Coupling oxidative signals to protein phosphorylation via methionine oxidation in Arabidopsis

Abstract

The mechanisms involved in sensing oxidative signalling molecules, such as H2O2, in plant and animal cells are not completely understood. In the present study, we tested the postulate that oxidation of Met (methionine) to MetSO (Met sulfoxide) 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 CDPKs (calcium-dependent protein kinases) and human AMPK (AMP-dependent protein kinase). To determine whether this effect may occur in vivo, we monitored the phosphorylation status of Arabidopsis leaf NR (nitrate reductase) on 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, phosphorylation of NR at the Ser534 site was sensitive to exogenous H2O2 and secondly, phosphorylation in normal darkened leaves was increased by overexpression of the cytosolic MetSO-repair enzyme PMSRA3 (peptide MetSO reductase A3). 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.

Reactive oxygen species and plant resistance to fungal pathogens

“…ROS can affect PTMs by at least two mechanisms: H 2 O 2 can modify the cysteine residues producing a sulfenic acid (-SOH) by the chemical reaction called sulfenylation (Scheler et al, 2013) and oxidation of methionine residues within a phosphorylation motif can inhibit phosphorylation of neighbouring peptides (Hardin et al, 2009). During the symbiotic interaction between Medicago truncatula and Sinorhizobium meliloti multiple proteins involved in nitrogen fixation are sulfenylated, highlighting the importance of ROS in the establishment and development of this plant-biotic interaction (Oger et al, 2012).…”

Convergent signaling pathways—interaction between methionine oxidation and serine/threonine/tyrosine O-phosphorylation

“…However, the common occurrence of Met adjacent to Ser/ Thr/Tyr residues (Rao et al 2013) and the frequent occurrence of MetSO (Ghesquière et al 2011;Liu et al 2013;Salvato et al 2014) near O-phosphorylation-sites (Song et al 2012) suggests that crosstalk between these two PTM's is widespread. (Hardin et al 2009;Miernyk et al 2009), oxidation of Met might directly inhibit O-phosphorylation of a nearby Ser/Thr/Tyr-residue by interfering with the kinase recognition/binding (Fig. 1).…”

“…Furthermore, msrA-null mutants showed lower degradation of α-synuclein protein. Two studies (Hardin et al 2009;Miernyk et al 2009) have specifically tested the effect of Met oxidation on the subsequent phosphorylation of nearby Ser residues. Using a multiple combinations of synthetic peptides and recombinant kinases, Hardin et al (2009) found that Met oxidation strongly inhibits phosphorylation in vitro.…”

“…Two studies (Hardin et al 2009;Miernyk et al 2009) have specifically tested the effect of Met oxidation on the subsequent phosphorylation of nearby Ser residues. Using a multiple combinations of synthetic peptides and recombinant kinases, Hardin et al (2009) found that Met oxidation strongly inhibits phosphorylation in vitro. They additionally found evidence for an in vivo effect of Met oxidation on the Ser534 phosphorylation of Arabidopsis thaliana leaf nitrate reductase (NR).…”

Advances in purification and separation of posttranslationally modified proteins

“…Sulfoxide formation can be enzymatically reversed by the action of specific reductases and it has been suggested that methionine could serve as an antioxidant that protects other amino acid residues from more deleterious damage [111]. While this possibility cannot be excluded, it has been shown that methionine oxidation can result in loss of a protein function or interference with other PTMs close to the oxidized site [112,113]. Although methionine oxidation is detected in biological samples relatively often, thorough proteomic methodology for its characterization is still in development.…”