2008 Annual Report
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.
Homozygous knockouts have been isolated for genes encoding several methionine sulfoxide reductase genes and seed supply is being increased. These plants, along with transgenics over expressing individual genes, will be used to evaluate the possible impact of methionine oxidation on protein phosphorylation in various cellular compartments. In addition, several candidate proteins have been identified that will be specifically examined for effects of oxidative signals on protein phosphorylation. Specific antibody tools are currently being developed. This work relates to NP302 Component 2A: Understanding plant growth and development, and Component 2B: Understanding plant interactions with the environment.