Identification of two isoenzymes of protein phosphatase 2C in both rabbit skeletal muscle and liver

Protein phosphatase 2C was isolated from rabbit skeletal muscle by a procedure that involved chromatography on DEAE-cellulose, precipitation with ammonium sulphate, gel-filtration on Sephadex G-100, affinity chromatography on thiophosphorylated myosin-P-light-chain--Sepharose and chromatography on Mono Q. The enzyme was purified about 35,000-fold and 0.3-0.4 mg was isolated from 2500 g skeletal muscle within 5 days. The final step resolved the activity into two peaks, termed protein phosphatases 2C1 and 2C2, that possessed identical substrate specificities and enzymatic properties. About 2.5-fold more protein phosphatase 2C2 was isolated than protein phosphatase 2C1. Protein phosphatases 2C1 and 2C2 migrated as single bands on SDS/polyacrylamide gels yielding apparent molecular masses of 44 kDa and 42 kDa, respectively, and the native proteins were both monomeric at pH 7.5 as judged by their elution from Sephadex G-100 and Sephacryl S200. Peptide maps of protein phosphatases 2C1 and 2C2, obtained after separate digestions with four different proteinases, were different, indicating that they are isoenzymes. Protein phosphatases 2C1 and 2C2 were purified from rabbit liver by the same procedure, and 0.2 mg (2C1 + 2C2) was isolated from 120 g hepatic tissue. Hepatic protein phosphatases 2C1 and 2C2 were also isolated in a molar ratio of about 1:2.5, and their enzymatic properties and apparent molecular masses in the presence and absence of SDS were identical to the skeletal muscle enzymes. Protein phosphatases 2C1 from muscle and liver displayed identical peptide maps, as did protein phosphatases 2C2 from these two tissues. It is concluded that the same two isoenzymes of protein phosphatase 2C are present in skeletal muscle and liver.     

中胚叶叉头-1在骨成形蛋白-2诱导肌胚细胞C2C12转化成骨细胞过程中的作用

为了研究中胚叶叉头-1(MFH-1)基因在骨骼形成和细胞分化中的作用,利用基因重组、杂交瘤技术制作MFH-1单克隆抗体, 利用蛋白质印迹和RNA印迹分析观察了骨成形蛋白-2 (BMP-2)诱导小鼠肌胚细胞C2C12表达MFH-1、产生碱性磷酸酶和骨钙蛋白.小鼠肌胚细胞C2C12低水平地表达内源性MFH-1蛋白以及导入小鼠MFH-1 cDNA的人膀胱癌细胞HTB9也表达小鼠MFH-1蛋白,这种蛋白质定位于细胞核中.用BMP-2处理后, MFH-1蛋白和mRNA在C2C12细胞中的表达显著地增加.用反义MFH-1序列转染小鼠肌胚细胞C2C12可降低内源性MFH-1水平, BMP-2不能诱导导入反义MFH-1序列的肌胚细胞C2C12产生MFH-1蛋白,也不能诱导碱性磷酸酶(ALP)活性和骨钙蛋白量的增加.结果表明, BMP-2诱导的MFH-1蛋白在调节肌胚细胞C2C12向成骨细胞分化方面起关键作用.     

Crystal structure of the protein serine/threonine phosphatase 2C at 2.0 A resolution.

Protein phosphatase 2C (PP2C) is a Mn2+- or Mg2+-dependent protein Ser/Thr phosphatase that is essential for regulating cellular stress responses in eukaryotes. The crystal structure of human PP2C reveals a novel protein fold with a catalytic domain composed of a central beta-sandwich that binds two manganese ions, which is surrounded by alpha-helices. Mn2+-bound water molecules at the binuclear metal centre coordinate the phosphate group of the substrate and provide a nucleophile and general acid in the dephosphorylation reaction. Our model presents a framework for understanding not only the classical Mn2+/Mg2+-dependent protein phosphatases but also the sequence-related domains of mitochondrial pyruvate dehydrogenase phosphatase, the Bacillus subtilus phosphatase SpoIIE and a 300-residue domain within yeast adenyl cyclase. The protein architecture and deduced catalytic mechanism are strikingly similar to the PP1, PP2A, PP2B family of protein Ser/Thr phosphatases, with which PP2C shares no sequence similarity, suggestive of convergent evolution of protein Ser/Thr phosphatases.     

The ABI1 and ABI2 protein phosphatases 2C act in a negative feedback regulatory loop of the abscisic acid signalling pathway

The Arabidopsis ABI1 and ABI2 genes encode two protein serine/threonine phosphatases 2C (PP2C). These genes have been originally identified by the dominant mutations abi1--1 and abi2--1, which reduce the plant's responsiveness to the hormone abscisic acid (ABA). However, recessive mutants of ABI1 were recently shown to be supersensitive to ABA, which demonstrated that the ABI1 phosphatase is a negative regulator of ABA signalling. We report here the isolation and characterisation of the first reduction-of-function allele of ABI2, abi2--1R1. The in vitro phosphatase activity of the abi2--1R1 protein is approximately 100-fold lower than that of the wild-type ABI2 protein. Abi2--1R1 plants displayed a wild-type ABA sensitivity. However, doubly mutant plants combining the abi2--1R1 allele and a loss-of-function allele at the ABI1 locus were more responsive to ABA than each of the parental single mutants. These data indicate that the wild-type ABI2 phosphatase is a negative regulator of ABA signalling, and that the ABI1 and ABI2 phosphatases have overlapping roles in controlling ABA action. Measurements of PP2C activity in plant extracts showed that the phosphatase activity of ABI1 and ABI2 increases in response to ABA. These results suggest that ABI1 and ABI2 act in a negative feedback regulatory loop of the ABA signalling pathway.     

The AKT3 potassium channel protein interacts with the AtPP2CA protein phosphatase 2C

The AKT3 potassium channel protein was identified as a strongly interacting partner of the Arabidopsis thaliana protein phosphatase 2C (AtPP2CA) in a yeast two-hybrid screen. A deletion analysis indicated that the catalytic domain of AtPP2CA was essential for the interaction with AKT3. Furthermore, the related PP2C phosphatase ABI1 did not interact with AKT3 in yeast.     

Protein phosphatase 2C, encoded by ptc1+, is important in the heat shock response of Schizosaccharomyces pombe.

Protein phosphatase 2C (PP2C), an Mg(2+)-dependent enzyme that dephosphorylates serine and threonine residues, defines one of the three major families of structurally unrelated eukaryotic protein phosphatases. Members of the two other families of protein phosphatases are known to have important cellular roles, but very little is known about the biological functions of PP2C. In this report we describe a genetic investigation of a PP2C enzyme in the fission yeast Schizosaccharomyces pombe. We discovered ptc1+ (phosphatase two C) as a multicopy suppressor gene of swo1-26, a temperature-sensitive mutation of a gene encoding the heat shock protein hsp90. The ptc1+ gene product is a 40-kDa protein with approximately 24% identity to a rat PP2C protein. Purified Ptc1 has Mg(2+)-dependent casein phosphatase activity, confirming that it is a PP2C enzyme. A ptc1 deletion mutant is viable and has approximately normal levels of PP2C activity, observations consistent with the fact that ptc1+ is a member of a multigene family. Although a ptc1 deletion mutant is viable, it has a greatly reduced ability to survive brief exposure to elevated temperature. Moreover, ptc1+ mRNA levels increase 5- to 10-fold during heat shock. These data, demonstrating that Ptc1 activity is important for survival of heat shock, provide one of the first genetic clues as to the biological functions of PP2C.     

Histone H1 phosphorylated by protein kinase C is a selective substrate for the assay of protein phosphatase 2A in the presence of phosphatase 1

A protein phosphatase assay, selective for protein phosphatase 2A, has been developed. Bovine histone H1 phosphorylated by protein kinase C and [gamma-32P]ATP, designated H1(C), was tested as the substrate for various preparations of protein phosphatases 1 and 2A. The phosphatase 2A preparations were 10-60-times more active with H1(C) as the substrate when compared to phosphorylase a. The phosphatase 1 enzymes showed very little dephosphorylation of the H1(C) substrate, the activity being less than 5% of the phosphorylase phosphatase activity. This preference and selectivity was demonstrated for purified phosphatase preparations in addition to fresh tissue extracts. The assay provides a rapid, simple assay for the routine analysis of phosphatase 2A in the presence of phosphatase 1, without the use of heat-stable inhibitor proteins.     

Protein phosphatase 2C is involved in the cAMP-dependent ciliary control in Paramecium caudatum

Forward swimming of the Triton-extracted model of Paramecium is stimulated by cAMP. Backward swimming of the model induced by Ca(2+) is depressed by cAMP. Cyclic AMP and Ca(2+) act antagonistically in setting the direction of the ciliary beat. Some ciliary axonemal proteins from Paramecium caudatum are phosphorylated in a cAMP-dependent manner. In the presence of cAMP, axonemal 29- and 65-kDa polypeptides were phosphorylated by endogenous A-kinase in vitro. These phosphoproteins, however, were not dephosphorylated after in vitro phosphorylation, presumably because of the low endogenous phosphoprotein phosphatase activity associated with isolated axonemes. We purified the protein phosphatase that specifically dephosphorylated the 29- and 65-kDa phosphoproteins from Paramecium caudatum. The molecular weight of the protein phosphatase was 33 kDa. The protein phosphatase had common characteristics as protein phosphatase 2C (PP2C). The characteristics of the protein phosphatase were the same as those of the PP2C from Paramecium tetraurelia (PtPP2C) [Grothe et al., 1998: J. Biol. Chem. 273:19167-19172]. We concluded that the phosphoprotein phosphatase is the PP2C from Paramecium caudatum (PcPP2C). The PcPP2C markedly accelerated the backward swimming of the Triton-extracted model in the presence of Ca(2+). On the other hand, the PcPP2C slightly depressed the forward swimming speed. This indicates that the PP2C plays a role in the cAMP-dependent regulation of ciliary movement in Paramecium caudatum through dephosphorylation of 29- and/or 65-kDa regulatory phosphoproteins by terminating the action of cAMP.     

Parallel purification of three catalytic subunits of the protein serine/threonine phosphatase 2A family (PP2A(C), PP4(C), and PP6(C)) and analysis of the interaction of PP2A(C) with alpha4 protein

The protein serine/threonine phosphatase (PP) type 2A family consists of three members: PP2A, PP4, and PP6. Specific rabbit and sheep antibodies corresponding to each catalytic subunit, as well as a rabbit antibody recognizing all three subunits, were utilized to examine the expression of these enzymes in select rat tissue extracts. PP2A, PP4, and PP6 catalytic subunits (PP2A(C), PP4(C), and PP6(C), respectively) were detected in all rat tissue extracts examined and exhibited some differences in their levels of expression. The expression of alpha4, an interacting protein for PP2A family members that may function downstream of the target of rapamycin (Tor), was also examined using specific alpha4 sheep antibodies. Like the phosphatase catalytic subunits, alpha4 was ubiquitously expressed with particularly high levels in the brain and thymus. All three PP2A family members, but not alpha4, bound to the phosphatase affinity resin microcystin-Sepharose. The phosphatase catalytic subunits were purified to apparent homogeneity (PP2A(C) and PP4(C)) or near homogeneity (PP6(C)) from bovine testes soluble extracts following ethanol precipitation and protein extraction. In contrast to PP2A(C), PP4(C) and PP6(C) exhibited relatively low phosphatase activity towards several substrates. Purified PP2A(C) and native PP2A in cellular extracts bound to GST-alpha4, and co-immunoprecipitated with endogenous alpha4 and ectopically expressed myc-tagged alpha4. The interaction of PP2A(C) with alpha4 was unaffected by rapamycin treatment of mammalian cells; however, protein serine/threonine phosphatase inhibitors such as okadaic acid and microcystin-LR disrupted the alpha4/PP2A complex. Together, these findings increase our understanding of the biochemistry of alpha4/phosphatase complexes and suggest that the alpha4 binding site within PP2A may include the phosphatase catalytic domain.     

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.     

Identification and characterization of PP2C phosphatase SjPtc1 in Schistosoma japonicum

Protein phosphorylation, regulated by protein kinases and protein phosphatases, is crucial for protein structure and function in eukaryotic organisms. Type 2C protein phosphatase (PP2C) belongs to the serine/threonine phosphatase family and its activities require the presence of a divalent magnesium or manganese ion. In the present study, a potential PP2C phosphatase (SjPtc1) was identified in Schistosoma japonicum. The SjPTC1 gene was found to be highly expressed in adult worms. A recombinant SjPtc1 protein showed typical PP2C phosphatase activity. Heterologous SjPTC1 expression reversed the sensitivity of yeast ptc1 null mutants toward H₂O₂, ZnCl₂, cisplatin, and rapamycin. Collectively, the results suggest that SjPtc1 may take part in the regulation of cellular responses to oxidative stress, DNA damage stress, and the TOR (target of rapamycin) signaling pathway.     

Molecular cloning and primary structure of a protein phosphatase 2C isoform.

Complementary DNA encoding the isoform of protein phosphatase 2C, termed PP2C2, has been isolated. The cDNA predicts a protein of 390 amino acid residues with a molecular mass of 42,888 Da. The protein displays 76% identity to the PP2C1 isoform.     

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.     

Developmental regulation of hexosamine biosynthesis by protein phosphatases 2A and 2C in Blastocladiella emersonii.

Extracts of the aquatic fungus Blastocladiella emersonii were found to contain protein phosphatases type 1, type 2A, and type 2C with properties analogous to those found in mammalian tissues. The activities of all three protein phosphatases are developmentally regulated, increasing during sporulation, with maximum level in zoospores. Protein phosphatases 2A and 2C, present in zoospore extracts, catalyze the dephosphorylation of L-glutamine:fructose-6-phosphate amidotransferase (EC 2.6.1.16, amidotransferase), a key regulatory enzyme in hexosamine biosynthesis. The protein phosphatase inhibitor okadaic acid induces encystment and inhibits germ tube formation but does not affect the synthesis of the chitinous cell wall. These results strongly suggest that phosphatase 2C is responsible for the dephosphorylation of amidotransferase in vivo. This dephosphorylation is inhibited by uridine-5'-diphospho-N-acetylglucosamine, the end product of hexosamine synthesis and the substrate for chitin synthesis. This result demonstrates a dual role of uridine-5'-diphospho-N-acetylglucosamine by inhibiting the activity of the phosphorylated form of amidotransferase and by preventing its dephosphorylation by protein phosphatases.     

A putative myristoylated 2C-type protein phosphatase, PP2C74, interacts with SnRK1 in Arabidopsis

N-myristoylation is a lipid modification of many signaling proteins in which myristate is added to an N-terminal glycine residue. Here we show that PP2C74, a putative myristoylated 2C-type protein phosphatase (PP2C) in Arabidopsis, is transcribed in various tissues and has protein phosphatase activity. GFP-fused PP2C74 localized to the plasma membrane, but not when a glycine residue at position 2, which is the putative myristoylation site, was substituted with an alanine residue. Yeast two-hybrid analysis and GST pull-down analysis showed that PP2C74 interacts with AKIN10, the catalytic α subunit of the SnRK1 protein kinase complex, the β subunits of which are known targets of myristoylation.     

Molecular characterization and expression analysis of a rice protein phosphatase 2C gene, OsBIPP2C1, and overexpression in transgenic tobacco conferred enhanced disease resistance and abiotic tolerance

A full-length cDNA of a rice protein phosphatase 2C gene, OsBIPP2C1 , was cloned and identified. OsBIPP2C1 is predicted to encode a 569 amino acid protein that contains phosphatase domain at its C-terminal and a relatively long N-terminal extension. Expression profiles of OsBIPP2C1 in rice seedlings upon treatments with disease resistance inducers, pathogen infection, and mechanical wounding as well as various environmental stress conditions were analyzed. Expression of OsBIPP2C1 was activated upon treatments with benzothiadiazole (BTH), salicylic acid, and hydrogen peroxide, which are signal molecules in plant disease resistance responses, and was induced during the first 48 h after inoculation with Magnaporthe grisea in BTH-treated rice seedlings. OsBIPP2C1 was also upregulated upon mechanical wounding and treatments with abscisic acid, high salt, low temperature, and drought stress. Transgenic tobacco plants overexpressing OsBIPP2C1 gene showed enhanced disease resistance against tobacco mosaic virus and Phytophthora paratisca and increased tolerance against salt and osmotic stresses. These results suggest that OsBIPP2C1 may play important roles in responses to biotic and abiotic stresses.     

Dephosphorylation of Microtubule Proteins by Brain Protein Phosphatases 1 and 2A, and Its Effect on Microtubule Assembly

Protein phosphatase C was purified 140-fold from bovine brain with 8% yield using histone H1 phosphorylated by the catalytic subunit of cyclic AMP-dependent protein kinase (cyclic AMP-kinase). Brain protein phosphatase C was considered to consist of 10 and 90%, respectively, of the catalytic subunits of protein phosphatases 1 and 2A on the basis of the effects of ATP and inhibitor-2. Protein phosphatase C dephosphorylated microtubule-associated protein 2 (MAP2), tau factor, and tubulin phosphorylated by a multifunctional Ca2+/calmodulin-dependent protein kinase (calmodulin-kinase) and the catalytic subunit of cyclic AMP-kinase. The properties of dephosphorylation of MAP2, tau factor, and tubulin were compared. The Km values were in the ranges of 1.6-2.7 microM for MAP2 and tau factor. The Km value for tubulin decreased from 25 to 10-12.5 microM in the presence of 1.0 mM Mn2+. No difference in kinetic properties of dephosphorylation was observed between the substrates phosphorylated by the two kinases. Protein phosphatase C did not dephosphorylate the native tubulin, but universally dephosphorylated tubulin phosphorylated by the two kinases. The holoenzyme of protein phosphatase 2A from porcine brain could also dephosphorylate MAP2, tau factor, and tubulin phosphorylated by the two kinases. The phosphorylation of MAP2 and tau factor by calmodulin-kinase separately induced the inhibition of microtubule assembly, and the dephosphorylation by protein phosphatase C removed its inhibitory effect. These data suggest that brain protein phosphatases 1 and 2A are involved in the switch-off mechanism of both Ca2+/calmodulin-dependent and cyclic AMP-dependent regulation of microtubule formation.