2009 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.
The mechanisms involved in sensing oxidative signaling molecules such as H2O2 in plant and animal cells are not completely understood. We are testing the postulate that oxidation of methionine (Met) to Met sulfoxide (MetSO) 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 calcium dependent protein kinases (CDPKs) and human AMP-dependent protein kinase (AMPK). To determine whether this effect may occur in vivo, we monitored the phosphorylation status of Arabidopsis leaf nitrate reductase (NR) serine-534 using modification-specific antibodies. NR was a candidate protein for this mechanism because methionine-538, located at the P + 4 position, serves as a hydrophobic recognition element for phosphorylation of serine-534 and its oxidation substantially inhibits phosphorylation of serine-534 in vitro. Two lines of evidence suggest that Met oxidation may inhibit phosphorylation of serine-534 in vivo. First, phosphorylation of NR at the serine-534 site was sensitive to exogenous H2O2 and second, phosphorylation in normal darkened leaves was increased by overexpression of the cytosolic MetSO-repair enzyme, PMSRA3. 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.