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 speculating that reactive oxygen species (ROS) may attenuate brassinosteroid (BR) hormone signaling by interfering with binding of the hormone to its receptor. It is known that BR binds to a specific region of the extracellular domain of the Arabidopsis BRASSINOSTEROID INSENSTIVE 1 (BRI1) receptor kinase that is unusually rich in methionine (Met) residues. Our hypothesis is that oxidation of these Met residue(s) alters the binding affinity of BL thereby modifying BR signaling. Transcriptome analysis of wild type Arabidopsis in response to exogenous hydrogen peroxide treatment followed with a BR treatment revealed that a number of genes were not up-regulated as expected consistent with the notion that BR signaling was altered by the exogenous peroxide. To specifically study the function of the five Met residues in the BRI1 receptor kinase we have generated site-directed mutants that are stably expressed in transgenic Arabidopsis plants. The Met residues have been substituted with the non-oxidizable hydrophobic residue, leucine, or the polar residue, glutamine, which is a mimic for oxidized form of Met. The impact of the directed mutations on BR signaling is being examined using several physiological and molecular read-outs of the signaling pathway. However, it is clear that one of the directed mutants (M661Q) shows no inhibition of root growth in response to exogenous BR, and another (M665Q) has enhanced overall plant growth suggesting that oxidation of specific Met residues in the hormone binding site can enhance or inhibit hormone binding.