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

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.Plants recognize potential microbial pathogens through microbial-associated molecular patterns (MAMPs; 5 1, 2).MAMP perception by defined host cell surface receptors initiates MAMP-triggered immunity, the first line of defense contributing to the arrest of microbial growth. The best characterized MAMP perception system in plants is the recognition of bacterial flagellin or flg22, the bioactive peptide from a highly conserved 22-amino-acid region of the protein (3), by the receptor kinase flagellin-sensitive 2 (FLS2) (4). Recognition of flg22 by FLS2 initiates intracellular signaling that results in a number of rapid responses thought to contribute to defense(s) against the invading microbe (5).Among the most studied of these early responses is the rapid activation of mitogen-activated protein kinases (MAPKs). MAPKs are the final components of signaling cascades, activated by upstream MAPK kinases (MAPKKs), which are also activated by other upstream kinases, MAPKK kinases (MAPKKKs) (6 -8). In Arabidopsis, flg22 has been shown to activate two MAPKs, AtMPK3 and AtMPK6 (9, 10). Moreover, plants with reduced levels of AtMPK6 displayed increased susceptibility to infection by virulent and avirulent strains of the bacterial pathogen, Pseudomonas syringae, indicating that signaling via AtMPK6 plays a critical role during defense responses (11).Although activation of MAPKs in response to a wide range of biotic and abiotic stimuli has been well characterized (8), relatively little is known about the substrates of these kin...

Mitogen-Activated Protein Kinases 3 and 6 Are Required for Full Priming of Stress Responses inArabidopsis thaliana

“…This may explain why knockout of only one of the two genes encoding MPK3 or MPK6 doesn't always attenuate IR (Figures 6A and 6B). Indeed, physiological interaction (Miles et al, 2005), substrate overlap (Feilner et al, 2005;Merkouropoulos et al, 2008), and overlapping functions (Wang et al, 2008) have been shown for MPK3 and MPK6. Moreover, the tobacco orthologs of Arabidopsis MPK3 and MPK6, WIPK and SA-induced protein kinase, interact with each other in planta (Liu et al, 2003).…”

Phosphorylation of the Nicotiana benthamiana WRKY8 Transcription Factor by MAPK Functions in the Defense Response

“…Similarly, SIPK/NTF4/WIPK-silenced plants showed a greater increase in susceptibility than WRKY8-silenced plants (Figure 4). Redundancy of genes encoding plant transcription factors complicates loss-of-function analyses that aim to identify the biological functions of large families that contain members with strongly conserved DNA binding domains (Mitsuda and Ohme-Takagi, 2009 (Lampard et al, 2008;Merkouropoulos et al, 2008;Yoo et al, 2008). Extensive in vitro analysis showed that MPK6 shares 40% of in vitro substrates with MPK3 (Popescu et al, 2009).…”

Phosphorylation of Arabidopsis MAP Kinase Phosphatase 1 (MKP1) Is Required for PAMP Responses and Resistance against Bacteria

“…All subsequent steps for IP were performed at 4°C. Homogenates were centrifuged at 16,000g for 15 min, and 1 mL of the resulting supernatant was mixed with 10 mL of MPK6-protein A Sepharose beads (Sigma) prepared by preincubating the beads with 1.5 mL of a polyclonal a-MPK6 antibody (Merkouropoulos et al, 2008) in IP buffer for 1.5 h. After 2 h, the beads were washed three times with IP buffer and once with kinase buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM MnCl 2 , 10 mM MgCl 2 , 10% glycerol, and 1 mM DTT). For kinase assays, purified MKP1 protein or MBP (Sigma) was mixed with a 10-mL aliquot of MPK6-bound protein A Sepharose beads, 0.3 mL of [ 32 P]g-ATP, and 1 mL of 250 mM ATP in a total reaction volume of 25 mL of kinase buffer.…”

“…Otherwise, protein extracts were separated using 8% mini-format (8.3 3 7.3 cm) SDS-PAGE. Proteins were transferred to PVDF, and immunoblotting was performed using primary mouse anti-myc antibody (Cell Signaling Technologies, Fisher) for detection of myc-tagged MKP1 protein, rabbit anti-phospho-p44/42 MAPK antibody (Cell Signaling Technologies) for detection of active MPK3/6, rabbit anti-Glu-Glu (EMD, Millipore) antibody for detection of Pyotagged MKP1, or previously described rabbit anti-MPK6 antibody for detection of MPK6 (Merkouropoulos et al, 2008). Chemiluminescence-based detection (Pierce) was performed using horseradish peroxidase-conjugated goat anti-rabbit antibody (Cell Signaling Technologies) or goat anti-mouse antibody (Sigma).…”