Project Number: 3611-21000-024-01
Project Type: Reimbursable

Start Date: Dec 01, 2007
End Date: Nov 30, 2011

Objective:
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.

Approach:
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.