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Annu. Rev. Plant Physiol. Plant Mol. Biol. 1996. 47:101–25 Copyright © 1996 by Annual Reviews Inc. All rights reserved PLANT PROTEIN PHOSPHATASES Robert D. Smith AgBiotech Center, Rutgers University, New Brunswick, New Jersey 08903-0231 John C. Walker Division of Biological Sciences, University of Missouri, Columbia, Missouri 65211 KEY WORDS: phosphorylation, signal transduction, protein kinase, metabolism, okadaic acid ABSTRACT Posttranslational modification of proteins by phosphorylation is a universal mechanism for regulating diverse biological functions. Recognition that many cellular proteins are reversibly phosphorylated in response to external stimuli or intracellular signals has generated an ongoing interest in identifying and characterizing plant protein kinases and protein phosphatases that modulate the phosphorylation status of proteins. This review discusses recent advances in our understanding of the structure, regulation, and function of plant protein phosphatases. Three major classes of enzymes have been reported in plants that are homologues of the mammalian type-1, -2A, and -2C protein serine/threonine phosphatases. Molecular genetic and biochemical studies reveal a role for some of these enzymes in signal transduction, cell cycle progression, and hormonal regulation. Studies also point to the presence of additional phosphatases in plants that are unrelated to these major classes. CONTENTS INTRODUCTION..................................................................................................................... PROTEIN PHOSPHATASE-1 ................................................................................................. Structure and Regulation.......................... ......................................................................... Heterologous Expression of Plant PP1............................................................................... PROTEIN PHOSPHATASE-2A............................................................................................... Structure and Regulation.......................... ......................................................................... Role in Cellular Metabolism..................... ......................................................................... PROTEIN PHOSPHATASE-2C............................................................................................... Structure, Regulation, and Function ................................................................................... 1040-2519/96/0601-0101$08.00 102 104 104 107 108 108 110 111 111 101 102 SMITH & WALKER OTHER PROTEIN PHOSPHATASES .................................................................................... 114 INHIBITOR STUDIES ............................................................................................................. 116 CONCLUDING REMARKS .................................................................................................... 119 INTRODUCTION It is well recognized that reversible phosphorylation of proteins controls many cellular processes in plants and animals. The phosphorylation status of proteins is regulated by the opposing activities of protein kinases and protein phosphatases. Phosphorylation of eukaryotic proteins occurs predominantly (97%) on serine and threonine residues and to a lesser extent on tyrosine residues (107). Phosphohistidine phosphorylation has also been reported in plants (50), fungi (86), and animals (25), but its relative contribution to the total phosphoamino acid content of eukaryotic cells is not known. In animals, protein phosphorylation plays well-known roles in diverse cellular processes such as glycogen metabolism, cell cycle control, and signal transduction (9, 23, 83, 106, 107). Clearly, there has been a similar interest in examining the role of protein phosphorylation in plant cellular regulation and in identifying the protein kinases and protein phosphatases that modulate the phosphorylation status of target substrate molecules. An increasing number of plant protein kinases have been reported in recent years. Some of these enzymes play pivotal roles in the control of plant defense mechanisms, signal transduction, and metabolism (11, 13, 50, 122). In plants, as in animals, recognition that a number of protein kinases respond directly to second messengers such as Ca2+ led to the view that protein kinases were primarily responsible for regulating the phosphorylation status of proteins and that protein phosphatases merely reverse the effects of protein kinases. Molecular genetic and biochemical studies have greatly advanced our knowledge of protein phosphatases and provide compelling evidence that these enzymes perform essential regulatory functions. The objective of this review is to examine recent advances in our knowledge of the structure and regulation of plant protein phosphatases as well as initial insights into their physiological roles. Protein phosphatase activities have been reported in most plant subcellular compartments, including mitochondria, chloroplast, nuclei, and cytosol, and are associated with various membrane and particulate fractions (50, 70). Some protein phosphatases are poorly characterized and may represent novel enzymes that are unique to plants, such as the chloroplast thylakoid protein phosphatase (124). Others have biochemical properties that are very similar to well-known mammalian protein phosphatases, such as the mitochondrial pyruvate dehydrogenase phosphatase (80) and cytosolic protein serine/threonine phosphatases (69). Because some plant and animal protein phosphatases are recognized to be evolutionarily conserved, biochemical characterization and molecular cloning of distinct phosphatases in plants that correspond to the PROTEIN PHOSPHATASES 103 mammalian type-1 (PP1) and type-2 (PP2) protein serine/threonine phosphatases have been achieved over a short period. Understandably, the plant PP1 and PP2 represent only a subset of the total phosphatases that will subsequently be discovered in plants, but their essential roles in diverse cellular processes are already becoming evident. Biochemical and genetic studies in plants implicate PP1 and/or PP2 activity in signal transduction, hormonal regulation, mitosis, and control of carbon and nitrogen metabolism. Classification of mammalian PP1 and PP2 is based on their unique substrate specificities and sensitivities to various inhibitors (52). PP1 dephosphorylates the β-subunit of mammalian phosphorylase kinase preferentially and is inhibited by the endogenous proteins, inhibitor-1 (I-1) and inhibitor-2 (I-2). PP2 has greater activity toward the α-subunit of phosphorylase kinase and is resistant to I-1 and I-2. PP2 is further divided into three subgroups, PP2A, PP2B, and PP2C, depending on their subunit structure, divalent cation requirements, and substrate specificities (23). PP2A is a heterotrimer of a catalytic C-subunit and two distinct regulatory A- and B-subunits and does not require divalent cations for activity. PP2B, a Ca2+-activated phosphatase, exists as a heterodimer consisting of a catalytic A-subunit and a regulatory B-subunit belonging to the EF-hand family of calcium-binding proteins. PP2C is found as a monomer and activity requires Mg2+. Mammalian PP1, PP2A, PP2B, and PP2C catalytic subunits are products of distinct genes, and examination of their primary structures indicates that PP1, PP2A, and PP2B are related enzymes (106). They share no structural homology with PP2C. The structure, regulation, and function of the protein serine/threonine phosphatases in animals and fungi have been the subject of many recent reviews (9, 23, 73, 83, 106, 107, 118). Protein phosphatases are inhibited by a number of natural toxins such as okadaic acid and cyclosporin A (21, 73). These compounds have proven extremely useful in analyzing the differing actions of specific classes of phosphatases in vitro, as well as in vivo, because many of them are readily taken up by animal and plant cells (73). The marine toxin okadaic acid, for instance, is a potent inhibitor of PP2A (IC50 ≈ 0.1–1.0 nM) and inhibits PP1 at 10- to 100-fold higher concentrations (IC50 ≈ 10–100 nM). Okadaic acid is marginally effective against PP2B at micromolar concentrations and has no effect on PP2C. The immunosuppressant cyclosporin A complexes with endogenous immunophilins and specifically targets PP2B for downregulation (103). PP2C is insensitive to these drugs. Increasing use of these drugs to study the role of reversible protein phosphorylation in diverse cellular processes has generated new insights into the regulation and physiological function of protein serine/ threonine phosphatases. Inhibitor studies also confirm the presence in plants and animals of protein phosphatases that do not belong to the PP1 and PP2 families of protein phosphatases. 104 SMITH & WALKER PROTEIN PHOSPHATASE-1 Structure and Regulation PP1 is a ubiquitous and highly conserved enzyme found in all eukaryotes. The native mammalian and fungal enzyme is a complex of a catalytic subunit and one or more regulatory subunits. Genetic studies have shown yeast PP1 catalytic subunit genes to be essential for cellular processes as diverse as glycogen accumulation, mitosis, and translational control (118). Regulatory subunits define specific functions of PP1 catalytic activity in vivo by controlling the subcellular location and substrate specificity of the enzyme complex. For instance, the Saccharomyces cerevisiae GAC1 protein, a homologue of the mammalian RG1 subunit, targets PP1 to glycogen particles and enhances dephosphorylation of glycogen-bound phosphoenzymes required for glycogen synthesis and accumulation (35). A nuclear-localized PP1 regulatory subunit, sds22+, which is essential for completion of mitosis in S. pombe, increases phosphohistone H1 phosphatase activity of PP1 and downregulates its activity against phosphorylase a (120). Other regulatory subunits, such as mammalian I-2, have been characterized biochemically. I-2 is hypothesized to function in the cell as a chaperone that binds and activates newly synthesized PP1 catalytic subunits via a process that requires phosphorylation of I-2 by glycogen synthase kinase-3 (GSK-3) in the presence of ATP-Mg (107). The active catalytic subunit may then complex with various targeting or regulatory subunits, thereby displacing I-2. On the basis of these and additional studies in animals and fungi, a targeting subunit has been hypothesized for regulation of PP1 in vivo (24, 45). The hypothesis is that distinct regulatory subunits are present in the cell that bind transiently to the catalytic subunit and dictate its subcellular location and substrate specificity. The hypothesis is supported by the findings that in S. pombe PP1 is present in the cell as high molecular complexes ranging in size from 80 to 200 kD, of which only the 80-kD complex has phosphorylase a phosphatase activity (59). Protein phosphatase activities have been reported in a number of plant species and in different tissue extracts through the use of mammalian phosphoprotein substrates. In Brassica napus seed extracts, over 60% of the phosphatase activity is inhibited by I-1 (IC50 = 0.6 nM), I-2 (IC50 = 2.0 nM), and okadaic acid (IC50 = 10 nM) and dephosphorylates the β-subunit of rabbit phosphorylase kinase preferentially, indicating that it belongs to the PP1 class of protein phosphatases (69). The PP1 activity is associated predominantly with membranes of the endoplasmic reticulum and other unidentified particulate fractions in B. napus seed extracts, and gel filtration analyses indicate it exists as a high molecular weight complex (70). PP1 is almost exclusively cytosolic in pea leaves and carrot cells and is associated with microsomes in wheat leaves (70). It has also been reported in isolated nuclei (104) and in PROTEIN PHOSPHATASES 105 plasma membranes (135). Very little is known about the structure or regulation of native PP1 in plants, nor have any physiological substrates been identified. Studies reveal a striking similarity between the biochemical properties and subcellular distribution of animal and plant PP1 and suggest the possibility that mechanisms for controlling PP1 activity and function may be equally well conserved. A key goal in attempting to understand PP1 function in plants is to identify and characterize regulatory subunits. CATALYTIC SUBUNIT PP1 catalytic subunit cDNA and genomic clones have been reported in several plant species (Table 1). Eight distinct isoforms have been identified in Arabidopsis (4, 34, 84, 114, 116), and additional related genes appear to be present in its genome (114, 116). The presence of PP1 multigenes is not unique to Arabidopsis, and they appear to be present in most plant species. The remarkable similarity (>70%) between plant and mammalian PP1 primary sequences and the sensitivity of a bacterially expressed recombinant maize ZmPP1 to I-2 (IC50 = 0.1 nM) and okadaic acid (IC50 = 200 nM) (113) confirms that the plant genes encode PP1 catalytic subunits. Unlike the native mammalian enzyme, however, the recombinant maize PP1 requires Mn2+ for activation (113). Mn2+-dependent activity is also observed in recombinant rabbit PP1 or when native PP1 is converted to a Mn2+-dependent form by incubation with NaF (1). The modified enzymes can be restored to their Mn2+-independent forms by incubation with I-2 and GSK-3/ATP-Mg, which supports the hypothesis that I-2 acts as a chaperone to activate PP1 in vivo and raises the possibility that plant PP1 catalytic subunits may need to be activated by a plant homologue of I-2. Multiple PP1 isogenes are present in most eukaryotes, with the exception of S. cerevisiae which contains a single PP1, GLC7 (118). The physiological advantage of having multiple PP1 isogenes remains unknown. Disruption of the two S. pombe PP1 genes is lethal, but deletion of either gene, singly, has no effect on cell viability (85), which indicates that the yeast PP1 isoforms have overlapping functions. In contrast, loss of one of four PP1 genes (30) in Drosophila inhibits chromosome separation (5), which suggests a selective role for this isoform in mitosis. Unique roles for some isogenes may be governed by their spatial and temporal expression. For instance, the rat PP1γ1 is predominantly expressed in brain tissues, whereas PP1γ2 is almost exclusively expressed in testes (108, 109). Upregulation of certain PP1 isogenes has been reported in plants as well. BoPP1 expression is enhanced at different stages of microspore development in B. oleracea, and mature trinucleate microspores contain a unique BoPP1 transcript not found at other stages of the plant life cycle (100). Arabidopsis AtPP1bg is constitutively expressed at low levels in all tissues with upregulation in male and female tissues (4). 106 SMITH & WALKER Table 1 Plant protein phosphatases Species Name Protein phosphatase-1 Catalytic subunit Arabidopsis TOPP1c TOPP2d TOPP3 TOPP4 TOPP5e TOPP6 AtPP1bgf TOPP8 Alfalfa PP1Ms B. napus PP1Bn B. oleracea BoPP1 Maize ZmPP1 AcetabuPP1Ac1 laria PP1Ac2 Protein phosphatase-2A Catalytic subunit Arabidopsis PP2A-1 PP2A-2 PP2A-3 PP2A-4 Alfalfa pp2aMs B. napus PP2ABn Sunflower PP2AHa Acetabularia PP2AAc Regulatory A-subunit Arabidopsis pDF1 pDF2 RCN1g Pea PP2A-Ps Regulatory B-subunit Arabidopsis AtBα Protein phosphatase-X Catalytic subunit Arabidopsis PPX-At1 PPX-At2 Protein phosphatase-2C Arabidopsis ABI1 KAPP PP2C-At Clone #AA %IDa Expressionb Database Reference(s) accession number cDNA cDNA cDNA cDNA cDNA cDNA cDNA cDNA cDNA cDNA cDNA cDNA genomic 318 312 322 321 312 — 322 — 321 — 316 316 319 72 76 73 72 76 — 71 — 74 72 71 70 71 R,L,S,F,C R,L,S,F,C R,L,S,F R,L,F R,L,S,F,C — F — R,L,S,N,FB,MF — R,L,Co,A,P R,S,L,H,T,C — M93408 M93409 M93410 M93411 M93412 — Z46253 — X80788 X57438 X63558 M60215 Z28627 genomic 319 71 — Z28632 cDNA cDNA cDNA cDNA cDNA cDNA cDNA genomic 306 306 308 313 313 – 305 307 82 82 81 81 79 72 — — R,L,S,F R,L,S,F R,L,S,F R,L,S,F R,L,S,FB,N, — — — M96732 M96733 M96734 M96841 X70399 X57439 Z26041 Z26654 3 3 3 16 90 74 cDNA cDNA genomic cDNA 587 — 588 — — — 58 55 — — seedlings E,R,S,L,C X82001 X82002 U21557 Z25888 112 112 54, 112 32 cDNA 513 46 R,L,S,Co,F,FB U18129 102 cDNA cDNA 305 307 83 83 R,L,S,F R,L,S,F Z22587 Z22596 89 89 genomic cDNA cDNA 434 582 399 35 19 35 R,L,S,Si R,L — X77116 U09505 D38109 64, 79 121 62 34, 84, 114 33, 114 114 114 114 116 4, 116 116 88 74 100 112 PROTEIN PHOSPHATASES 107 Functional differences between PP1 isoforms may also arise from structural variations in their primary sequence that control activity, substrate specificity, and/or regulatory subunit interactions. Phosphorylation of S. pombe PP1, dis2+, at a conserved cdc2 protein kinase consensus phosphorylation site [S/TP-X-Z: X = polar and Z = basic; (82)] downregulates its activity in vitro (141). In contrast, S. pombe PP1, sds21+, lacks a cdc2 phosphorylation motif and is neither phosphorylated nor downregulated by cdc2 protein kinases (141). Therefore, isoforms that harbor the phosphorylation recognition site may be selectively targeted for inactivation by cdc2-like protein kinases. The phosphorylation site is found at a conserved position in about half of the known plant PP1 catalytic subunits (116), but whether they are subject to this form of regulation in vivo remains unknown. Heterologous Expression of Plant PP1 One approach that has been taken to address the functional importance of structural variations in the primary sequences of the catalytic subunits is to express individual PP1 isoforms in heterologous systems and ask whether they can fully complement the multiple functions of their host PP1. Expression of distinct plant PP1 clones in fungal systems reveals that isoforms differ in their ability to complement various PP1 functions (Table 2). A lethal trait caused by disruption of S. cerevisiae PP1, glc7- (14), is rescued by Arabidopsis TOPP2 but not TOPP1 (116). Failure of TOPP1 to complement the lethal trait cannot be attributed to improper transcription or translation because TOPP1 protein is detected in wild-type cells expressing the TOPP1 clone. However, only S. cerevisiae strains expressing TOPP2 have increased PP1 activity, which suggests that TOPP1 may be downregulated in yeast cells by an unknown posttranslational mechanism. TOPP2 is unable to suppress an S. cerevisiae glc7-1 glycogen deficiency phenotype (14, 116, 123), which indicates that TOPP2 can dephosphorylate substrates that are essential for completion of mitosis but is unable to dephosphorylate a substrate (glycogen synthase) of GLC7 required for glycogen accumulation in yeast. Glycogen accumulation is restored a Percent identity between plant PP1 catalytic subunits and rabbit PP1α, plant PP2A catalytic subunits and rabbit PP2α, plant PP2A regulatory A-subunits and human PR65α, plant PP2A B-regulatory subunit and human B-subunit, plant PPX catalytic subunits and rabbit PPX, plant PP2C catalytic domains and rat PP2C. b Expression of plant genes in different tissues: R, root; L, leaf; S, stem; F, flower; FB, flower bud; Si, silique; N, node; MF, mature flower; Co, coleoptile; C, cell culture. c TOPP1 independently isolated and named PP1-At (84), and PP1A-At2 (33). d TOPP2 independently isolated and named PP1A-At1 (34). e TOPP5 independently isolated and named PP1A-At3 (34). f AtPP1bg independently isolated and named TOPP7 (116). g RCN1 independently isolated and named regA (112). 108 SMITH & WALKER Table 2 Complementation of fungal mutations by plant PP1 clones S. cerevisiae TOPP1 TOPP2 TOPP3 AtPP1bg PP1Ms glc7-1 No (116) No (116) No (116) glc7No (116) Yes (116) No (116) S. pombe dis2-11 Yes (84) No (34) wee1- Aspergillus nidulans bimG11 Yes (34) Yes (4) No (88) in glc7-1 strains, however, by expressing a chimeric PP1 consisting of the N-terminal 1-93 amino acid residues of GLC7 fused to residues 98-312 of TOPP2 (116). This suggests that unique structural sequences located at the N-terminus may be important for controlling substrate specificity and/or regulatory subunit interactions. Functional differences among plant PP1 isoforms have also been reported in S. pombe and Aspergillus (Table 2). A cold-sensitive dis2-11 mutation that blocks exit from mitosis is suppressed by TOPP1 but not TOPP2 (34, 84). However, TOPP2 is able to restore a temperature-sensitive S. pombe cdc25ts/wee1- double mutation (34) that inhibits entry into mitosis (10), indicating that failure of TOPP2 to complement the dis2-11 mutation is not due to the absence of active protein phosphatase activity but probably results from a failure of TOPP2 to dephosphorylate substrates of dis2+/sds21+ that are required for entering mitosis. Furthermore, expression of Arabidopsis AtPP1bg in a temperature-sensitive Aspergillus bimG11 PP1 mutant supports vegetative growth but not conidia development at nonpermissive temperatures (4). These results demonstrate that plant PP1 isoforms have limited ability to functionally complement fungal PP1 enzymes despite their remarkable structural and biochemical similarities and suggest that even small structural differences in their primary sequences may have profound effects on their activity and function. PROTEIN PHOSPHATASE-2A Structure and Regulation Native PP2A in animals is found either as a heterodimer of a 36-kD catalytic C-subunit and a 65-kD regulatory A-subunit, or as a heterotrimer in which a variable regulatory B-subunit (50–70 kD) complexes to the core heterodimer (23, 77, 106). In S. cerevisiae, a PP2A A-subunit encoded by tpd3 is essential for cytokinesis (134), and a B-subunit encoded by cdc55 is required for cellular morphogenesis (44). Single A-subunit and B-subunit genes are present in Drosophila and both appear to be essential for pattern formation (132). Ge- PROTEIN PHOSPHATASES 109 netic and biochemical studies indicate that regulatory A- and B-subunits control substrate specificity of PP2A (77). PP2A has been reported in a number of plant species and is found in most tissues (53, 66, 69–71, 91, 104). Like PP1, PP2A is found in many subcellular locations including the nucleus (104) and cytosol and is associated with various membranes and insoluble fractions (70, 135). Neither PP1 nor PP2A is found in chloroplasts (70, 125). Native PP2A from B. napus seed extracts dephosphorylates the α-subunit of phosphorylase kinase preferentially and is potently inhibited by okadaic acid (IC50 = 0.1 nM) in vitro (53, 69). A partially purified PP2A catalytic subunit from maize seedlings readily dephosphorylates phosphocasein, phosphohistone H1, and phosphorylase a; displays no appreciable activity toward pNPP; and fails to bind to heparin-Sepharose, a distinct binding characteristic of PP1 (53). These results indicate that plant PP2A is biochemically nearly indistinguishable from mammalian PP2A. Four genes encoding PP2A catalytic subunits (Csubunit) have been isolated from Arabidopsis, and Southern blot analyses indicate a fifth isoform may be present in the genome (3, 16). C-subunit clones have also been identified in alfalfa (90), B. napus (74), sunflower, and the green alga Acetabularia cleftonii (Table 1). Plant and mammalian PP2A share about 80% identity. Transcripts corresponding to each of the Arabidopsis PP2A catalytic subunits are found in all tissues examined, although the level of expression of some isogenes appears to be developmentally regulated (16, 89). Thus, unique roles for some PP2A genes may result from enhanced expression in some tissues. Differential regulation of the C-subunits may also occur from posttranslational modifications (98). Phosphorylation of tyrosine (17, 18) or threonine (27, 41) residues downregulates in vitro activity of mammalian PP2A. Tyrosine and threonine residues are found at a similar position in all plant PP2A isoforms. Methylation of the C-terminal leucine, conserved in all PP2A, may provide an additional level of regulation (107). CATALYTIC SUBUNIT REGULATORY SUBUNITS Three A-subunit clones have been isolated from Arabidopsis (112), and a partial cDNA clone has also been identified in pea (32) (Table 1). The plant A-subunits are approximately 58% identical to the human regulatory A-subunit. In addition, a B-subunit homologue has been reported in Arabidopsis that shares 46% identity with the human Bα-subunit (102). Expression of the A-subunit genes has not been thoroughly examined (112), and the B-subunit appears to be uniformly expressed throughout the plant (102). Interactions between plant catalytic and regulatory subunits have been examined, but the presence of multiple catalytic and regulatory subunits in plants suggests that plants may contain a number of different PP2A complexes with distinct physiological functions. 110 SMITH & WALKER Insight into a potential role for PP2A in plants comes from the recent discovery that an Arabidopsis polar auxin transport mutant rcn1 encodes a PP2A regulatory A-subunit homologue (54) that shares 58% identity with the human PR65α (54). RCN1 rescues the S. cerevisiae temperature-sensitive tpd3 mutant, which indicates that it is a functional A-subunit homologue capable of controlling the activity of the yeast PP2A catalytic subunit. A T-DNA insertion into the coding region of RCN1 results in normal expression of a truncated transcript that may encode a protein lacking a significant portion of its C-terminus. This may represent a loss-of-function mutation because the C-terminal end of the mammalian regulatory A-subunit is known to be required for interaction with the catalytic domain (99). The rcn1 recessive mutation causes an altered morphological response in seedlings to N-1-naphthylphthalamic acid (NPA), a polar auxin transport inhibitor (54). Auxin transport in stems of rcn1 mutants also shows increased sensitivity to NPA. These results point to a unique role for RCN1 in controlling the activity of an endogenous PP2A toward a key phosphosubstrate(s) involved in polar auxin transport. Role in Cellular Metabolism Control of key metabolic enzymes by reversible phosphorylation has been studied extensively in plants (13, 93). Activation of sucrose phosphate synthase (SPS), nitrate reductase (NR), and phosphoenolpyruvate carboxylase (PEPC) in light is associated with a decrease in the phosphorylation status of SPS and NR and an increase in phosphorylation of PEPC (50). Phosphorylation of quinate dehydrogenase (QDH) and hydroxymethylglutaryl-CoA reductase kinase (HMG-CoA reductase kinase) and dephosphorylation of HMGCoA reductase also activates these enzymes in vivo (70, 75, 95). Several lines of evidence point to PP2A activity in dephosphorylating these enzymes. Dephosphorylation-mediated changes in activity of each enzyme are blocked by PP1/PP2A inhibitors, such as okadaic acid, microcystin-LR, and calyculin A (15, 39, 47, 55, 56, 60, 78, 110). Okadaic acid also prevents in vivo activation of SPS (47, 110) and NR (46, 48) in spinach leaves. Furthermore, addition of mammalian PP2A catalytic subunit to cell extracts enhances SPS (110) and NR activities (71) and downregulates PEPC (78) and QDH (70). In contrast, addition of the mammalian PP1 catalytic subunit does not alter their activities. These studies suggest that PP2A, and not PP1, is responsible for dephosphorylating these enzymes in vivo. SPS, PEPC, and NR are key enzymes that control nitrogen and carbon assimilation in plants. Because these pathways compete for carbon skeletons and energy sources, mechanisms must exist to regulate their relative activities in response to changing environmental and metabolic conditions. Coordination of these pathways could result from tight control of SPS, NR, and PEPC activities in the cytosol, which raises the possibility that their respective pro- PROTEIN PHOSPHATASES 111 tein kinases and phosphatases respond differentially to signals in the cell. Emerging evidence indicates that the SPS PP2A may be distinct from the PP2A that dephosphorylates NR. Activation of SPS, for instance, is inhibited by Pi, sulfate, and tungstate, but NR activation is unaffected by these compounds (49). In contrast, NR activation is inhibited by Mg2+ and stimulated by 5′-AMP (55, 56). These results could be explained by the presence of multiple PP2A enzymes in the cytosol that respond differentially to effectors such as Pi, shown to be a potent inhibitor of the SPS phosphatase (47, 139). Alternatively, these effectors may interact directly with the protein substrates to modulate activation. Light does not appear to control the protein kinase and phosphatase activities directly because feeding mannose to excised leaves in the dark activates SPS and NR (49, 119). However, a recent study suggests that the NR PP2A may be light-activated by a process that requires de novo protein synthesis, because it can be blocked with cycloheximide (46). PROTEIN PHOSPHATASE-2C PP2C is the least well-characterized member of the protein serine/threonine phosphatases (106). PP2C demonstrates high activity toward enzymes of the cholesterol biosynthetic pathway in mammals (106, 107), and genetic studies in fission yeast implicate a possible role for PP2C in growth (76). Physiological substrates for PP2C remain to be identified in animals or plants. PP2C demonstrates relatively high activity toward phosphocasein, a commonly used substrate for measuring PP2C activity in vitro. Mg2+-dependent and okadaicinsensitive phosphocasein phosphatase activity has been reported in several plant species (70, 75), but its distribution in plants appears more restrictive than for PP1 and PP2A. Activity is predominantly cytosolic in carrot cells, cauliflower inflorescence, and leaves from pea and wheat (70, 75). However, PP2C activity was reported to be absent in maize seedlings (53) and B. napus seed extracts (69). Cauliflower PP2C and PP2A readily dephosphorylate HMG-CoA reductase kinase, a key enzyme in isoprenoid biosynthesis (75). There is presently no indication whether the reductase kinase is a physiological substrate for either phosphatase. Structure, Regulation, and Function Three novel PP2C phosphatases have been cloned from Arabidopsis (Figure 1). Each contains a catalytic domain that is structurally related (20–35% identical) to mammalian PP2C. In addition, the plant genes contain N-terminal extensions of variable lengths that share no homology with one another, or to protein sequences in the data banks (Figure 1). These unique structural domains among plant PP2C are not found in any of the known fungal or animal PP2C. One of the Arabidopsis phosphatases, PP2C-At, was identified via a 112 SMITH & WALKER Figure 1 Structural organization of rat and Arabidopsis thaliana type-2C protein phosphatases. Conserved catalytic domains are indicated as PP2C. The putative EF-hand calcium binding found in ABI1 is shown as a solid box. The box containing horizontal lines in KAPP designates a signal anchor. genetic screen for genes that complement the sterile phenotype of the S. pombe pde1 mutant, which is defective in cAMP phosphodiesterase, a component of the cAMP-dependent protein kinase cascade (62). Among three distinct cDNA clones isolated from this screen, a PP2C that shares 35% identity with the rat PP2C was recovered. Expression of PP2C-At in S. pombe pde1 mutants also restores expression of a transcription factor (ste11) that is required for sexual development and whose expression is inhibited by the activity of a cAMP-protein kinase (PKA). This suggests that the plant PP2C-At may be counteracting the activity of a PKA in S. pombe. Its role in Arabidopsis, however, remains unknown. Abscisic acid (ABA) regulates multiple functions in plants including embryo maturation, seed dormancy, stomatal closure, and mitosis in root meristems. Several abscisic acid–insensitive (abi) Arabidopsis mutants that are unresponsive to elevated concentrations of ABA have been extensively characterized (61). The dominant mutant, abi1, has been cloned and found to encode a PP2C homologue with two distinct domains: an N-terminal domain containing a putative Ca2+-binding site and a C-terminal PP2C catalytic domain that is 35% identical to the rat PP2C (64, 79). This represents a novel putative Ca2+-regulated protein serine/threonine phosphatase that is structurally unrelated to the PP2B class of Ca2+-dependent phosphatases. Bacterially expressed ABI1 is Mg2+-dependent, but Ca2+ regulation of ABI activity has not been reported (79). The presence of a putative Ca2+-binding site suggests that ABI1 may be responsive to ABA-mediated changes in cellular Ca2+ levels. The involvement of Ca2+ in ABA-dependent stomatal closure is ABSCISIC ACID RESPONSE GENE—ABI1 PROTEIN PHOSPHATASES 113 well established (40), and recent observations indicate that the abi1-1 mutant protein interferes with ABA-dependent regulation of K+ channels in guard cells of transgenic tobacco (38). A Ca2+-activated phosphatase activity has previously been reported in Vicia faba guard cells that is inhibited by cyclosporin A-cyclophilin protein complexes (CyP-CsA complex) (67). Cyclosporin A is an immunosuppressant that binds to endogenous cyclophilin proteins and inhibits Ca2+/calmodulin-activated PP2B (105). It is interesting to note that the CyP-CsA complex blocks Ca2+-induced inactivation of inward K+ channel activity in V. faba guard cells (67), which suggests that a PP2B homologue, or possibly ABI1, may be involved in the control of K+ channel activity. Future studies may resolve whether ABI is a target for CyP-CsA. The lesion in the abi1-1 mutant is a single nucleotide substitution that converts Gly-180 to Asp within the catalytic domain (64, 79). The mutation does not alter the ubiquitous expression of ABI1 (64). The dominant nature of the mutation could possibly be explained by an alteration in the response of abi1 to ABA-mediated cellular changes in Ca2+ levels. Constitutive activation of the phosphatase, for instance, could inhibit ABA-stimulated signaling pathways. Alternatively, protein dephosphorylation may be required to turn on the pathways, and activation of ABI1 by Ca2+ may be altered in the mutant protein phosphatase. KINASE-ASSOCIATED PROTEIN PHOSPHATASE—KAPP Additional evidence that type-2C protein phosphatases are involved in plant signaling pathways comes from the identification of a third novel PP2C in Arabidopsis, termed “KAPP” for kinase-associated protein phosphatase (121). KAPP was isolated via an in vitro protein interaction screen for proteins that interact with RLK5, a membrane-bound receptor-like protein kinase (RLK) (137). The predicted structure of KAPP indicates that it contains three distinct domains, an N-terminal signal anchor, a kinase interaction domain (KI), and a C-terminal PP2C domain (121). A bacterially expressed KAPP fusion protein demonstrates Mg2+-dependent and okadaic-insensitive activity in vitro, consistent with the classification of this enzyme as a PP2C (121). The signal anchor predicts that KAPP is membrane localized, and translational insertion of KAPP into membrane vesicles, in vitro, supports this hypothesis (JM Stone & JC Walker, personal communication). These results suggest that PP2C is not exclusively cytosolic in the cell. Furthermore, ubiquitous expression of KAPP in Arabidopsis suggests that PP2C distribution in plants is more extensive than previously indicated from PP2C activities measured in cell extracts. A possible explanation for this difference is that native KAPP may have negligible activity toward nonphysiological substrates commonly used to measure PP2C activity. The KI domain is both necessary and sufficient for interaction with the receptor-like protein kinase, RLK5 (121). However, interaction between 114 SMITH & WALKER RLK5 and KAPP occurs only upon autophosphorylation of the receptor kinase domain. In vitro dephosphorylation of RLK5 with recombinant maize PP1 blocks binding by the KI domain. This indicates that sequences bearing phosphoamino acids may act as high-affinity binding sites for KAPP. RLK5 binding to KAPP is akin to the interaction between autophosphorylated protein tyrosine kinases and proteins containing src-homology-2 (SH2) domains in mammalian systems (133). However, unlike SH2 domains that bind exclusively to sites bearing phosphotyrosyl residues, the KI domain may belong to a novel class of protein-protein interacting domains that bind to sequences containing either a phosphoserine or phosphothreonine residue. The KI domain bears no sequence homology with SH2 domains. By analogy with the regulation of SH2-phosphotyrosine phosphatase (SH2PTP) activity in animals, it is possible that binding of KAPP to autophosphorylated RLK5 modulates KAPP phosphatase activity in vivo. Phosphorylation of the KI domain by RLK5, which has been observed in vitro, may have additional effects on its binding affinity to RLK5 or on KAPP phosphatase activity. It is not known whether RLK5 is a physiological substrate for KAPP. The structure of KAPP and its interaction with RLK5 suggests that KAPP may control early steps of the RLK5 signaling pathway. RLK5 is a member of a family of related protein kinases that participate in diverse biological functions including self-incompatibility, defense responses, and plant development (121). KAPP and related proteins, therefore, may represent a novel class of protein phosphatases in plants that are early components of RLK-mediated signaling pathways. It will be of interest to learn whether KAPP acts as a positive or a negative regulator of the pathway and whether it interacts specifically with RLK5 or additional phosphorylated receptor and nonreceptor protein kinases in the cell. OTHER PROTEIN PHOSPHATASES Molecular cloning studies have revealed the presence, in animals and fungi, of a number of protein serine/threonine phosphatases that are structurally related to PP1, PP2A, and PP2B phosphatases but that cannot be placed in any of these classes because of unique structural and/or biochemical features (107). Among these is the rabbit PPX (26), a PP2A-related protein phosphatase that localizes to centrosomes and is thought to play a role in nucleation of microtubules (12). The substrate specificity of rabbit PPX and its sensitivity to various phosphatase inhibitors is similar to PP2A; however, PPX fails to bind PP2A regulatory A-subunits (12). Two cDNA clones showing 83% amino acid identity to the rabbit PPX have been isolated from Arabidopsis by hybridization (Table 1) (89). The Arabidopsis genes, ppx1 and ppx2, are PROTEIN PHOSPHATASE-X PROTEIN PHOSPHATASES 115 expressed at low levels in flowers, leaves, stems, and roots. The function of ppx1 and ppx2 in Arabidopsis is not known. The plant mitochondrial pyruvate dehydrogenase complex (PDC) is regulated in part by reversible phosphorylation of one of its subunits, pyruvate dehydrogenase E1α (PDH) (80). Phosphorylation by an intrinsic protein kinase inactivates PDH in the light. Conversely, dephosphorylation of PDH in the dark by a loosely associated Mg2+-dependent protein serine/threonine phosphatase activates the complex (81). Calmodulin, monovalent cations, and polyamines do not affect the phosphatase, and inorganic phosphate (Pi) is the only known metabolite that has a slight inhibitory effect at physiological concentrations (92). The plant PDH-phosphatase is similar in its biochemical properties to the mammalian phosphatase with the exception of its response to Ca2+, which enhances Mg2+-activation of the mammalian PDHphosphatase (28) but antagonizes the plant enzyme (81). Structural characterization of the bovine PDH-phosphatase indicates that it is a novel PP2C that contains a Ca2+-binding site (Kd ≈ 8 µM) (63). Recombinant bovine PDH-phosphatase expressed in Escherichia coli is Mg2+-dependent and Ca2+-stimulated. The similarity of the plant and mammalian PDH-phosphatases suggests the plant enzyme may also be a PP2C homologue, but unlike the mammalian enzyme the plant phosphatase may lack a Ca2+ regulatory domain. MITOCHONDRIAL PDC-PHOSPHATASE Phosphorylation of chloroplast proteins has been studied extensively over the past decade, but knowledge of the requisite protein kinases and phosphatases is limited (2, 7). Proteins are phosphorylated on serine or threonine almost exclusively, with the exception of pyruvate, Pi dikinase, which contains a phosphorylated histidine residue (97). Phosphorylation of thylakoid proteins occurs by light- and redox-dependent kinase(s), whereas redox-independent dephosphorylation in the dark is catalyzed by a membrane-bound protein phosphatase (or phosphatases), which is sensitive to sodium fluoride and molybdate ions (6, 20, 111, 124). The chloroplast thylakoid phosphatase is probably unrelated to the cytosolic serine/threonine phosphatases, because it fails to dephosphorylate phosphohistone or phosphorylase a and is not inhibited by okadaic acid or microcystin-LR (70, 125). A detailed characterization of the thylakoid phosphatase has been hampered by the lack of a suitable substrate (142). An alternate approach has been the use of synthetic peptides (126), which reveals that at least one thylakoidbound phosphatase is able to dephosphorylate multiple thylakoid phosphoproteins in vitro (20). CHLOROPLAST THYLAKOID PROTEIN PHOSPHATASE Phosphotyrosine content of proteins in eukaryotes accounts for only a fraction (<3%) of the total phosphoamino acids in the cell, yet reversible phosphorylation on tyrosine residues is essential for PROTEIN TYROSINE PHOSPHATASE 116 SMITH & WALKER cellular growth and differentiation in animals (51, 127). Phosphotyrosine residues have also been reported in plants (19, 131), but little is known of the physiological role of reversible tyrosine phosphorylation in plants. Dual specificity protein kinases that phosphorylate on serine, threonine, and tyrosine residues have been reported in plants (8, 87). Some of these kinases are homologues of mammalian GSK-3, which requires phosphorylation of a specific tyrosine residue for full activation. A tyrosine residue is found at a conserved location on the plant GSK-3 homologues, suggesting that phosphorylation on the residues may also control activity in vivo. A homologue of mammalian mitogen-activated protein kinases (MAPK) has been identified in tobacco and is transiently activated and tyrosine phosphorylated in response to a fungal elicitor (128). Activation of MAPK in animals requires phosphorylation of both a threonine residue and a tyrosine residue. It is not known whether the tobacco MAPK is also phosphorylated on threonine/serine residues and whether this is important for activation. Downregulation of the tobacco MAPK is associated with tyrosine dephosphorylation and indicates that plants may contain endogenous protein tyrosine phosphatases and/or dual specificity phosphatases that can dephosphorylate both phosphoserine/threonine and phosphotyrosine residues (22). Addition of calyculin A to elicitor-treated tobacco sustains the activation and tyrosine phosphorylation of the protein kinase (128), which indicates that a PP1/PP2A may also be involved in turning off the kinase because protein tyrosine phosphatases and dual-specificity phosphatases are resistant to calyculin A. One possible explanation for these results is that a PP1/PP2A is activating an intermediate protein tyrosine phosphatase or a dual-specificity phosphatase. Inactivation of the tobacco kinase may also require dephosphorylation of phosphoserine/threonine residues directly by a PP1/ PP2A. Identifying homologues of mammalian protein tyrosine phosphatases in plants has been hampered by the lack of structural information on putative tyrosine phosphatases and because phosphotyrosine phosphatase activity in plants is often attributed to acid phosphatases (36, 53). A 90-kD protein was partially purified from pea nuclei that specifically dephosphorylates phosphotyrosine-containing peptides, is inactive toward phosphoserine and phosphothreonine residues, and has little activity toward pNPP, a common substrate used to measure acid phosphatase activity (42). The pea nuclear phosphatase is biochemically more similar to mammalian protein tyrosine phosphatases than the acid or serine/threonine phosphatases, although its exact classification awaits structural characterization. INHIBITOR STUDIES The discovery of protein phosphatase inhibitors has significantly advanced our understanding of the biological processes that are controlled by reversible PROTEIN PHOSPHATASES 117 protein phosphorylation and has aided in the identification of physiological substrates for specific classes of protein phosphatases. Most notable among these inhibitors is okadaic acid, a marine toxin that inhibits PP2A (IC50 = 0.1–1.0 nM) at concentrations 10- to 100-fold lower than PP1 (IC50 = 10–100 nM). Okadaic acid is readily taken up by cells, which provides a useful molecular probe to study the role of PP1 and PP2A in many cellular processes. In vivo sensitivity of PP1 and PP2A to okadaic acid, however, is decreased by high protein concentrations in the cell (107). Thus, while okadaic acid sensitivity of a specific cellular event is indicative of PP1/PP2A involvement, the concentration of okadaic acid used may not distinguish between these two classes of phosphatases. Insight into the differing actions of PP1 and PP2A in particular cellular events has been furthered by the additional use of chemically distinct toxins such as calyculin A, microcystin-LR, and tautomycin (21, 73). Calyculin A and microcystin-LR inhibit PP1 and PP2A with equal potency (IC50 ≈ 0.1–0.3 nM), whereas tautomycin inhibits PP1 (IC50 = 0.16 nM) at concentrations fivefold lower than PP2A (IC50 = 8 nM). Comparison of the biological effects of these cell-permeable compounds with those of okadaic acid can provide an initial evaluation of the relative contribution of each phosphatase activity. For example, the use of calyculin A and okadaic acid to study light-dependent signaling pathways in maize protoplasts suggests that a PP1 is involved in activating multiple transcription factors involved in lightinduced gene expression in plants (104). The addition of nodularin, acanthifolicin, cantharidin, and the herbicide endothal to the growing arsenal of PP1/PP2A inhibitors should provide additional confidence in our abilities to study the differing actions of PP1 and PP2A in intact cells. However, identification of novel protein phosphatases that are sensitive to these same compounds may ultimately limit identification of specific phosphatases based on inhibitor studies alone and will require additional verification by genetic and/ or biochemical experiments. Nonetheless, these inhibitors provide a very powerful approach for the initial assessment of the role of protein phosphorylation in controlling numerous cellular events. More than 40 publications describing the effects of these toxins on diverse biological processes in plants have appeared within the last four years. A summary of many of these studies is outlined in Table 3 and implicates PP1/PP2A or related phosphatase activities in hormonal- (31, 94), carbohydrate- (129), light- (104), and elicitor-mediated signaling pathways; cell cycle regulation (43, 140, 143); growth and development (104, 115); ion channel control (65, 67, 130, 144); pollination (57, 101); and cellular metabolism (15, 39, 46, 47, 60, 70, 71, 75, 78, 110, 138). A different class of phosphatase inhibitors is found in microbially derived immunosuppressants cyclosporin A and FK506, which, upon binding to endogenous immunophilin proteins, cyclophilin and FK506-binding protein, form inhibitory complexes that target Ca2+/calmodulin-activated PP2B (102). Their 118 SMITH & WALKER Table 3 Use of phosphatase inhibitors in plants Biological Species function Signal transduction Auxin N. plumbaginifolia Sugar Potato Tissuea Ib Response C O LP O, C, M Blocks feedback inhibition of auxin- 31 stimulated gene expression Blocks expression of sporamin, 129 β-amylase, ADP-glucose phosphorylase small subunit. Increases expression of sucrose synthase 68 Increases expression of αmy3 Rice C O Light Maize P O, C Ethylene Tobacco L O Pathogens Tomato C O Tomato C C Tobacco C C Potato T O Parsley P O Soybean C, Ct O, C, M Soybean C O Regulation of membrane channels GC K+ chan- Vicia faba nel Vicia faba GC Vicia faba Anion Tobacco channel Control of enzyme activity SPS Spinach NR Spinach Pea B. campestris PEPCK Cucumber Refs Blocks expression of lightinducible genes Increases expression of ethyleneinduced PR genes Blocks elicitor-induced changes in NADH oxidase and ascorbate perodixase activities Causes growth medium alkalinization, hyperphosphorylation of cellular proteins and increased ACC-S activity Causes sustained activation of dualspecificity kinase Increases expression of PR-10a and binding of PBF-1 Blocks elicitor-induced furanocoumarin accumulation Increases PAL expression, medium alkalinization and production of isoflavonoid phytoalexins Increases expression of PAL, CHS, and HRGP 104 67, 130 94 136 33, 117 128 29 96 72 37 MC O, C O Blocks Ca2+-induced inactivation of inward current Blocks inward current; no effect on outward current Increases outward current Alters voltage gating and kinetics L L R L O, M O, M O C Blocks light-activation Blocks light-activation Blocks light-activation Blocks light-activation 47, 110 46, 71 39 60 Ct M Blocks dephosphorylation 138 F, Cs O, C 65 66 144 PROTEIN PHOSPHATASES 119 Table 3 (continued) Tissuea Ib Response Maize QDH Carrot HMGCauliflower CoA red Growth and development Root Arabidopsis L C I O O, M O Blocks inactivation 78 Blocks inactivation 70 Blocks activation of HMG-CoA red 75 R, RH O, C ChlorMaize oplast Cell cycle Tobacco Tradescantia Ca2+ C. roseus uptake Pollination B. napus B. napus; B. oleracea; B. campestris L O C SH O, C O, M C O Inhibits cell division and cortical cell elongation. Blocks root hair development Blocks light-induced chlorophyll accumulation Causes cell cycle arrest Alters metaphase transit times and chromosome separation Blocks Ca2+ uptake and callose syn Pi F, FB O, M O, M Biological function Species Refs 115 104 43, 143 140 58 Blocks pollen growth 57 Inhibits pollen tube growth in cross- 101 pollinated flowers. Alters selfincompatability in flower buds a Tissues: A, aleurone; C, cell culture; Ct, cotyledon; F, flower; FB, flower bud; GC, guard cell; I, inflorescence; L, leaf; MC, mesophyll cell; LP, leaf petiole; P, protoplast; Pi, pistil; PM, plasma membrane; S, seed; SH, stamen hair cell; T, tuber. b Phosphatase inhibitor: O, okadaic acid; C, calyculin A; M, microcystin-LR; F, FK506 binding protein-FK506 complex; Cs, cyclosporin-cyclophilin complex. limited use in plants has been discussed above and suggests that a Ca2+-activated protein phosphatase, possibly a PP2B homologue or ABI1, is involved in regulating K+ channels in guard cells of V. faba (67). CONCLUDING REMARKS The objective of this review was to provide an overview of recent advances in the study of protein phosphatases in plants. Progress has been made in cloning a number of plant homologues belonging to three major classes of the mammalian protein serine/threonine phosphatases, and a handful of physiological substrates for PP2A have been identified. Still, very little is known about the structure and regulation of the native enzymes or their physiological roles. Molecular genetic and inhibitor studies have provided powerful avenues to begin addressing these areas, but progress on understanding the role of specific protein phosphatases in plants will depend, in large part, on the identification of physiological substrates and the purification of native enzyme complexes using traditional biochemical methods. Future studies will no doubt reveal that 120 SMITH & WALKER many novel protein phosphatases exist in plants that do not belong to the archetypal classes of serine/threonine protein phosphatases. 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