The Arabidopsis Mitogen-Activated Protein Kinase Kinase Kinase 20 (MKKK20) Acts Upstream of MKK3 and MPK18 in Two Separate Signaling Pathways Involved in Root Microtubule Functions

Abstract

Mitogen-activated protein kinase (MAPK) signaling networks represent important means of signal transduction in plants and other eukaryotes, controlling intracellular signaling by linking perception of environmental or developmental cues to downstream targets. In the Arabidopsis MEKK subfamily, the MKKK19, 20, and 21 form a highly supported clade with the Solanaceous Fertilization-Related Kinases. In Arabidopsis, little is known about this group, except for MKKK20, which is involved in osmotic stress. Using a directed MKKK-MKK yeast two-hybrid (Y2H) screen, MKKK20 was found to interact only with MKK3, while a MKKK20 large-scale Y2H screen retrieved MPK18 as a direct interactant. In vitro phosphorylation assays showed that MKKK20 phosphorylates both MKK3 and MPK18. However, when all three kinases are combined, no synergistic effect is observed on MPK18 phosphorylation, suggesting a direct access to MPK18, consistent with the absence of interaction between MKK3 and MPK18 in protein–protein interaction assays. Since mpk18 mutant plants were previously shown to be defective in microtubule-related functions, phenotypes of mkkk20 single and mkkk20/mpk18 double mutants were investigated to determine if MKKK20 acts upstream of MPK18. This was the case, as mkkk20 root length was shorter than WT in media containing microtubule-disrupting drugs as previously observed for mpk18 plants. Surprisingly, mkk3 plants were also similarly affected, suggesting the presence of two non-complementary pathways involved in Arabidopsis cortical microtubule function, the first including MKKK20, MKK3 and an unknown MPK; the second, a non-canonical MAPK cascade made of MKKK20 and MPK18 that bypasses the need for an MKK intermediate.

The Arabidopsis Mitogen-Activated Protein Kinase Kinase Kinase 20 (MKKK20) C-terminal domain interacts with MKK3 and harbors a typical DEF mammalian MAP kinase docking site

“…concentration of 3-AT (3-amino-1,2,4-triazole), retaining only high-affinity binding interactions. Has shown previously 8 , full length MKKK20 and MKK3 interacted strongly ( Figure 1A, yeast colony 6; Figure 1B, yeast colony 2). MKKK20 was then dissected in three sections: N-terminal, Mid and C-terminal.…”

“…We recently showed that mkkk20, mkk3 and mpk18 mutant plants showed microtubule defect phenotypes and were involved in two separate signaling pathways, a canonical one with MKKK20-MKK3 and an unknown downstream MPK(s), and a non-canonical one with direct MKKK20-MPK18 interaction and [T-D-Y] phosphorylation of the catalytic domain activation loop of MPK18 8 . Here, MKKK20 and MKK3 were dissected into nine and five fragments, respectively, and used for pairwise interactions in a yeast two hybrid system, including a complete bait and prey plasmid swap for a total of 104 interactions tested at high stringency with a 100 mM CONTACT Daniel P. Matton dp.matton@umontreal.ca Institut de Recherche en Biologie Végétale, Département de Sciences Biologiques, Université de Montréal, Montréal, Canada Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/kpsb.…”

“…The presence of the DEF domain is most probably not fortuitous since none of the other 18 Arabidopsis MEKKs have an FXF [P/D/E)] motif, like MKKK5 (FRFN motif), or have a [S/T]-P phosphoacceptor site too far from the FXF motif (MKKK14 FLFG and MKKK15 FFFS, from 100 and 42, amino acids respectively), or being imbedded in the kinase domain, like MKKK11 (FAFK motif). Interestingly, although MKKK20 was found to strongly autophosphorylate while expressed in bacteria, phosphorylation was only observed in the protein kinase domain (amino acids 3-268) in around 25 Ser (S), Thr (T) and Tyr (Y) sites 8 . The only two SP phosphoacceptor sites were found in Box I and were not phosphorylated, suggesting that the MKKK20 DEF domain might be a bona fide docking site for an upstream protein kinase.…”

“…Contrary to the MKKs and MPKs that have well conserved docking domains, MAPKKKs (MKKKs or MEKKs) docking interaction sites are scarce in animals literature 6 , and have yet to be uncovered in plants, a surprising situation considering that numerous MKKKs interact directly with MKKs in heterologous systems, like in Y2H screens 7,8 , and phosphorylate MKKs in kinase assays 8 , suggesting that scaffolds might not be required for their specific interactions. With 21 MEKKs in A. thaliana and much more in other species 9 , a major question is how these MEKKs achieve the specificity required to interact with their ten downstream MEKs.…”

Activation of the plant mevalonate pathway by extracellular ATP

“…5d). The cytoplasmic kinase, mitogen-activated protein kinase kinase 3 (MKK3) 44 , was used as a negative control in these assays and showed no interaction with P2K1 ( Fig. 5).…”