Novel MAP kinase substrates identified by solid-phase phosphorylation screening in Arabidopsis thaliana

AtMPK6‐induced phosphorylation of AtERF72 enhances its DNA binding activity and interaction with TGA4/OBF4 in Arabidopsis

“…We previously identified 29 putative substrates of rice blast‐ and wound‐induced MAP kinase1 (OsBWMK1) and several transcription factors and transcriptional regulators as potential AtMPK substrates in Arabidopsis (Park et al 2016). Among these substrates, AtERF72 functions as a transcriptional activator of the ethylene and defence response pathway, and overexpression of AtERF72 is associated with resistance to heat and oxidative stress treatments (Allen et al 1998; Ogawa et al 2005).…”

“…A total of 10 μg of total proteins extracted from wild‐type plants treated by wounding or with 1 m m H 2 O 2 were separated on 10% SDS polyacrylamide gels embedded with either MBP, GST, GST‐AtERF72, GST‐AtERF72 T146A or GST‐AtERF72 S151A as MPK substrate. Protein denaturation, renaturation and kinase activity in gels were performed as described previously (Park et al 2016). For termination of the kinase reaction, the gel was washed five times with washing solution (5% TCA [Trichloroacetic acid] and 1% pyrophosphoric acid) and once with 5% glycerol.…”

“…After each treatment, plants were immediately frozen in liquid nitrogen and used for protein extraction. Protein extracts were prepared as described previously (Park et al 2016).…”

“…We previously isolated AtERF72 as a common substrate for AtMPK3, AtMPK4 and AtMPK6 by solid-phase phosphorylation screening using a lambda phage expression library constructed from a mixture of mRNAs isolated from Arabidopsis seedlings treated with salt, pathogen and mechanical wounding stress (Park et al 2016). However, the phosphorylation of AtERF72 was significantly decreased in atmpk6 mutant plants (Park et al 2016), indicating that AtERF72 is a major target substrate of AtMPK6 in the plant. Therefore, we focused on the phosphorylation effect on the activity of AtERF72 by AtMPK6 in this study.…”

“…Subsequently, various Arabidopsis proteins, including MKS1 (Andreasson et al 2005), MKP1 (Park et al 2011), WRKY1 (Menke et al 2005), EIN3 (Yoo et al 2008), VIP1 (Djamei et al 2007), PPS3 (Katou et al 2005), AtPHOS32 (Merkouropoulos et al 2008) and MAP65‐1 (Sasabe et al 2006), were identified as MPK substrates. Recently, a large number of plant MPK substrates has been reported using high‐throughput approaches (Feilner et al 2005; Popescu et al 2009; Sörensson et al 2012; Hoehenwarter et al 2013; Park et al 2016). Although many MPK substrates have been isolated in plants, further biochemical analysis of these substrates is needed to advance our understanding of the mechanism underlying MPK‐induced phosphorylation.…”