An Arabidopsis Protein 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

doi:10.1074/jbc.m800735200

Abstract

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.

Highlights

microbial-associated molecular patterns (MAMPs) perception by defined host cell surface receptors initiates MAMP-triggered immunity, the first line of defense contributing to the arrest of microbial growth
mitogen-activated protein kinases (MAPKs) are the final components of signaling cascades, activated by upstream MAPK kinases (MAPKKs), which are activated by other upstream kinases, MAPKK kinases (MAPKKKs) (6 – 8)
MAPKs in plants have been demonstrated to be tions, demonstrating that the nickel binding activity by which activated in response to many biotic and abiotic stresses [8], we these proteins were initially identified is intrinsic to the protein. currently know very little about the substrates of this family of

Summary

Introduction

MAMP perception by defined host cell surface receptors initiates MAMP-triggered immunity, the first line of defense contributing to the arrest of microbial growth. 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.

Results

Conclusion