Purification and characterization of two protein kinases acting on the aquaporin SoPIP2;1

Aquaporins are water channel proteins that facilitate the movement of water and other small solutes across biological membranes. Plants usually have large aquaporin families, providing them with many ways to regulate the water transport. Some aquaporins are regulated post-translationally by phosphorylation. We have previously shown that the water channel activity of SoPIP2;1, an aquaporin in the plasma membrane of spinach leaves, was enhanced by phosphorylation at Ser115 and Ser274. These two serine residues are highly conserved in all plasma membrane aquaporins of the PIP2 subgroup. In this study we have purified and characterized two protein kinases phosphorylating Ser115 and Ser274 in SoPIP2;1. By anion exchange chromatography, the Ser115 kinase was purified from the soluble protein fraction isolated from spinach leaves. The Ca2+-dependent Ser274 kinase was purified by peptide affinity chromatography using plasma membranes isolated from spinach leaves. When characterized, the Ser115 kinase was Mg2+-dependent, Ca2+-independent and had a pH-optimum at 6.5. In accordance with previous studies using the oocyte expression system, site-directed mutagenesis and kinase and phosphatase inhibitors, the phosphorylation of Ser274, but not of Ser115, was increased in the presence of phosphatase inhibitors while kinase inhibitors decreased the phosphorylation of both Ser274 and Ser115. The molecular weight of the Ser274 kinase was approximately 50 kDa. The identification and characterization of these two protein kinases is an important step towards elucidating the signal transduction pathway for gating of the aquaporin SoPIP2;1.     

Structural and Functional Analysis of SoPIP2;1 Mutants Adds Insight into Plant Aquaporin Gating

Plant plasma membrane aquaporins facilitate water flux into and out of plant cells, thus coupling their cellular function to basic aspects of plant physiology. Posttranslational modifications of conserved phosphorylation sites, changes in cytoplasmic pH and the binding of Ca²⁺ can regulate water transport activity by gating the plasma membrane aquaporins. A structural mechanism unifying these diverse biochemical signals has emerged for the spinach aquaporin SoPIP2;1, although several questions concerning the opening mechanism remain. Here, we describe the X-ray structures of the S115E and S274E single SoPIP2;1 mutants and the corresponding double mutant. Phosphorylation of these serines is believed to increase water transport activity of SoPIP2;1 by opening the channel. However, all mutants crystallised in a closed conformation, as confirmed by water transport assays, implying that neither substitution fully mimics the phosphorylated state. Nevertheless, a half-turn extension of transmembrane helix 1 occurs upon the substitution of Ser115, which draws the C^α atom of Glu31 10 Å away from its wild-type conformation, thereby disrupting the divalent cation binding site involved in the gating mechanism. Mutation of Ser274 disorders the C-terminus but no other significant conformational changes are observed. Inspection of the hydrogen-bond interactions within loop D suggested that the phosphorylation of Ser188 may also produce an open channel, and this was supported by an increased water transport activity for the S188E mutant and molecular dynamics simulations. These findings add additional insight into the general mechanism of plant aquaporin gating.     

Specific plasma membrane aquaporins of the PIP1 subfamily are expressed in sieve elements and guard cells

BACKGROUND INFORMATION: Transmembrane water flow is aided by water-specific channel proteins, aquaporins. Plant genomes code for approx. 35 expressed and functional aquaporin isoforms. Plant aquaporins fall into four different subfamilies of which the PIPs (plasma membrane intrinsic proteins) constitute the largest and evolutionarily most conserved subfamily with 13 members in Arabidopsis and maize. Furthermore, the PIPs can be divided into two phylogenetic groups, PIP1 and PIP2, of which the PIP1 isoforms are most tightly conserved, sharing >90% amino acid sequence identity. As the nomenclature implies, the majority of PIPs have been shown to be localized at the plasma membrane. Recently, two highly abundant plasma membrane aquaporins, SoPIP2;1 and SoPIP1;2, have been purified and structurally characterized. RESULTS: We report the cloning of a cDNA encoding SoPIP1;2 and show that there are at least five additional sequences homologous with SoPIP2;1 and SoPIP1;2 in the spinach genome. To understand their role in planta, we have investigated the cellular localization of the aquaporin homologues SoPIP1;2 and SoPIP1;1. By Western- and Northern-blot analyses and by immunocytochemical detection at the light and electron microscopic levels, we show that SoPIP1;2 is highly expressed in phloem sieve elements of leaves, roots and petioles and that SoPIP1;1 is present in stomatal guard cells. CONCLUSIONS: Localization of the two abundant aquaporin isoforms suggests roles for specific PIPs of the PIP1 subgroup in phloem loading, transport and unloading, and in stomatal movements.     

Water transport activity of the plasma membrane aquaporin PM28A is regulated by phosphorylation.

PM28A is a major intrinsic protein of the spinach leaf plasma membrane and the major phosphoprotein. Phosphorylation of PM28A is dependent in vivo on the apoplastic water potential and in vitro on submicromolar concentrations of Ca2+. Here, we demonstrate that PM28A is an aquaporin and that its water channel activity is regulated by phosphorylation. Wild-type and mutant forms of PM28A, in which putative phosphorylation sites had been knocked out, were expressed in Xenopus oocytes, and the resulting increase in osmotic water permeability was measured in the presence or absence of an inhibitor of protein kinases (K252a) or of an inhibitor of protein phosphatases (okadaic acid). The results indicate that the water channel activity of PM28A is regulated by phosphorylation of two serine residues, Ser-115 in the first cytoplasmic loop and Ser-274 in the C-terminal region. Labeling of spinach leaves with 32P-orthophosphate and subsequent sequencing of PM28A-derived peptides demonstrated that Ser-274 is phosphorylated in vivo, whereas phosphorylation of Ser-115, a residue conserved among all plant plasma membrane aquaporins, could not be demonstrated. This identifies Ser-274 of PM28A as the amino acid residue being phosphorylated in vivo in response to increasing apoplastic water potential and dephosphorylated in response to decreasing water potential. Taken together, our results suggest an active role for PM28A in maintaining cellular water balance.     

Multiple phosphorylations in the C-terminal tail of plant plasma membrane aquaporins: role in subcellular trafficking of AtPIP2;1 in response to salt stress

Aquaporins form a family of water and solute channel proteins and are present in most living organisms. In plants, aquaporins play an important role in the regulation of root water transport in response to abiotic stresses. In this work, we investigated the role of phosphorylation of plasma membrane intrinsic protein (PIP) aquaporins in the Arabidopsis thaliana root by a combination of quantitative mass spectrometry and cellular biology approaches. A novel phosphoproteomics procedure that involves plasma membrane purification, phosphopeptide enrichment with TiO(2) columns, and systematic mass spectrometry sequencing revealed multiple and adjacent phosphorylation sites in the C-terminal tail of several AtPIPs. Six of these sites had not been described previously. The phosphorylation of AtPIP2;1 at two C-terminal sites (Ser(280) and Ser(283)) was monitored by an absolute quantification method and shown to be altered in response to treatments of plants by salt (NaCl) and hydrogen peroxide. The two treatments are known to strongly decrease the water permeability of Arabidopsis roots. To investigate a putative role of Ser(280) and Ser(283) phosphorylation in aquaporin subcellular trafficking, AtPIP2;1 forms mutated at either one of the two sites were fused to the green fluorescent protein and expressed in transgenic plants. Confocal microscopy analysis of these plants revealed that, in resting conditions, phosphorylation of Ser(283) is necessary to target AtPIP2;1 to the plasma membrane. In addition, an NaCl treatment induced an intracellular accumulation of AtPIP2;1 by exerting specific actions onto AtPIP2;1 forms differing in their phosphorylation at Ser(283) to induce their accumulation in distinct intracellular structures. Thus, the present study documents stress-induced quantitative changes in aquaporin phosphorylation and establishes for the first time a link with plant aquaporin subcellular localization.     

Reconstitution of water channel function of an aquaporin overexpressed and purified from Pichia pastoris

The aquaporin PM28A is one of the major integral proteins in spinach leaf plasma membranes. Phosphorylation/dephosphorylation of Ser274 at the C-terminus and of Ser115 in the first cytoplasmic loop has been shown to regulate the water channel activity of PM28A when expressed in Xenopus oocytes. To understand the mechanisms of the phosphorylation-mediated gating of the channel the structure of PM28A is required. In a first step we have used the methylotrophic yeast Pichia pastoris for expression of the pm28a gene. The expressed protein has a molecular mass of 32462 Da as determined by matrix-assisted laser desorption ionization-mass spectrometry, forms tetramers as revealed by electron microscopy and is functionally active when reconstituted in proteoliposomes. PM28A was efficiently solubilized from urea- and alkali-stripped Pichia membranes by octyl-beta-D-thioglucopyranoside resulting in a final yield of 25 mg of purified protein per liter of cell culture.     

Phosphorylation of aquaporin PvTIP3;1 defined by mass spectrometry and molecular modeling

The water channel protein PvTIP3;1 (alpha-TIP) is a member of the Major Intrinsic Protein membrane channel family. The in vitro activity of this aquaporin is dependent on phosphorylation, and the protein is phosphorylated in vivo by a membrane-associated Ca(2+)-dependent kinase. Mutagenesis studies have implicated three serine residues as kinase targets, but only phosphorylation of Ser7 has been observed in vivo. An atomic model of PvTIP3;1 generated by homology modeling suggested that Ser7 is the only residue that would be sterically accessible to kinases. To further explain the phosphorylation of PvTIP3;1, we overexpressed this aquaporin in the methylotrophic yeast Pichia pastoris and purified the hexahistidine-tagged protein by immobilized metal affinity chromatography. Mass spectrometry confirmed that a fraction of recombinant PvTIP3;1 was phosphorylated. Phosphatase and kinase treatments indicated that Ser7 was the only residue that could be phosphorylated. In addition, mass spectrometry indicated that the native and expressed proteins are N-terminally acetylated. This is the first demonstration that a full-length, recombinant aquaporin can be produced in yeast and authentically phosphorylated in vitro. Characterization of phosphorylation-mediated gating in PvTIP3;1 will serve as a paradigm for understanding gating mechanisms of other channels.     

The response of Arabidopsis root water transport to a challenging environment implicates reactive oxygen species- and phosphorylation-dependent internalization of aquaporins

Aquaporins, which facilitate the diffusion of water across biological membranes, are key molecules for the regulation of water transport at the cell and organ levels. We recently reported that hydrogen peroxide (H₂O₂) acts as an intermediate in the regulation of Arabidopsis root water transport and aquaporins in response to NaCl and salicylic acid (SA).¹ Its action involves signaling pathways and an internalization of aquaporins from the cell surface. The present addendum connects these findings to another recent work which describes multiple phosphorylations in the C-terminus of aquaporins expressed in the Arabidopsis root plasma membrane.² A novel role for phosphorylation in the process of salt-induced relocalization of AtPIP2;1, one of the most abundant root aquaporins, was unraveled. Altogether, the data delineate reactive oxygen species (ROS)-dependent signaling mechanisms which, in response to a variety of abiotic and biotic stresses, can trigger phosphorylation-dependent PIP aquaporin intracellular trafficking and root water transport downregulation.Key words: reactive oxygen species, aquaporin, phosphorylation, cell signaling, stress, protein relocalization, root water transportPlants can regulate their water uptake capacity i.e. their root hydraulic conductivity (Lp_r) on a short term (minutes to hour) basis through regulation of plasma membrane (PM) aquaporins of the Plasma membrane Intrinsic Protein (PIP) subfamily.³ It has been known for a long time that salt stress (NaCl), as many other abiotic stresses such as cold, anoxia or nutrient deprivation, induces an inhibition of Lp_r in many plant species.³ In the recent study by Boursiac et al. (2008),¹ we identified SA as a new inhibitory increased the accumulation of ROS in roots, it was hypothesized that H₂O₂ or other ROS may have a central role in the regulation of root water transport in response to various biotic or abiotic stimuli. When Arabidopsis roots were treated with mM concentrations of exogenous H₂O₂, Lp_r was inhibited within minutes by up to 90%. These findings are consistent with previous reports showing that ROS can downregulate water transport in cucumber and maize roots or in the algae Chara corallina.^4–7 H₂O₂ and possibly other derived ROS may modulate the Lp_r through signaling mechanisms or by a direct oxidative gating of aquaporins. The latter hypothesis, which has been favored in previous studies by Steudle and colleagues,^6,7 was investigated by Boursiac et al., by functionally expressing aquaporins in Xenopus oocytes and by testing their sensitivity to external H₂O₂. The results show that Arabidopsis aquaporins are insensitive to direct oxidation by H₂O₂ or hydroxyl radicals. Thus, these and complementary pharmacological analyses on excised roots rather support a role for H₂O₂ as a second messenger that connects environmental stimulus perception to water transport regulation in plant roots. The additional finding that H₂O₂ can be transported by aquaporins^8,9 opens the possibility of intricate loop mechanisms whereby these proteins may interfere with their own regulation. For example, active PIP aquaporins could facilitate the diffusion within the cell of NADPH-oxidase derived apoplastic H₂O₂, which in turn would activate signaling pathways acting on PIP activity and/or subcellular localization.In a previous study, we monitored the subcellular localization of AtPIP1;2 and AtPIP2;1, two of the most abundant PIPs in roots, by expression in transgenic Arabidopsis of fusions with the green fluorescent protein (GFP).¹⁰ We observed that a 100 mM NaCl treatment induced in 2–4 hours an increased intracellular labeling which was interpreted as an intracellular relocalization of the two aquaporins.¹⁰ In our more recent study, both a 150 mM NaCl and a 0.5 mM SA treatments induced an intracellular labeling by GFP-PIP1;2 and PIP2;1-GFP fusions, with a “fuzzy” pattern or at the level of spherical bodies. Preventing the NaCl- or SA-dependent accumulation of ROS with exogenous catalase was able to almost completely counteract the effects of the two stimuli on the localization pattern of the PIP2;1-GFP fusion. In addition, the inhibition of Lp_r by SA was also counteracted at 33% by the catalase treatment. Altogether, the data stress the importance of an ROS-induced relocalization of aquaporins in the regulation of root water transport. Yet, we still miss quantitative data and complementary pharmacological evidence to determine the exact contribution of aquaporin relocalization with respect to other aquaporin regulatory mechanisms.Another recent work by our group has, however, provided deeper insights into the mechanisms of stress-induced relocalization of aquaporins in plants.² Our group identified by mass spectrometry multiple adjacent phosphorylation sites (up to 4 in the case of AtPIP2;4) in the C-terminus of aquaporins expressed at the root plasma membrane.² Phosphorylation of AtPIP2;1, which shows a simpler profile with only two sites at Ser280 and Ser283, was studied in closer detail by site-directed mutagenesis and expression in transgenic Arabidopsis of GFP-PIP2;1 fusions. A Ser283Ala mutation, which mimics a constitutively dephosphorylated Ser283, induced a marked intracellular accumulation of GFP-PIP2;1 in resting conditions. Because no phenotype was observed after a Ser280Ala mutation, the data suggest a specific role for Ser283 phosphorylation in the proper targeting of the protein. When plants were treated by 100 mM NaCl for 2 to 4 hours, the wild type (WT) and Ser280Ala mutant forms of GFP-PIP2;1 showed similar intracellular staining, in both “fuzzy” structures or spherical bodies. On the contrary, the Ser283Ala mutant did not label any spherical body. Interestingly, a Ser283Asp mutation that mimics a constitutively phosphorylated Ser283 resulted in a salt-induced labeling of spherical bodies similar to the one observed with WT GFP-PIP2;1 whereas no “fuzzy” staining was observed. Therefore, the phosphorylation status of Ser283 seems to determine the redistribution of AtPIP2;1 towards fuzzy structures (non-phosphorylated Ser283) or spherical bodies (phosphorylated Ser283). Although the nature of these intracellular structures remains to be identified, we now consider the possibility that the spherical bodies correspond to the late endosome/prevacuolar compartment that orientates aquaporins towards a degradation pathway whereas the fuzzy structures may act as a storage compartment for subsequent relocalization of PIP aquaporins to the PM, and rapid recovery of the PM water permeability. Although we favor the idea that the intracellular labeling shown by GFP-PIP2;1 in response to salt originates from aquaporins relocalized from the PM, newly synthesized proteins may also contribute to this pattern.Prak et al., also developed an absolute quantification method to show that the phosphorylation profile of AtPIP2;1 at the root plasma membrane was altered upon 100 mM NaCl and 2 mM H₂O₂ treatments. Whereas NaCl decreased the abundance of phosphorylated Ser283, H₂O₂ enhanced the overall phosphorylation of the AtPIP2;1 C-terminus. These observations add another level of complexity to the mechanisms of stimulus-induced and phosphorylation- dependent relocalisation of plant aquaporins uncovered in our group. Although one of the primary effects of NaCl is undoubtedly an accumulation of ROS, the difference in phosphorylation patterns observed in response to H₂O₂ and NaCl treatments may come from quantitative and kinetic differences in ROS patterns between the two treatments or from additional regulations activated by salt.We note that phosphorylation of PIP aquaporins had already been investigated in detail.^11–13 In particular, studies with spinach SoPIP2;1 has pointed to two phosphorylation sites, Ser115 in the first cytoplasmic loop (loop B) and Ser274 at the C-terminus, as important for modulating the water transport activity of this aquaporin after expression in Xenopus oocytes. A role for these two sites in aquaporin gating was also deduced from the atomic structure of SoPIP2;1.¹⁴ Whereas Ser280 in AtPIP2;1 corresponds to Ser274 in SoPIP2;1, the functional role of sites equivalent to Ser283 in AtPIP2;1 had not been considered previously in any other PIP. To our knowledge, the study by Prak et al., provides the first evidence in plants for a role of phosphorylation on the relocalization of aquaporins and highlights the importance of multiple phosphorylations sites in the C-terminus of aquaporins, as has been recently shown in human Aquaporin-2.^15,16Overall, the advance provided by our two recent studies delineates a working model (Fig. 1), whereby multiple abiotic and biotic stresses, which all induce an accumulation of ROS, activate common signaling pathways to downregulate root water transport. We have provided evidence that some of these pathways are calcium- and/ or protein kinase-dependent. One regulatory mechanism triggered by these pathways is the relocalization of aquaporins into intracellular “fuzzy” structures or bigger spherical bodies. For AtPIP2;1, the sorting between these structures is determined in part by the phosphorylation status of Ser283, which ultimately may control the cellular fate of the protein for degradation or remobilization to the PM. A coming challenge will be to determine how this and other cellular mechanisms quantitatively contribute to the integrated regulation of water transport at the cell and tissue (whole root) levels. Another avenue for future research will be to identify the molecular components involved in upstream ROS-dependent cell signaling and aquaporin phosphorylation. These studies will tell us how the regulation of root water uptake in parallel to the regulation of transpiration allows the plant to preserve its water status when it is continuously challenged by multiple stresses.Open in a separate windowFigure 1Tentative model of regulation of root hydraulic conductivity (Lp_r) through reactive oxygen species (ROS) signaling. Multiple biotic and abiotic stimuli such as NaCl or salicylic acid can induce an intra- and/or extracellular accumulation of ROS by acting on their production, degradation or transport. The stimulus-induced ROS in turn activate signaling pathways involving protein kinases and cytosolic calcium. These events result in changes in the phosphorylation and subcellular localization patterns of plasma membrane (PM) aquaporins (PIPs). In particular, endocytosis can direct PIPs towards various intracellular compartments for subsequent recycling at the PM or degradation. Phosphorylation can interfere with this routing process, but also determines the intrinsic water transport activity (gating) of PM localized PIPs. The possibility exists that signaling components directly act on PIP gating, recycling or degradation through phosphorylation- and endocytosis-independent pathways (not shown). In addition, transport of H₂O₂ by PIP aquaporins may provide retroactive effects of aquaporins on upstream signaling events. Aquaporin activity at the PM determines root cell water permeability, which contributes to most of Lp_r in Arabidopsis. The overall scheme shows how stress-induced ROS signaling results in an inhibition of PIP aquaporin activity and, as a consequence, in an overall downregulation of Lp_r.     

The calcium‐dependent protein kinase CPK7 acts on root hydraulic conductivity

The hydraulic conductivity of plant roots (Lp_r) is determined in large part by the activity of aquaporins. Mechanisms occurring at the post‐translational level, in particular phosphorylation of aquaporins of the plasma membrane intrinsic protein 2 (PIP2) subfamily, are thought to be of critical importance for regulating root water transport. However, knowledge of protein kinases and phosphatases acting on aquaporin function is still scarce. In the present work, we investigated the Lp_r of knockout Arabidopsis plants for four Ca²⁺‐dependent protein kinases. cpk7 plants showed a 30% increase in Lp_r because of a higher aquaporin activity. A quantitative proteomic analysis of wild‐type and cpk7 plants revealed that PIP gene expression and PIP protein quantity were not correlated and that CPK7 has no effect on PIP2 phosphorylation. In contrast, CPK7 exerts a negative control on the cellular abundance of PIP1s, which likely accounts for the higher Lp_r of cpk7. In addition, this study revealed that the cellular amount of a few additional proteins including membrane transporters is controlled by CPK7. The overall work provides evidence for CPK7‐dependent stability of specific membrane proteins.     

Structure and stability of the spinach aquaporin SoPIP2;1 in detergent micelles and lipid membranes

Background

SoPIP2;1 constitutes one of the major integral proteins in spinach leaf plasma membranes and belongs to the aquaporin family. SoPIP2;1 is a highly permeable and selective water channel that has been successfully overexpressed and purified with high yields. In order to optimize reconstitution of the purified protein into biomimetic systems, we have here for the first time characterized the structural stability of SoPIP2;1.

Methodology/Principal Finding

We have characterized the protein structural stability after purification and after reconstitution into detergent micelles and proteoliposomes using circular dichroism and fluorescence spectroscopy techniques. The structure of SoPIP2;1 was analyzed either with the protein solubilized with octyl-β-D-glucopyranoside (OG) or reconstituted into lipid membranes formed by E. coli lipids, diphytanoylphosphatidylcholine (DPhPC), or reconstituted into lipid membranes formed from mixtures of 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPE), 1-palmitoyl-2oleoyl-phosphatidylethanolamine (POPE), 1-palmitoyl-2-oleoyl-phosphatidylserine (POPS), and ergosterol. Generally, SoPIP2;1 secondary structure was found to be predominantly α-helical in accordance with crystallographic data. The protein has a high thermal structural stability in detergent solutions, with an irreversible thermal unfolding occurring at a melting temperature of 58°C. Incorporation of the protein into lipid membranes increases the structural stability as evidenced by an increased melting temperature of up to 70°C.

Conclusion/Significance

The results of this study provide insights into SoPIP2;1 stability in various host membranes and suggest suitable choices of detergent and lipid composition for reconstitution of SoPIP2;1 into biomimetic membranes for biotechnological applications.     

Regulatory phosphorylation of Sorghum leaf phosphoenolpyruvate carboxylase. Identification of the protein-serine kinase and some elements of the signal-transduction cascade.

The phosphoenolpyruvate (PPrv) carboxylase isozyme involved in C4 photosynthesis undergoes a day/night reversible phosphorylation process in leaves of the C4 plant, Sorghum. Ser8 of the target enzyme oscillates between a high (light) and a low (dark) phosphorylation status. Both in vivo and in vitro, phosphorylation of dark-form carboxylase was accompanied by an increase in the apparent Ki of the feedback inhibitor L-malate and an increase in Vmax. Feeding detached leaves various photosynthetic inhibitors, i.e. 3-(3,4-dichlorophenyl)-1,1-dimethylurea, gramicidin and DL-glyceraldehyde, prevented PPrv carboxylase phosphorylation in the light, thus suggesting that the cascade involves the photosynthetic apparatus as the light signal receptor, and presumably has the electron transfer chain and the Calvin-Benson cycle as components in the signal-transduction chain. Two protein-serine kinases capable of phosphorylating PPrv carboxylase in vitro have been partially purified from light-adapted leaves. One was isolated on a calmodulin-Sepharose column; it was calcium-dependent but did not require calmodulin for activity. The other was purified on a blue-dextran-agarose column and the only Me2+ required for activity was Mg2+. In reconstituted phosphorylation assays, only the latter caused the expected decrease in malate sensitivity of PPrv carboxylase suggesting that this protein is the genuine PPrv-carboxylase-kinase. Desalted extracts from light-adapted leaves possessed a considerably greater phosphorylation capacity with immunopurified dephosphorylated PPrv carboxylase as substrate than did dark extracts. This light stimulation was insensitive to type 2A protein phosphatase inhibitors, okadaic acid and microcystin-LR, which suggests that the kinase is a controlled step in the cascade which leads to phosphorylation of PPrv carboxylase. The higher phosphorylation capacity of light-adapted leaf tissue was nullified by pretreatment with the cytosolic protein synthesis inhibitor, cycloheximide. Thus, protein turnover is involved as part of the mechanism controlling the activity of the kinase purified on blue-dextran-agarose. However, no information is available with respect to the specific nature of the link between the above-mentioned light transducing steps and the protein kinase that achieves the physiological response. Finally, the in vivo phosphorylation site (Ser8) in the N-terminal region of the C4 type Sorghum PPrv carboxylase is also present in a non-photosynthetic form of the Sorghum enzyme (Ser7), as deduced by cDNA sequence analysis.     

Two SNF1-related protein kinases from spinach leaf phosphorylate and inactivate 3-hydroxy-3-methylglutaryl-coenzyme A reductase, nitrate reductase, and sucrose phosphate synthase in vitro.

We resolved from spinach (Spinacia oleracea) leaf extracts four Ca2+-independent protein kinase activities that phosphorylate the AMARAASAAALARRR (AMARA) and HMRSAMSGLHLVKRR (SAMS) peptides, originally designed as specific substrates for mammalian AMP-activated protein kinase and its yeast homolog, SNF1. The two major activities, HRK-A and HRK-C (3-hydroxy-3-methylglutaryl-coenzyme A reductase kinase A and C) were extensively purified and shown to be members of the plant SnRK1 (SNF1-related protein kinase 1) family using the following criteria: (a) They contain 58-kD polypeptides that cross-react with an antibody against a peptide sequence characteristic of the SnRK1 family; (b) they have similar native molecular masses and specificity for peptide substrates to mammalian AMP-activated protein kinase and the cauliflower homolog; (c) they are inactivated by homogeneous protein phosphatases and can be reactivated using the mammalian upstream kinase; and (d) they phosphorylate 3-hydroxy-3-methylglutaryl-coenzyme A reductase from Arabidopsis at the inactivating site, serine (Ser)-577. We propose that HRK-A and HRK-C represent either distinct SnRK1 isoforms or the same catalytic subunit complexed with different regulatory subunits. Both kinases also rapidly phosphorylate nitrate reductase purified from spinach, which is associated with inactivation of the enzyme that is observed only in the presence of 14-3-3 protein, a characteristic of phosphorylation at Ser-543. Both kinases also inactivate spinach sucrose phosphate synthase via phosphorylation at Ser-158. The SNF1-related kinases therefore potentially regulate several major biosynthetic pathways in plants: isoprenoid synthesis, sucrose synthesis, and nitrogen assimilation for the synthesis of amino acids and nucleotides.     

The 5A structure of heterologously expressed plant aquaporin SoPIP2;1

SoPIP2;1 is one of the major integral proteins in spinach leaf plasma membranes. In the Xenopus oocyte expression system its water channel activity is regulated by phosphorylation at the C terminus and in the first cytosolic loop. To assess its structure, SoPIP2;1 was heterologously expressed in Pichia pastoris as a His-tagged protein and in the non-tagged form. Both forms were reconstituted into 2D crystals in the presence of lipids. Tubular crystals and double-layered crystalline sheets of non-tagged SoPIP2;1 were observed and analyzed by cryo-electron microscopy. Crystalline sheets were highly ordered and diffracted electrons to a resolution of 2.96A. High-resolution projection maps of tilted specimens provided a 3D structure at 5A resolution. Superposition of the SoPIP2;1 potential map with the atomic model of AQP1 demonstrates the generally well conserved overall structure of water channels. Differences concerning the extracellular loop A explain the particular crystal contacts between oppositely oriented membrane sheets of SoPIP2;1 2D crystals, and may have a function in rapid volume changes observed in stomatal guard cells or mesophyll protoplasts. This crystal packing arrangement provides access to the phosphorylated C terminus as well as the loop B phosphorylation site for studies of channel gating.     

The major integral proteins of spinach leaf plasma membranes are putative aquaporins and are phosphorylated in response to Ca2+ and apoplastic water potential.

We show that homologs of the major intrinsic protein (MIP) family are major integral proteins of the spinach leaf plasma membrane and constitute approximately 20% of integral plasma membrane protein. By using oligonucleotide primers based on partial amino acid sequences for polymerase chain reaction and screening of a spinach leaf cDNA library, we obtained two full-length clones of MIP homologs (pm28a and pm28b). One of these clones, pm28a, was sequenced, and it encodes a protein (PM28A) of 281 amino acids with a molecular mass of 29.9 kD. DNA gel blots indicated that PM28A is the product of a single gene, and RNA gel blots showed that pm28a is ubiquitously expressed in the plant. In vivo phosphorylation of the 28-kD polypeptide(s), corresponding to PM28A and PM28B, was dependent on apoplastic water potential, suggesting a role in regulation of cell turgor for these putative aquaporins. In vitro, only one of the homologs, PM28A, was phosphorylated. Phosphorylation of PM28A occurred on Ser-274, seven amino acids from the C terminus of the protein, within a consensus phosphorylation site (Ser-X-Arg) for vertebrate protein kinase C. In vitro phosphorylation of PM28A was due to a plasma membrane-associated protein kinase and was strictly dependent on submicromolar concentrations of Ca2+.     

Calmodulin-dependent and independent NAD kinase activities from cytoplasmic and chloroplastic fractions of spinach (Spinacia oleracea L.)

NAD kinase activity has been found in a soluble, cytoplasmic fraction and in the chloroplasts prepared from green spinach leaves. A small amount of both the cytoplasmic and the chloroplastic NAD kinase activities was retained on a calmodulin-Sepharose affinity column. The cytoplasmic NAD kinase eluted from the affinity column was found to be enhanced by calmodulin in a Ca²⁺-dependent manner. The chloroplastic enzyme which is located exclusively in the stroma and not in the envelope and thylakoid fractions was not affected by Ca²⁺ and calmodulin. The stromal fraction of purified chloroplasts contained only a negligible amount of calmodulin, most probably due to cytoplasmic contamination. Based on these data, two different mechanisms for the light-dependent modulation of spinach NAD kinase activity are suggested.     

Inhibition of neuronal nitric-oxide synthase by calcium/ calmodulin-dependent protein kinase IIalpha through Ser847 phosphorylation in NG108-15 neuronal cells

We have previously demonstrated that phosphorylation of neuronal nitric-oxide synthase (nNOS) at Ser(847) by Ca(2+)/calmodulin-dependent protein kinases (CaM kinases) attenuates the catalytic activity of the enzyme in vitro (Hayashi Y., Nishio M., Naito Y., Yokokura H., Nimura Y., Hidaka H., and Watanabe Y. (1999) J. Biol. Chem. 274, 20597-20602). In the present study we determined that CaM kinase IIalpha (CaM-K IIalpha) can directly phosphorylate nNOS on Ser(847), leading to a reduction of nNOS activity in cells. The phosphorylation abilities of purified CaM kinase Ialpha (CaM-K Ialpha), CaM-K IIalpha, and CaM-kinase IV (CaM-K IV) on Ser(847) were analyzed using the synthetic peptide nNOS-(836-859) (Glu-Glu-Arg-Lys-Ser-Tyr-Lys-Val-Arg-Phe-Asn-Ser-Val-Ser-Ser-Tyr-Ser- Asp-Ser-Arg-Lys-Ser-Ser-Gly) from nNOS as substrate. The relative V(max)/K(m) ratios of CaM kinases for nNOS-(836-859) were found to be as follows: CaM-K IIalpha, 100; CaM-K Ialpha, 54.5; CaM-K IV, 9.1. Co-transfection of constitutively active CaM-K IIalpha1-274 but not inactive CaM-K IIalpha1-274, generated by mutation of Lys(42) to Ala, with nNOS into NG108-15 cells, resulted in increased Ser(847) phosphorylation in the presence of okadaic acid, an inhibitor of protein phosphatase (PP)1 and PP2A, with a concomitant inhibition of NOS enzyme activity. In addition, this latter decrease could be reversed by treatment with exogenous PP2A. Cells expressing mutant nNOS (S847A) proved resistant to phosphorylation and a decrease of NOS activity. Thus, our results indicate that Ca(2+) triggers cross-talk signal transduction between CaM kinase and NO and CaM-K IIalpha phosphorylating nNOS on Ser(847), which in turn decreases the gaseous second messenger NO in neuronal cells.     

Identification of phosphorylation sites in aminoglycoside phosphotransferase VIII from Streptomyces rimosus

We demonstrate for the first time the role of phosphorylation in the regulation of activities of enzymes responsible for inactivation of aminoglycoside antibiotics. The aminoglycoside phosphotransferase VIII (APHVIII) from the actinobacterial strain Streptomyces rimosus ATCC 10970 is an enzyme regulated by protein kinases. Two serine residues in APHVIII are shown to be phosphorylated by protein kinases from extracts of the kanamycin-resistant strain S. rimosus 683 (a derivative of strain ATCC 10970). Using site-directed mutagenesis and molecular modeling, we have identified the Ser146 residue in the activation loop of the enzyme as the key site for Ca²⁺-dependent phosphorylation of APHVIII. Comparison of the kanamycin kinase activities of the unphosphorylated and phosphorylated forms of the initial and mutant APHVIII shows that the Ser146 modification leads to a 6–7-fold increase in the kanamycin kinase activity of APHVIII. Thus, Ser146 in the activation loop of APHVIII is crucial for the enzyme activity. The resistance of bacterial cells to kanamycin increases proportionally. From the practical viewpoint, our results increase prospects for creation of highly effective test systems for selecting inhibitors of human and bacterial serine/threonine protein kinases based on APHVIII constructs and corresponding human and bacterial serine/threonine protein kinases.     

Involvement of aquaporin in thromboxane A2 receptor-mediated, G12/13/RhoA/NHE-sensitive cell swelling in 1321N1 human astrocytoma cells

The physiological role of the thromboxane A₂ (TXA₂) receptor expressed on glial cells remains unclear. We previously reported that 1321N1 human astrocytoma cells pretreated with dibutyryl cyclic AMP (dbcAMP) became swollen in response to U46619, a TXA₂ analogue. In the present study, we examined the detailed mechanisms of TXA₂ receptor-mediated cell swelling in 1321N1 cells. The cell swelling caused by U46619 was suppressed by expression of p115-RGS, an inhibitory peptide of Gα_12/13 pathway and C3 toxin, an inhibitory protein for RhoA. The swelling was also inhibited by treatment with Y27632, a Rho kinase inhibitor and 5-(ethyl-N-isopropyl)amiloride (EIPA), a Na⁺/H⁺-exchanger inhibitor. Furthermore, cell swelling was suppressed by the pretreatment with aquaporin inhibitors mercury chloride or phloretin in a concentration-dependent manner, suggesting that aquaporins are involved in U46619-induced 1321N1 cell swelling. In fact, U46619 caused [³H]H₂O influx into the cells, which was inhibited by p115-RGS, C3 toxin, EIPA, mercury chloride and phloretin. This is the first report that the TXA₂ receptor mediates water influx through aquaporins in astrocytoma cells via TXA₂ receptor-mediated activation of Gα_12/13, Rho A, Rho kinase and Na⁺/H⁺-exchanger.