A Novel Tobacco Mitogen-activated Protein (MAP) Kinase Kinase, NtMEK1, Activates the Cell Cycle-regulated p43Ntf6 MAP Kinase

Ornella Calderini,Nathalie Glab,Catherine Bergounioux,Erwin Heberle-Bors,Cathal Wilson

doi:10.1074/jbc.m010621200

Abstract

Two-hybrid screening of a tobacco BY-2 cell suspension cDNA library using the p43(Ntf6) mitogen-activated protein (MAP) kinase as bait resulted in the isolation of a cDNA encoding a protein with features characteristic of a MAP kinase kinase (MEK), which has been called NtMEK1. Two-hybrid interaction analysis and pull-down experiments showed a physical interaction between NtMEK1 and the tobacco MAP kinases p43(Ntf6) and p45(Ntf4), but not p43(Ntf3). In kinase assays NtMEK1 preferentially phosphorylated p43(Ntf6). Functional studies in yeast showed that p43(Ntf6) could complement the yeast MAP kinase mutant mpk1 when co-expressed with NtMEK1, and that this complementation depended on the kinase activity of p43(Ntf6). Expression analysis showed that the NtMEK1 and ntf6 genes are co-expressed both in plant tissues and following the induction of cell division in leaf pieces. These data suggest that NtMEK1 is an MEK for the p43(Ntf6) MAP kinase.

Highlights

Eukaryotic cells respond to a variety of extracellular stimuli via activation of specific MAP1 kinase cascades
Functional studies in yeast showed that p43Ntf6 could complement the yeast mitogen-activated protein (MAP) kinase mutant mpk1 when co-expressed with NtMEK1, and that this complementation depended on the kinase activity of p43Ntf6
In synchronized tobacco cell cultures, the p43Ntf6 MAP kinase [9] is activated at a late stage in mitosis, around the anaphase/early telophase transition, and localizes in the middle of two microtubule arrays characteristic of the phragmoplast [10], a plant-specific structure involved in laying down the new cell wall

Summary

Introduction

Eukaryotic cells respond to a variety of extracellular stimuli via activation of specific MAP1 kinase cascades. Functional studies in yeast showed that p43Ntf6 could complement the yeast MAP kinase mutant mpk1 when co-expressed with NtMEK1, and that this complementation depended on the kinase activity of p43Ntf6. Two-hybrid interaction analysis between NtMEK1 and the MAP kinases p43Ntf3, p45Ntf4, and p43Ntf6 (and the LOF and GOF versions of p43Ntf6) was tested on medium lacking histidine and containing 3-AT, and ␤-galactosidase activity was measured in liquid assays (CLONTECH).

Results

Conclusion