Activation of AtMPK9 through autophosphorylation that makes it independent of the canonical MAPK cascades

Abstract

Mitogen activated protein kinases (MAPKs) are part of conserved signal transduction modules in eukaryotes that are typically organised into three-tiered kinase cascades. The activation of MAPKs in these pathways is fully dependent on the bisphosphorylation of the TXY motif in the T-loop by the pertinent dual-specificity MAPK kinases (MAPKKs). The plant AtMPK9 is a member of an atypical class of MAPKs. Representatives of this MAPK family have TDY phosphoacceptor site, a long C-terminal extension, and lack the common MAPKK binding docking motif. Here, we present multiple in vitro and in vivo data that AtMPK9 is activated independently of any upstream MAPKKs but it is activated through autophosphorylation. We mapped the autophosphorylation sites by mass spectrometry to the TDY motif and to the C-terminal regulatory extension. We mutated the phosphoacceptor sites on the TDY, which confirmed the requirement for bisphorylation at this site for full kinase activity. Next, we demonstrated that the kinase inactive mutant form of AtMPK9 is not transphosphorylated on the TDY site when mixed with an active AtMPK9, implying that the mechanism of the autocatalytic phosphorylation is intramolecular. Furthermore, we show that in vivo AtMPK9 is activated by salt and is regulated by okadaic acid-sensitive phosphatases. We conclude that the plant AtMPK9 shows similarities to the mammalian atypical MAPKs, ERK7/8 in terms of MAPKK-independent activation mechanism.

Regulation of Stomatal Immunity by Interdependent Functions of a Pathogen-Responsive MPK3/MPK6 Cascade and Abscisic Acid

“…It was reported previously that MPK9 and MPK12 function downstream of ROS and modulate SLAC1 activity in guard cell ABA signaling (Jammes et al, 2009(Jammes et al, , 2011. Based on genetic and biochemical analyses, MKK4 and MKK5 are unable to activate MPK9 and MPK12 in vivo and in vitro (Asai et al, 2002;Lee et al, 2009;Nagy et al, 2015). As a result, MPK9/MPK12 and MPK3/MPK6 are likely to function as two independent MAPK cascades in stomatal movement.…”

A review of redox signaling and the control of MAP kinase pathway in plants

“…It has been shown that AtMPK12 but not AtMPK9 can interact with MAPK phosphatase IBR5 [162]. Recent research indicates that AtMPK9 can be activated by autophosphorylation that may be independent of any upstream MAPKKKs or MKKs [163]. Furthermore, AtMPK12 allele with an amino acid substitution alone could impair the ABA inhibition of opening [164].…”

The MPK8‐TCP14 pathway promotes seed germination in Arabidopsis

“…Noteworthy, S 102 was followed by an alanine residue instead of a proline. Similarly, MPK9, another D‐type MPK, was able to autophosphorylate on S 464 that is also followed by an alanine (Nagy et al ., ). As the other TCP14 phosphoresidues detected were followed by various amino acids but proline, the sequences targeted by MPK8 and other D‐type MPKs might significantly diverge from the canonical [S/T‐P] motif, and the identification of additional substrates of these MPKs would help unravel their motif specificity.…”