|Hardin, Shane -|
|Oh, Man Ho|
|Jain, Vanita -|
Submitted to: Biochemical Journal
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: June 16, 2009
Publication Date: July 1, 2009
Citation: Hardin, S.C., Larue, C.T., Oh, M., Huber, S.C., Jain, V. 2009. Coupling Oxidative Signals to Protein Phosphorylation via Methionine Oxidation in Arabidopsis. Biochemical Journal. 422(2):305-312. Interpretive Summary: Plants and animals use molecules such as hydrogen peroxide, which is a mild oxidant, as signaling molecules to regulate many aspects of cellular function. However, it is not completely known how peroxide molecules function at the biochemical level. One effect of peroxide could be the oxidation of methionine residues in proteins. In the present study, we tested the postulate that methionine oxidation in proteins could affect the modification of nearby residues by a process known as phosphorylation, which involves the covalent attachment of of a phosphate molecule to proteins by enzymes known as protein kinases. The potential for this to occur was demonstrated in vitro using plant protein kinases and synthetic peptides containing methionine residues as substrates. Importantly, compelling evidence suggests that phosphorylation of nitrate reductase, which is a key enzyme of nitrate assimilation, may be inhibited by peroxide in vivo. These results establish a new mechanism to explain how protein phosphorylation may be regulated in crop plants, and in particular may provide a missing link between stress conditions, which all produce peroxide, and plant response to the stress.
Technical Abstract: The mechanisms involved in sensing oxidative signaling molecules such as H2O2 in plant and animal cells are not completely understood. In the present study, we tested 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) in vitro. To determine whether this effect may occur in vivo, we monitored the phosphorylation status of Arabidopsis leaf nitrate reductase (NR) Ser534 using modification-specific antibodies. NR was a candidate protein for this mechanism because Met538, located at the P + 4 position, serves as a hydrophobic recognition element for phosphorylation of Ser534 and its oxidation substantially inhibits phosphorylation of Ser534 in vitro. Two lines of evidence suggest that Met oxidation may inhibit phosphorylation of NR-Ser534 in vivo. First, feeding exogenous H2O2 to Arabidopsis seedlings in liquid culture increased phosphorylation of NR at the Ser534 site at low H2O2 concentrations (5 mM) and then progressively inhibited phosphorylation as the concentration of H2O2 was increased. The effect was selective, as activation of MAPKs and phosphorylation of the proteome generally remained high at concentrations of H2O2 that reduced NR phosphorylation. Second, in experiments with plants grown in soil, overexpression of the cytosolic MetSO-repair enzyme, PMSRA3, increased phosphorylation of NR at the Ser534 site in darkened leaves compared to wild type plants. 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.