title: Quantitative phosphoproteomic analysis of plasma membrane proteins reveals regulatory mechanisms of plant innate immune responses.
abstract: Advances in proteomic techniques have allowed the large-scale identification of phosphorylation sites in complex protein samples, but new biological insight requires an understanding of their in vivo dynamics. Here, we demonstrate the use of a stable isotope-based quantitative approach for pathway discovery and structure-function studies in Arabidopsis cells treated with the bacterial elicitor flagellin. The quantitative comparison identifies individual sites on plasma membrane (PM) proteins that undergo rapid phosphorylation or dephosphorylation. The data reveal both divergent dynamics of different sites within one protein and coordinated regulation of homologous sites in related proteins, as found for the PM H(+)-ATPases AHA1, 2 and 3. Strongly elicitor-responsive phosphorylation sites may reflect direct regulation of protein activity. We confirm this prediction for RbohD, an NADPH oxidase that mediates the rapid production of reactive oxygen species (ROS) in response to elicitors and pathogens. Plant NADPH oxidases are structurally distinct from their mammalian homologues, and regulation of the plant enzymes is poorly understood. On RbohD, we found both unchanging and strongly induced phosphorylation sites. By complementing an RbohD mutant plant with non-phosphorylatable forms of RbohD, we show that only those sites that undergo differential regulation are required for activation of the protein. These experiments demonstrate the potential for use of quantitative phosphoproteomics to determine regulatory mechanisms at the molecular level and provide new insights into innate immune responses.
authors.affiliation: The Sainsbury Laboratory, Norwich Research Park, Colney Lane, Norwich, UK.
authors.forename: Thomas S
authors.initials: TS
authors.lastname: Nühse
authors.forename: Andrew R
authors.initials: AR
authors.lastname: Bottrill
authors.forename: Alexandra M E
authors.initials: AM
authors.lastname: Jones
authors.forename: Scott C
authors.initials: SC
authors.lastname: Peck
mesh_terms: D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000276:immunology* / Q000378:metabolism; D005408:Flagellin / Q000276:immunology*; D007113:Immunity, Innate / Q000502:physiology*; D007553:Isotope Labeling; D008565:Membrane Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D019255:NADPH Oxidases / Q000378:metabolism; D010766:Phosphorylation; D010770:Phosphotransferases / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology
chemical_list: D008565:Membrane Proteins; D017382:Reactive Oxygen Species; D005408:Flagellin; D019255:NADPH Oxidases; D010770:Phosphotransferases