An Arabidopsis protein phosphorylated in response to microbial elicitation, AtPHOS32, is a substrate of MAP kinases 3 and 6.
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 flg22 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.
Merkouropoulos
Georgios
G
The Sainsbury Laboratory, John Innes Centre, Colney Lane, Norwich NR4 7UH, United Kingdom.
Andreasson
Erik
E
Hess
Daniel
D
Boller
Thomas
T
Peck
Scott C
SC
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000378:metabolism* / Q000382:microbiology; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology*; D017124:Conserved Sequence; D019143:Evolution, Molecular; D005408:Flagellin / Q000378:metabolism; D013058:Mass Spectrometry; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism*; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D008954:Models, Biological; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010750:Phosphoproteins / Q000737:chemistry / Q000502:physiology*; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid
D029681:Arabidopsis Proteins; C529267:PHOS32 protein, Arabidopsis; C529268:PHOS34 protein, Arabidopsis; D010750:Phosphoproteins; D005408:Flagellin; C458516:AtMPK3 protein, Arabidopsis; C436293:MPK6 protein, Arabidopsis; D020928:Mitogen-Activated Protein Kinases; D020929:Mitogen-Activated Protein Kinase Kinases