Coupling oxidative signals to protein phosphorylation via methionine oxidation in Arabidopsis.

The mechanisms involved in sensing oxidative signalling molecules, such as H2O2, in plant and animal cells are not completely understood. In the present study, we tested the postulate that oxidation of Met (methionine) to MetSO (Met sulfoxide) can couple oxidative signals to changes in protein phosphorylation. We demonstrate that when a Met residue functions as a hydrophobic recognition element within a phosphorylation motif, its oxidation can strongly inhibit peptide phosphorylation in vitro. This is shown to occur with recombinant soybean CDPKs (calcium-dependent protein kinases) and human AMPK (AMP-dependent protein kinase). To determine whether this effect may occur in vivo, we monitored the phosphorylation status of Arabidopsis leaf NR (nitrate reductase) on Ser534 using modification-specific antibodies. NR was a candidate protein for this mechanism because Met538, located at the P+4 position, serves as a hydrophobic recognition element for phosphorylation of Ser534 and its oxidation substantially inhibits phosphorylation of Ser534 in vitro. Two lines of evidence suggest that Met oxidation may inhibit phosphorylation of NR-Ser534 in vivo. First, phosphorylation of NR at the Ser534 site was sensitive to exogenous H2O2 and secondly, phosphorylation in normal darkened leaves was increased by overexpression of the cytosolic MetSO-repair enzyme PMSRA3 (peptide MetSO reductase A3). These results are consistent with the notion that oxidation of surface-exposed Met residues in kinase substrate proteins, such as NR, can inhibit the phosphorylation of nearby sites and thereby couple oxidative signals to changes in protein phosphorylation.

Light and metabolic signals control the selective degradation of sucrose synthase in maize leaves during deetiolation.

The content and activity of Suc (Suc) synthase (SUS) protein is high in sink organs but low in source organs. In this report, we examined light and metabolic signals regulating SUS protein degradation in maize (Zea mays) leaves during deetiolation. We found that SUS protein accumulated in etiolated leaves of the dark-grown seedlings but was rapidly degraded upon exposure to white, blue, or red light. This occurred concurrent with the accumulation of photosynthetic enzymes, such as Rubisco and Rubisco activase, and enzymes of Suc biosynthesis such as Suc-phosphate synthase. Deetiolation-induced SUS degradation was not inhibited by the proteasome inhibitor MG132. Moreover, neither full-length nor truncated SUS phosphorylated at the serine-170 site was found in the crude 26S proteasome fraction (150,000g postmicrosomal pellet) isolated in the presence of MG132. However, SUS degradation was strongly inhibited by feeding cycloheximide or amino acids to detached leaves, while Suc feeding had no effect. Of the amino acids tested, exogenous glutamate had the greatest effect. Collectively, these results demonstrate that SUS protein degradation during deetiolation: (1) is selective; (2) can be triggered by either blue- or red light-mediated signaling pathways; (3) does not involve the 26S proteasome; and (4) is inhibited by free amino acids. These findings suggest that SUS degradation is important to supply residues for the synthesis of other proteins required for autotrophic metabolism.

The three maize sucrose synthase isoforms differ in distribution, localization, and phosphorylation.

Although sucrose synthase (SUS) is widely appreciated for its role in plant metabolism and growth, very little is known about the contribution of each of the SUS isoforms to these processes. Using isoform-specific antibodies, we evaluated the three known isoforms individually at the protein level. SUS1 and SUS-SH1 proteins have been studied previously; however, SUS2 (previously known as SUS3) has only been studied at the transcript level. Using SUS2 isoform-specific antibodies, we determined that this isoform is present in several maize tissues. The intracellular localization of all SUS isoforms was studied by cellular fractionation of leaves and developing kernels. Interestingly, SUS1 and SUS-SH1 were associated with membranes while SUS2 was not. The lack of membrane-associated SUS2 indicates that it might have a unique role in cytoplasmic sucrose metabolism. Using co-immunoprecipitation with kernel extracts, it was also established that SUS2 exists predominantly as a hetero-oligomer with SUS1, while SUS-SH1 forms only homo-oligomers. Using sequence-specific and phospho-specific antibodies, we have established for the first time that SUS-SH1 is phosphorylated in vivo at the Ser10 site in kernels, similar to the SUS1 Ser15 site. In midveins, additional evidence suggests that SUS can be phosphorylated at a novel C-terminal threonine site. Together, these results show that the isoforms of SUS are important in both cytosolic and membrane-associated sucrose degradation, but that their unique attributes most probably impart isoform-specific functional roles.

Determination of structural requirements and probable regulatory effectors for membrane association of maize sucrose synthase 1.

Sucrose (Suc) synthase (SUS) cleaves Suc to form UDP glucose and fructose, and exists in soluble and membrane-associated forms, with the latter proposed to channel UDP glucose to the cellulose-synthase complex on the plasma membrane of plant cells during synthesis of cellulose. However, the structural features responsible for membrane localization and the mechanisms regulating its dual intracellular localization are unknown. The maize (Zea mays) SUS1 isoform is likely to have the intrinsic ability to interact directly with membranes because we show: (1) partial membrane localization when expressed in Escherichia coli, and (2) binding to carbonate-stripped plant microsomes in vitro. We have undertaken mutational analyses (truncations and alanine substitutions) and in vitro microsome-binding assays with the SUS1 protein to define intrinsic membrane-binding regions and potential regulatory factors that could be provided by cellular microenvironment. The results suggest that two regions of SUS1 contribute to membrane affinity: (1) the amino-terminal noncatalytic domain, and (2) a region with sequence similarity to the C-terminal pleckstrin homology domain of human pleckstrin. Alanine substitutions within the pleckstrin homology-like domain of SUS1 reduced membrane association in E. coli and with plant microsomes in vitro without reducing enzymatic activity. Microsomal association of wild-type SUS1 displayed cooperativity with SUS1 protein concentration and was stimulated by both lowering the pH and adding Suc. These studies offer insight into the molecular level regulation of SUS1 localization and its participation in carbon partitioning in plants. Moreover, transgenics with active SUS mutants altered in membrane affinity may be of technological utility.

Identification of a new motif for CDPK phosphorylation in vitro that suggests ACC synthase may be a CDPK substrate.

1-Amino-cyclopropane-1-carboxylate synthase (ACS) catalyzes the rate-determining step in the biosynthesis of the plant hormone ethylene, and there is evidence for regulation of stability of the protein by reversible protein phosphorylation. The site of phosphorylation of the tomato enzyme, LeACS2, was recently reported to be Ser460, but the requisite protein kinase has not been identified. In the present study, a synthetic peptide based on the known regulatory phosphorylation site (KKNNLRLS460FSKRMY) in LeACS2 was found to be readily phosphorylated in vitro by several calcium-dependent protein kinases (CDPKs), but not a plant SNF1-related protein kinase or the kinase domain of the receptor-like kinase, BRI1, involved in brassinosteroid signaling. Studies with variants of the LeACS2-Ser460 peptide establish a fundamentally new phosphorylation motif that is broadly targeted by CDPKs: phi -1-[ST]0- phi +1-X-Basic+3-Basic+4, where phi is a hydrophobic residue. Database analysis using the new motif predicts a number of novel phosphorylation sites in plant proteins. Finally, we also demonstrate that CDPKs and SnRK1s do not recognize motifs presented in the reverse order, indicating that side chain interactions alone are not sufficient for substrate recognition.

Numerous posttranslational modifications provide opportunities for the intricate regulation of metabolic enzymes at multiple levels.

The metabolic plasticity displayed by plants during normal development, and in response to environmental fluctuations and stressors, is essential for their growth and survival. The capacity to regulate metabolic enzymes intricately arises in part from posttranslational modifications that can affect enzymatic activity, intracellular localization, protein-protein interactions, and stability. Protein phosphorylation and thiol/disulfide redox modulation are important modifications in plants, and it is likely that O-glycosylation and S-nitrosylation will also emerge as important mechanisms. Recent advances in the field of proteomics, in particular the development of novel and specific chemistries for the detection of a diverse number of modifications, are rapidly expanding our awareness of possible modifications and our understanding of the enzymes whose functions are likely to be regulated posttranslationally.

Phosphorylation of the amino terminus of maize sucrose synthase in relation to membrane association and enzyme activity.

Sucrose synthase (SUS) is phosphorylated on a major, amino-terminal site located at Ser-15 (S15) in the maize (Zea mays) SUS1 protein. Site- and phospho-specific antibodies against a phosphorylated S15 (pS15) peptide allowed direct analysis of S15 phosphorylation in relation to membrane association. Immunoblots of the maize leaf elongation zone, divided into 4-cm segments, demonstrated that the abundance of soluble (s-SUS) and membrane (m-SUS) SUS protein showed distinct positional profiles. The content of m-SUS was maximal in the 4- to 8-cm segment where it represented 9% of total SUS and occurred as a peripheral membrane protein. In contrast, s-SUS was highest in the 12- to 16-cm segment. Relative to s-SUS, m-SUS was hypophosphorylated at S15 in the basal 4 cm but hyperphosphorylated in apical segments. Differing capabilities of the anti-pS15 and anti-S15 peptide antibodies to immunoprecipitate SUS suggested that phosphorylation of S15, or exposure of unphosphorylated SUS to slightly acidic pH, altered the structure of the amino terminus. These structural changes were generally coincident with the increased sucrose cleavage activity that occurs at pH values below 7.5. In vitro S15 phosphorylation of the S170A SUS protein by a maize calcium-dependent protein kinase (CDPK) significantly increased sucrose cleavage activity at low pH. Collectively, the results suggest that (1) SUS membrane binding is controlled in vivo; (2) relative pS15 content of m-SUS depends on the developmental state of the organ; and (3) phosphorylation of S15 affects amino-terminal conformation in a way that may stimulate the catalytic activity of SUS and influence membrane association.

Proteasome activity and the post-translational control of sucrose synthase stability in maize leaves.

The serine-170 (S170) calcium-dependent protein kinase phosphorylation site of maize (Zea mays L.) sucrose synthase (SUS) (EC 2.4.1.13) has been implicated in the post-translational regulation of SUS protein stability. To clarify the proteolytic process and the role of phosphorylation, SUS degradation and proteasome activities were studied in the maize leaf elongation zone. Size-exclusion chromatography resolved two peaks of proteasome-like proteolytic activity. The large molecular mass ( approximately 1350 kDa) peak required Mg(2+) and ATP for maximal activity and was inhibited by the proteasome inhibitors MG132 and NLVS. Anion-exchange chromatography resolved a similar proteolytic activity that was activated by ATP, characteristics that are consistent with those of a 26S-proteasome. Appropriately, immunoblotting revealed the presence of a 26S-proteasome subunit and highly ubiquitinated proteins within the active fractions eluted from both columns. The smaller molecular mass ( approximately 600 kDa) peak represented only 40% of the total proteasome-like activity and is likely a maize 20S-proteasome as it was activated in vitro by low levels of sodium dodecyl sulfate (SDS). S170 phosphorylated SUS (pS170-SUS) was detected as both high molecular mass (HMM) forms and proteolytic fragments that co-eluted with 26S-proteasome activities on both size-exclusion and anion-exchange columns. Conditions that maintained maximal 26S-proteasome activity reduced the amounts of pS170-SUS recovered. In vitro, the 26S-proteasome degraded SUS and proteasome-specific inhibitors reduced SUS proteolysis. HMM-SUS conjugates were produced in vitro and immunoprecipitations suggested that some SUS might be ubiquitinated in vivo. The results suggest that S170 phosphorylation promotes the formation of HMM, ubiquitin-SUS conjugates that can be targeted for 26S-proteasome-dependent degradation.

Phosphorylation of sucrose synthase at serine 170: occurrence and possible role as a signal for proteolysis.

Sequence analysis identified serine 170 (S170) of the maize (Zea mays L.) SUS1 sucrose synthase (SUS) protein as a possible, second phosphorylation site. Maize leaves contained two calcium-dependent protein kinase activities and a calcium-independent kinase activity with characteristics of an sucrose non-fermenting 1 (SNF1)-related protein kinase. Phosphorylation of the novel S170 and the known serine 15 (S15) site by these protein kinases was determined in peptide substrates and detected in SUS1 protein substrates utilizing sequence- and phosphorylation-specific antibodies. We demonstrate phosphorylation of S170 in vitro and in vivo. The calcium-dependent protein kinases phosphorylated both S170 and S15, whereas SNF1-related protein kinase activity was restricted to S15. Calcium-dependent protein-kinase-mediated S170 and S15 phosphorylation kinetics were determined in wild-type and mutant SUS1 substrates. These analyses revealed that kinase specificity for S170 was threefold lower than that for S15, and that phosphorylation of S170 was stimulated by prior phosphorylation at the S15 site. The SUS-binding peptides encoded by early nodulin 40 (ENOD40) specifically antagonized S170 phosphorylation in vitro. A model wherein S170 phosphorylation functions as part of a mechanism targeting SUS for proteasome-mediated degradation is supported by the observations that SUS proteolytic fragments: (i) were detected and possessed relatively high phosphorylated-S170 (pS170) stoichiometry; (ii) were spatially coincident with proteasome activity within developing leaves; and (iii) co-sedimented with proteasome activity. In addition, full-length pS170-SUS protein was less stable than S170-SUS in cultured leaf segments and was stabilized by proteasome inhibition. Post-translational control of SUS protein level through pS170-promoted proteolysis may explain the specific and significant decrease in SUS abundance that accompanies the sink-to-source transition in developing maize leaves.

A novel C-terminal proteolytic processing of cytosolic pyruvate kinase, its phosphorylation and degradation by the proteasome in developing soybean seeds.

Cytosolic pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40) is an important glycolytic enzyme, but the post-translational regulation of this enzyme is poorly understood. Sequence analysis of the soybean seed enzyme suggested the potential for two phosphorylation sites: site-1 (FVRKGS220DLVN) and site-2 (VLTRGGS407TAKL). Sequence- and phosphorylation state-specific antipeptide antibodies established that cytosolic pyruvate kinase (PyrKinc) is phosphorylated at both sites in vivo. However, by SDS-PAGE, the phosphorylated polypeptides were found to be smaller (20-51 kDa) than the full length (55 kDa). Biochemical separations of seed proteins by size exclusion chromatography and sucrose-density gradient centrifugation revealed that the phosphorylated polypeptides were associated with 26S proteasomes. The 26S proteasome particle in developing seeds was determined to be of approximately 1900 kDa. In vitro, the 26S proteasome degraded associated PyrKinc polypeptides, and this was blocked by proteasome-specific inhibitors such as MG132 and NLVS. By immunoprecipitation, we found that some part of the phosphorylated PyrKinc was conjugated to ubiquitin and shifted to high molecular mass forms in vivo. Moreover, recombinant wild-type PyrKinc was ubiquitinated in vitro to a much greater extent than the S220A and S407A mutant proteins, suggesting a link between phosphorylation and ubiquitination. In addition, during seed development, a progressive accumulation of a C-terminally truncated polypeptide of approximately 51 kDa was observed that was in parallel with a loss of the full-length 55 kDa polypeptide. Interestingly, the C-terminal 51 kDa truncation showed not only pyruvate kinase activity but also activation by aspartate. Collectively, the results suggest that there are two pathways for PyrKinc modification at the post-translational level. One involves partial C-terminal truncation to generate a 51 kDa pyruvate kinase subunit which might have altered regulatory properties and the other involves phosphorylation and ubiquitin conjugation that targets the protein to the 26S proteasome for complete degradation.