AnArabidopsisProtein Phosphorylated in Response to Microbial Elicitation, AtPHOS32, Is a Substrate of MAP Kinases 3 and 6

Georgios Merkouropoulos, Erik Andreasson, Daniel Hess, Thomas Boller, Scott C. Peck
2008 Journal of Biological Chemistry  
Although mitogen-activated protein kinases (MAPKs) have been shown to be activated by a wide range of biotic and abiotic stimuli in diverse plant species, few in vivo substrates for these kinases have been identified. While studying proteins that are differentially phosphorylated upon treatment of Arabidopsis suspension cultures with the general bacterial elicitor peptide flagellin-22 (flg22), we identified two proteins with endogenous nickel binding properties that become phosphorylated after
more » ... lg22 elicitation. These highly related proteins, AtPHOS32 and AtPHOS34, show similarity to bacterial universal stress protein A. We identified one of the phosphorylation sites on AtPHOS32 by nanoelectrospray ionization tandem mass spectrometry. Phosphorylation in a phosphoSer-Pro motif indicated that this protein may be a substrate of MAPKs. Using in vitro kinase assays, we confirmed that AtPHOS32 is a substrate of both AtMPK3 and AtMPK6. Specificity of phosphorylation was demonstrated by site-directed mutagenesis of the first phosphorylation site. In addition, immunosubtraction of both MAPKs from protein extracts removed detectable kinase activity toward AtPHOS32, indicating that the two MAPKs were the predominate kinases recognizing the motif in this protein. Finally, the target phosphorylation site in AtPHOS32 is conserved in AtPHOS34 and among apparent orthologues from many plant species, indicating that phosphorylation of these proteins by AtMPK3 and AtMPK6 orthologues has been conserved throughout evolution. Plants recognize potential microbial pathogens through microbial-associated molecular patterns (MAMPs; 5 1, 2). . 5 The abbreviations used are: MAMP, microbial-associated molecular pattern; MALDI-TOF, matrix-assisted laser desorption ionization-time of flight; MS, mass spectrometry; nanoESI, nano-electrospray ionization; nanoESI-MS/ MS, nanoESI tandem mass spectrometry; MAP, mitogen-activated protein; MAPK, mitogen-activated protein kinase; USPA, universal stress protein A; Ni-NTA, nickel-nitrilotriacetic acid.
doi:10.1074/jbc.m800735200 pmid:18285339 fatcat:o52sltrkjzhopgwbugjmxpkzpq