Physical and functional interaction of the Arabidopsis K(+) channel AKT2 and phosphatase AtPP2CA

The AKT2 K(+) channel is endowed with unique functional properties, being the only weak inward rectifier characterized to date in Arabidopsis. The gene is expressed widely, mainly in the phloem but also at lower levels in leaf epiderm, mesophyll, and guard cells. The AKT2 mRNA level is upregulated by abscisic acid. By screening a two-hybrid cDNA library, we isolated a protein phosphatase 2C (AtPP2CA) involved in abscisic acid signaling as a putative partner of AKT2. We further confirmed the interaction by in vitro binding studies. The expression of AtPP2CA (beta-glucuronidase reporter gene) displayed a pattern largely overlapping that of AKT2 and was upregulated by abscisic acid. Coexpression of AtPP2CA with AKT2 in COS cells and Xenopus laevis oocytes was found to induce both an inhibition of the AKT2 current and an increase of the channel inward rectification. Site-directed mutagenesis and pharmacological analysis revealed that this functional interaction involves AtPP2CA phosphatase activity. Regulation of AKT2 activity by AtPP2CA in planta could allow the control of K(+) transport and membrane polarization during stress situations.     

ABA-hypersensitive germination3 encodes a protein phosphatase 2C (AtPP2CA) that strongly regulates abscisic acid signaling during germination among Arabidopsis protein phosphatase 2Cs

The phytohormone abscisic acid (ABA) regulates physiologically important developmental processes and stress responses. Previously, we reported on Arabidopsis (Arabidopsis thaliana) L. Heynh. ahg mutants, which are hypersensitive to ABA during germination and early growth. Among them, ABA-hypersensitive germination3 (ahg3) showed the strongest ABA hypersensitivity. In this study, we found that the AHG3 gene is identical to AtPP2CA, which encodes a protein phosphatase 2C (PP2C). Although AtPP2CA has been reported to be involved in the ABA response on the basis of results obtained by reverse-genetics approaches, its physiological relevance in the ABA response has not been clarified yet. We demonstrate in vitro and in vivo that the ahg3-1 missense mutation causes the loss of PP2C activity, providing concrete confirmation that this PP2C functions as a negative regulator in ABA signaling. Furthermore, we compared the effects of disruption mutations of eight structurally related PP2C genes of Arabidopsis, including ABI1, ABI2, HAB1, and HAB2, and found that the disruptant mutant of AHG3/AtPP2CA had the strongest ABA hypersensitivity during germination, but it did not display any significant phenotypes in adult plants. Northern-blot analysis clearly showed that AHG3/AtPP2CA is the most active among those PP2C genes in seeds. These results suggest that AHG3/AtPP2CA plays a major role among PP2Cs in the ABA response in seeds and that the functions of those PP2Cs overlap, but their unique tissue- or development-specific expression confers distinct and indispensable physiological functions in the ABA response.     

AtPP2CG1, a protein phosphatase 2C, positively regulates salt tolerance of Arabidopsis in abscisic acid-dependent manner

AtPP2CG1 (Arabidopsis thaliana protein phosphatase 2C G Group 1) was predicted as an abiotic stress candidate gene by bioinformatic analysis in our previous study. The gene encodes a putative protein phosphatase 2C that belongs to Group G of PP2C. There is no report of Group G genes involved in abiotic stress so far. Real-time RT-PCR analysis showed that AtPP2CG1 expression was induced by salt, drought, and abscisic acid (ABA) treatment. The expression levels of AtPP2CG1 in the ABA synthesis-deficient mutant abi2-3 were much lower than that in WT plants under salt stress suggesting that the expression of AtPP2CG1 acts in an ABA-dependent manner. Over-expression of AtPP2CG1 led to enhanced salt tolerance, whereas its loss of function caused decreased salt tolerance. These results indicate that AtPP2CG1 positively regulates salt stress in an ABA-dependent manner. Under salt treatment, AtPP2CG1 up-regulated the expression levels of stress-responsive genes, including RD29A, RD29B, DREB2A and KIN1. GUS activity was detected in roots, leaves, stems, flower, and trichomes of AtPP2CG1 promoter-GUS transgenic plants. AtPP2CG1 protein was localized in nucleus and cytoplasm via AtPP2CG1:eGFP and YFP:AtPP2CG1 fusion approaches.     

Antisense inhibition of protein phosphatase 2C accelerates cold acclimation in Arabidopsis thaliana

Two related protein phosphatases 2C, ABI1 and AtPP2CA have been implicated as negative regulators of ABA signalling. In this study we characterized the role of AtPP2CA in cold acclimation. The pattern of expression of AtPP2CA and ABI1 was studied in different tissues and in response to abiotic stresses. The expression of both AtPP2CA and ABI1 was induced by low temperature, drought, high salt and ABA. The cold and drought-induced expression of these genes was ABA-dependent, but divergent in various ABA signalling mutants. In addition, the two PP2C genes exhibited differences in their tissue-specific expression as well as in temporal induction in response to low temperature. To elucidate the function of AtPP2CA in cold acclimation further, the corresponding gene was silenced by antisense inhibition. Transgenic antisense plants exhibited clearly accelerated development of freezing tolerance. Both exposure to low temperature and application of ABA resulted in enhanced freezing tolerance in antisense plants. These plants displayed increased sensitivity to ABA both during development of frost tolerance and during seed germination, but not in their drought responses. Furthermore, the expression of cold-and ABA-induced genes was enhanced in transgenic antisense plants. Our results suggest that AtPP2CA is a negative regulator of ABA responses during cold acclimation.     

Oxidative stress, heat shock and drought differentially affect expression of a tobacco protein phosphatase 2C

A protein phosphatase 2C (PP2C)-homologous cDNA was isolated from Nicotiana tabacum (NtPP2C1). The deduced protein sequence of 416 amino acids showed the highest degree of similarity to the PP2C of Arabidopsis thaliana (AtPP2CA) implicated in abscisic acid signalling. The expression of NtPP2C1 was strongly induced by drought, but repressed by oxidative stress and heat shock. It is suggested that NtPP2C1 operates at the junction of drought, heat shock and oxidative stress.     

Characterization of a phosphatase 2C protein as an interacting partner of the histone acetyltransferase GCN5 in Arabidopsis

Arabidopsis GCN5 is a major histone acetyltransferase. The mutation of the gene induces pleiotropic effects on plant development, and affects the expression of a large number of genes. The mechanism of action of this protein in controlling plant chromatin structure and genome expression is not understood. In this work, we report the identification of a number of potential protein interacting partners of GCN5 in Arabidopsis. In particular, GCN5 was shown to interact specifically with a phosphatase 2C protein (AtPP2C-6-6). GCN5 phosphorylated by activities in cellular extracts could be dephosphorylated by AtPP2C-6-6 in vitro. Analysis of T-DNA insertion mutants revealed a positive role of AtPP2C-6-6 in salt induction of stress-inducible genes, while the gcn5 mutation seemed to have no effect on the induction but showed up-regulation of a subset of the stress-inducible genes under non-induced conditions. In addition, the gcn5 mutation seriously reduced acetylation of histone H3K14 and H3K27, whereas the T-DNA insertions of the AtPP2C6-6 gene enhanced the acetylation of these lysine residues. Taken together, the present data suggest that AtPP2C-6-6 may function as a negative regulator of GCN5 activity in Arabidopsis.     

Heat-induced chaperone activity of serine/threonine protein phosphatase 5 enhances thermotolerance in Arabidopsis thaliana

? This study reports that Arabidopsis thaliana protein serine/threonine phosphatase 5 (AtPP5) plays a pivotal role in heat stress resistance. A high-molecular-weight (HMW) form of AtPP5 was isolated from heat-treated A. thaliana suspension cells. AtPP5 performs multiple functions, acting as a protein phosphatase, foldase chaperone, and holdase chaperone. The enzymatic activities of this versatile protein are closely associated with its oligomeric status, ranging from low oligomeric protein species to HMW complexes. ? The phosphatase and foldase chaperone functions of AtPP5 are associated primarily with the low-molecular-weight (LMW) form, whereas the HMW form exhibits holdase chaperone activity. Transgenic over-expression of AtPP5 conferred enhanced heat shock resistance to wild-type A. thaliana and a T-DNA insertion knock-out mutant was defective in acquired thermotolerance. A recombinant phosphatase mutant (H290N) showed markedly increased holdase chaperone activity. ? In addition, enhanced thermotolerance was observed in transgenic plants over-expressing H290N, which suggests that the holdase chaperone activity of AtPP5 is primarily responsible for AtPP5-mediated thermotolerance. ? Collectively, the results from this study provide the first evidence that AtPP5 performs multiple enzymatic activities that are mediated by conformational changes induced by heat-shock stress.     

The protein phosphatase 7 regulates phytochrome signaling in Arabidopsis

The psi2 mutant of Arabidopsis displays amplification of the responses controlled by the red/far red light photoreceptors phytochrome A (phyA) and phytochrome B (phyB) but no apparent defect in blue light perception. We found that loss-of-function alleles of the protein phosphatase 7 (AtPP7) are responsible for the light hypersensitivity in psi2 demonstrating that AtPP7 controls the levels of phytochrome signaling. Plants expressing reduced levels of AtPP7 mRNA display reduced blue-light induced cryptochrome signaling but no noticeable deficiency in phytochrome signaling. Our genetic analysis suggests that phytochrome signaling is enhanced in the AtPP7 loss of function alleles, including in blue light, which masks the reduced cryptochrome signaling. AtPP7 has been found to interact both in yeast and in planta assays with nucleotide-diphosphate kinase 2 (NDPK2), a positive regulator of phytochrome signals. Analysis of ndpk2-psi2 double mutants suggests that NDPK2 plays a critical role in the AtPP7 regulation of the phytochrome pathway and identifies NDPK2 as an upstream element involved in the modulation of the salicylic acid (SA)-dependent defense pathway by light. Thus, cryptochrome- and phytochrome-specific light signals synchronously control their relative contribution to the regulation of plant development. Interestingly, PP7 and NDPK are also components of animal light signaling systems.     

Protein phosphatase 3 differentially modulates vascular endothelial growth factor- and fibroblast growth factor 2-stimulated cell proliferation and signaling in ovine fetoplacental artery endothelial cells

A critical process for vascular endothelial growth factor (VEGF)- and fibroblast growth factor 2 (FGF2)-regulated cellular function is reversible protein phosphorylation, which is tightly controlled by a balance of protein kinases and phosphatases. We have reported that in ovine fetoplacental artery endothelial (OFPAE) cells, VEGF and FGF2 stimulate cell proliferation in part via activation of mitogen-activated protein kinase kinase 1/2 (MAP2K1/2)/mitogen-activated protein kinase 3/1 (MAPK3/1) and phosphoinositide 3-kinase (PI3K)/v-akt murine thymoma viral oncogene homolog 1 (AKT1) pathways. In the present study, we examined if protein phosphatase 3 (PPP3) mediated VEGF- and FGF2-stimulated OFPAE cell proliferation via modulating activation of MAPK3/1 and AKT1. Small interfering RNA (siRNA) targeting human PPP3 catalytic subunit alpha (PPP3CA) was used to suppress PPP3CA protein expression in OFPAE cells. Compared with the scrambled siRNA, PPP3CA siRNA decreased PPP3CA protein levels by approximately 97% without altering protein levels of protein phosphatase 2 catalytic subunit alpha, total MAPK3/1, total AKT1, or glyceraldehyde-3-phosphate dehydrogenase. Knockdown of PPP3CA protein expression enhanced VEGF-stimulated, but not FGF2-stimulated, cell proliferation. Knockdown of PPP3CA protein expression did not significantly affect VEGF-induced MAPK3/1 and AKT1 phosphorylation but attenuated FGF2-induced MAPK3/1 and AKT1 phosphorylation. Thus, to our knowledge, the present study is the first to demonstrate successful knockdown of PPP3CA protein expression in any cell model using a single pair of double-strained siRNA. Moreover, specific knockdown of PPP3CA protein expression enhances VEGF-stimulated, but not FGF2-stimulated, OFPAE cell proliferation and attenuates FGF2-induced, but not VEGF-induced, MAPK3/1 and AKT1 activation. Thus, PPP3CA differentially modulates the VEGF- and FGF2-stimulated cell proliferation and signaling cascades in OFPAE cells. These data also suggest that signaling molecules other than MAPK3/1 and AKT1 play an important role in VEGF- and FGF2-stimulated cell proliferation after knockdown of PPP3CA in OFPAE cells.     

Serine/threonine protein phosphatase 5 (PP5) interacts with substrate under heat stress conditions and forms protein complex in Arabidopsis

Protein phosphatase 5 plays a pivotal role in signal transduction in animal and plant cells, and it was previously shown that Arabidopsis protein phosphatase 5 (AtPP5) performs multiple enzymatic activities that are mediated by conformational changes induced by heat shock stress. In addition, transgenic overexpression of AtPP5 gene conferred enhanced heat shock resistance compared with wild-type plant. However, the molecular mechanism underlying this enhanced heat shock tolerance through functional and conformational changes upon heat stress is not clear. In this report, AtPP5 was shown to preferentially interact with its substrate, MDH, under heat stress conditions. In addition, in co-IP analysis, AtPP5 was observed to form a complex with AtHsp90 in Arabidopsis. These results suggest that AtPP5 may enhance thermotolerance via forming multi-chaperone complexes under heat shock conditions in Arabidopsis. Finally, we show that AtPP5 is primarily localized in the cytoplasm of Arabidopsis.     

Identification of a glycogen synthase phosphatase from yeast Saccharomyces cerevisiae as protein phosphatase 2A.

A glycogen synthase phosphatase was purified from the yeast Saccharomyces cerevisiae. The purified yeast phosphatase displayed one major protein band which coincided with phosphatase activity on nondenaturing polyacrylamide gel electrophoresis. This phosphatase had a molecular mass of about 160,000 Da determined by gel filtration and was comprised of three subunits, termed A, B, and C. The subunit molecular weights estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were 60,000 (A), 53,000 (B), and 37,000 (C), indicating that this yeast glycogen synthase phosphatase is a heterotrimer. On ethanol treatment, the enzyme was dissociated to an active species with a molecular weight of 37,000 estimated by gel filtration. The yeast phosphatase dephosphorylated yeast glycogen synthase, rabbit muscle glycogen phosphorylase, casein, and the alpha subunit of rabbit muscle phosphorylase kinase, was not sensitive to heat-stable protein phosphatase inhibitor 2, and was inhibited 90% by 1 nM okadaic acid. Dephosphorylation of glycogen synthase, phosphorylase, and phosphorylase kinase by this yeast enzyme could be stimulated by histone H1 and polylysines. Divalent cations (Mg2+ and Ca2+) and chelators (EDTA and EGTA) had no effect on dephosphorylation of glycogen synthase or phosphorylase while Mn2+ stimulated enzyme activity by approximately 50%. The specific activity and kinetics for phosphorylase resembled those of mammalian phosphatase 2A. An antibody against a synthetic peptide corresponding to the carboxyl terminus of the catalytic subunit of rabbit skeletal muscle protein phosphatase 2A reacted with subunit C of purified yeast phosphatase on immunoblots, whereas the analogous peptide antibody against phosphatase 1 did not. These data show that this yeast glycogen synthase phosphatase has structural and catalytic similarity to protein phosphatase 2A found in mammalian tissues.     

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.     

Loss of chloroplast‐localized protein phosphatase 2Cs in Arabidopsis thaliana leads to enhancement of plant immunity and resistance to Xanthomonas campestris pv. campestris infection

Protein phosphatases (PPs) counteract kinases in reversible phosphorylation events during numerous signal transduction pathways in eukaryotes. PP2Cs, one of the four major classes of the serine/threonine‐specific PP family, are greatly expanded in plants. Thus, PP2Cs are thought to play a specific role in signal transduction pathways. Some rice PP2Cs classified in subgroup K are responsive to infection by the compatible Xanthomonas oryzae pv. oryzae, the causal agent of bacterial blight. In Arabidopsis thaliana, orthologous PP2C genes (AtPP2C62 and AtPP2C26) classified to subgroup K are also responsive to Xanthomonas campestris pv. campestris (Xcc, causal agent of black rot) infection. To elucidate the function of these subgroup K PP2Cs, atpp2c62‐ and atpp2c26‐deficient A. thaliana mutants were characterized. A double mutant plant which was inoculated with a compatible Xcc showed reduced lesion development, as well as the suppression of bacterial multiplication. AtPP2C62 and AtPP2C26 localized to the chloroplast. Furthermore, the photosynthesis‐related protein, chaperonin‐60, was indicated as the potential candidate for the dephosphorylated substrate catalysed by AtPP2C62 and AtPP2C26 using two‐dimensional isoelectric focusing sodium dodecylsulfate‐polyacrylamide gel electrophoresis (2D‐IDF‐SDS‐PAGE). Taken together, AtPP2C62 and AtPP2C26 are suggested to be involved in both photosynthesis and suppression of the plant immune system. These results imply the occurrence of crosstalk between photosynthesis and the plant defence system to control productivity under pathogen infection.