1a. Objectives (from AD-416)
Characterize the effect of methionine oxidation on phosphorylation of synthetic peptides. Determine the impact of methionine oxidation on the phosphoproteome in vivo. Develop novel motif antibodies to identify specific phosphoproteins that may be sensitive to methionine oxidation in vivo.
1b. Approach (from AD-416)
We will test the emerging concept that reversible methionine oxidation participates in cellular responses to mild oxidants such as H2O2, by attenuating the phosphorylation of key cellular proteins. To do this, we will employ both in vitro and in vivo approaches in the proposed studies. First, we will build on the observation that methionine oxidation can inhibit peptide phosphorylation by soybean protein kinases in vitro. We will consider canonical and non-canonical motifs targeted by calcium-dependent protein kinases and SNF1-related protein kinases, and also determine the biochemical basis for the inhibition. Second, we will examine the in vivo significance of methionine oxidation using transgenic Arabidopsis with altered expression of methionine sulfoxide repair enzymes (cytosolic PMSRA3 or plastid-targeted MsrB1/2 and PMSRA4), and assess the impact on the leaf and root phosphoproteome. In addition to this broad discovery mode approach to identify phosphoproteins sensitive to methionine oxidation, we will also employ a candidate protein approach. We will specifically focus on selected metabolic enzymes that have a methionine residue within known phosphorylation motifs, such as nitrate reductase and chloroplast elongation factor EF-Tu, which was identified in preliminary studies as an abundant phosphoprotein that is sensitive to methionine oxidation in vivo. In addition, we will also develop targeted-phosphospecific antibodies that may help to identify low abundance proteins that are phosphorylated and are sensitive to oxidative signals in vivo.
3. Progress Report
We are working with three candidate proteins: 1) nitrate reductase (NR); 2) the pyruvate dehydrogenase complex (PDC); and 3) sucrose-phosphate synthase (SPS). Previous results indicated that phosphorylation of NR at the regulatory Ser534 site that is required for binding of a 14-3-3 inhibitor protein was inhibited by oxidation of Met538. To determine whether this occurs in vivo, we are generating transgenic Arabidopsis plants expressing directed mutants of NR in the nia2 null background. Of particular interest are the transgenics expressing the NR (M538L) and (M538Q) directed mutants. We are speculating that the response of these directed mutants to hypoxic conditions in vivo (e.g., flooding) will differ, and if so would provide the most direct evidence that this mechanism plays a role in plant response to abiotic stress. NR is recognized to be important for plant tolerance to low O2 but until now the basis for the activation of the enzyme has been unclear. The other candidate protein for this mechanism is PDC, which contains a conserved Met residue at the +1 position of the regulatory Ser29 (site 1) of the E1a subunit of the complex. When synthetic peptides including site 1 were treated with H2O2, the Met residue was oxidized to methionine sulfoxide (MetSO), and the peptides were no longer phosphorylated by E1a-kinase. Isolated mitochondria were incubated under state III or IV conditions, lysed, the pyruvate dehydrogenase complex (PDC) immunoprecipitated, and tryptic peptides analyzed by MALDI-TOF mass spectrometry. In all instances both Met and MetSO site 1 tryptic-peptides were detected. Similar results were obtained when suspension-cultured cells were incubated with chemical agents known to stimulate production of reactive oxygen species within the mitochondria. Treatment with these agents had no effect upon the amount of total PDC, but decreased the proportion of P-PDC. We propose that the redox-state of the Met residue adjacent to phosphorylation site 1 of pyruvate dehydrogenase contributes to overall regulation of PDC activity in vivo. The third candidate protein that we are exploring is sucrose-phosphate synthase (SPS), which has a known regulatory phosphorylation site (Ser152) that is involved in light-dark modulation of activity. Arabidopsis has four SPS genes, and the three that are predominantly expressed (at the transcript level at least) all have Met residues at key positions in the vicinity of the phosphorylated Ser residue. We have produced homozygous knockouts for each of the four genes and it appears that just one of these knockouts has a phenotype: increased starch production under normal conditions and decreased maximum assimilation capacity at saturating external [CO2]. This will provide the material to test the functional role of Met oxidation of this enzyme in vivo.