weight) and hearts were examined after 1, 2, 3, 4 and 8 weeks. Some of the 4-week diabetic animals received subcutaneous injections of insulin (3 U/day) for a further period of 4 weeks. Cardiac dysfunction was evident after 2 weeks of inducing diabetes and deteriorated further with time. A significant increase in protein phosphatase activity appeared after 1 week and persisted until 8 weeks. Increased protein phosphatase activity in the diabetic heart was associated with a corresponding increase in the protein contents of both phosphatase 1 and phosphatase 2A. Insulin treatment partly prevented the changes observed in diabetic animals. The results suggest that increased protein phosphatase activities and subsequent enhanced protein dephosphorylation may play a role in diabetes-induced cardiac dysfunction.     

A type 2A protein phosphatase dephosphorylates the elongation factor 2 and is stimulated by the phorbol ester TPA in mouse epidermis in vivo

Mouse epidermal cytosol contains a protein phosphatase with Mr 38,000, which dephosphorylates the elongation factor 2 (EF-2) of protein biosynthesis and is stimulated after topical application of TPA to mouse skin [(1988) Biochem. Biophys. Res. Commun. 153, 1129-1135]. Dephosphorylation of EF-2 by this phosphatase is inhibited by okadaic acid at concentrations as low as 10(-8) M, but not by heparin up to concentrations of 600.micrograms/ml. The catalytic subunit of protein phosphatase 2A (PP2Ac) with EF-2 as a substrate exhibits the same sensitivity towards okadaic acid and insensitivity towards heparin as the EF-2 phosphatase of epidermal cytosol. The catalytic subunit of protein phosphatase 1 (PP1c) is strongly suppressed by heparin and less sensitive towards okadaic acid than PP2Ac. PP2Ac is around 50 times more efficient in dephosphorylating EF-2 than PP1c. These data indicate that the TPA-stimulated EF-2 phosphatase in epidermal cytosol is a type 2A protein phosphatase.     

Multiplicity of protein serine-threonine phosphatases in PC12 pheochromocytoma and FTO-2B hepatoma cells

Protein purification and molecular cloning have defined five classes of protein serine-threonine phosphatase catalytic subunits referred to as types 1, 2A, 2B (calcineurin), 2C, and X. Protein serine-threonine phosphatases 1, 2A, 2B, and X appear to have significant sequence homologies, whereas the 2C enzyme is more divergent. We have used the polymerase chain reaction to define the multiplicity of the closely related types 1, 2A, 2B, and X phosphatase catalytic subunits in two clonal cell lines, rat PC12 pheochromocytoma and rat FTO-2B hepatoma. RNAs for all four related phosphatase types were expressed in both cell lines. In addition to the phosphatase X enzyme, four phosphatase 1, two phosphatase 2A, and three phosphatase 2B isoforms were identified in PC12 and FTO-2B cells. The results indicate a large multiplicity of protein serine-threonine phosphatases within clonal cells of different tissue origin, suggesting that their role in cell regulation will be as divergent as that for the protein serine-threonine kinases.     

The protein phosphatases involved in cellular regulation. 1. Classification and substrate specificities

The protein phosphatase activities involved in regulating the major pathways of intermediary metabolism can be explained by only four enzymes which can be conveniently divided into two classes, type-1 and type-2. Type-1 protein phosphatases dephosphorylate the beta-subunit of phosphorylase kinase and are potently inhibited by two thermostable proteins termed inhibitor-1 and inhibitor-2, whereas type-2 protein phosphatases preferentially dephosphorylate the alpha-subunit of phosphorylase kinase and are insensitive to inhibitor-1 and inhibitor-2. The substrate specificities of the four enzymes, namely protein phosphatase-1 (type-1) and protein phosphatases 2A, 2B and 2C (type-2) have been investigated. Eight different protein kinases were used to phosphorylate 13 different substrate proteins on a minimum of 20 different serine and threonine residues. These substrates include proteins involved in the regulation of glycogen metabolism, glycolysis, fatty acid synthesis, cholesterol synthesis, protein synthesis and muscle contraction. The studies demonstrate that protein phosphatase-1 and protein phosphatase 2A have very broad substrate specificities. The major differences, apart from the site specificity for phosphorylase kinase, are the much higher myosin light chain phosphatase and ATP-citrate lyase phosphatase activities of protein phosphatase-2A. Protein phosphatase-2C (an Mg2+-dependent enzyme) also has a broad specificity, but can be distinguished from protein phosphatase-2A by its extremely low phosphorylase phosphatase and histone H1 phosphatase activities, and its slow dephosphorylation of sites (3a + 3b + 3c) on glycogen synthase relative to site-2 of glycogen synthase. It has extremely high hydroxymethylglutaryl-CoA (HMG-CoA) reductase phosphatase and HMG-CoA reductase kinase phosphatase activity. Protein phosphatase-2B (a Ca2+-calmodulin-dependent enzyme) is the most specific phosphatase and only dephosphorylated three of the substrates (the alpha-subunit of phosphorylase kinase, inhibitor-1 and myosin light chains) at a significant rate. It is specifically inhibited by the phenathiazine drug, trifluoperazine. Examination of the amino acid sequences around each phosphorylation site does not support the idea that protein phosphatase specificity is determined by the primary structure in the immediate vicinity of the phosphorylation site.