Abstract
Mitogen-activated protein kinase (MAPK) cascades, which are the highly conserved signalling modules in eukaryotic organisms, have been shown to play important roles in regulating growth, development, and stress responses. The structures of various MAPKs from yeast and animal have been solved, and structure-based mutants were generated for their function analyses, however, the structures of plant MAPKs remain unsolved. Here, we report the crystal structure of Arabidopsis MPK6 at a 3.0 Å resolution. Although MPK6 is topologically similar to ERK2 and p38, the structures of the glycine-rich loop, MAPK insert, substrate binding sites, and L16 loop in MPK6 show notable differences from those of ERK2 and p38. Based on the structural comparison, we constructed MPK6 mutants and analyzed their kinase activity both in vitro and in planta. MPK6F364L and MPK6F368L mutants, in which Phe364 and Phe368 in the L16 loop were changed to Leu, respectively, acquired higher intrinsic kinase activity and retained the normal MAPKK activation property. The expression of MPK6 mutants with basal activity is sufficient to induce camalexin biosynthesis; however, to induce ethylene and leaf senescence, the expression of MPK6 mutants with higher activity is required. The results suggest that these mutants can be used to analyze the specific biological functions of MPK6.
Highlights
Mitogen-activated protein kinase (MAPK) cascades, which are composed of MAPK kinase kinase, MAPK kinase and MAPK, are highly conserved signalling modules in eukaryotic organisms
The protein sequence alignment shows that MPK6 has an extended N terminus compared with a variety of mammalian MAPKs whose structures have been reported (Fig. 1)
The results suggest that MPK6 and its mutants, except for MPK6K92R, can be fully activated by upstream MAPKK, and the conformation changes in the L16 loop and MAP kinase insert (MKI) alter the kinase activity of MPK6
Summary
Mitogen-activated protein kinase (MAPK) cascades, which are composed of MAPK kinase kinase, MAPK kinase and MAPK (called MAPKKK, MAPKK, and MAPK), are highly conserved signalling modules in eukaryotic organisms. MAPKKs are activated by MAPKKK via the phosphorylation of the two serine and threonine residues in their conserved S/T-X3–5-S/T motif. MAPKKs are dual-specificity kinases that activate MAPKs by phosphorylating the threonine and tyrosine residues in their conserved TXY motif. The activated MAPKs have been shown to regulate specific cellular processes primarily through the phosphorylation of different substrates; several MAPKs (such as ERK1 and ERK2 in animals) were shown to perform their function only by binding to targets in an kinase activity-independent manner[1]. Several structural elements within MAPKs that participated in the regulation of kinase activity and interaction with their substrates were defined. Using recombinant proteins and Arabidopsis transgenic plants, we analyzed the kinase activity of the MPK6 mutants and their biological function. To induce ethylene and leaf senescence, the expression of MPK6 mutants with higher activity is required
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