Defining the dynamic interplay of a MAPK signaling cascade involved in root and pollen tube growth

BENHAMMAN, R; DRORY, SD; PEREZ, J; BAI, F; MATTON, DP

Portland, Oregon

Congreso; Annual meeting of the American Society of Plant Biologists; 2014

American Society of Plant Biologists

The pivotal role played by protein phosphorylation in eukaryotic signal transduction is well illustrated by the wide range of phosphorylation cascades that involve Mitogen-Activated Protein Kinases (MAPK). Although this should also apply to plants, which display a larger MAPK repertoire than other eukaryotes, up to now only a handful of plant MAPK cascades (MAP3K-MAP2K-MAPK) have been characterized. Furthermore, the paucity of information relative to plant MAP kinase interacting domains precludes the assembly of MAPK networks through predictive methods other than co-expression analyses and protein interaction assays. Here we describe a new and complete MAPK cascade that affects root and pollen tube growth through the modulation of cortical microtubule function. Starting from the MKKK20, an Arabidopsis ortholog of the Fertilization-related kinases (FRK) of the Solanaceae family, downstream MAP2K and MAPK were isolated using protein interaction assays and validated through enzyme-substrate relationships. Genetic evidences confirmed the cascade since T-DNA insertional mutant lines for all members of the cascade displayed identical phenotypes under specific conditions. Enzymatic assays followed by phosphopeptide analysis revealed that the MKKK20 autophosphorylates on serine, threonine and tyrosine residues, most probably through intermolecular transphosphorylation. Phosphopeptide patterns obtained from fully active MKKK20 also revealed the importance of specific amino acid residues in MKKK20 activation. Interestingly, the upstream MAP3K (MKKK20) could phosphorylate both the MAP2K and the MAPK, suggesting a possible scaffold effect from one of the kinases. Kinase interacting domains, downstream targets as well as phenotypical analyses of the kinases double and triple mutants on root and pollen tube growth will also be presented.