Isolation of ATMEKK1 (a MAP kinase kinase Kinase) - Interacting proteins and analysis of a MAP kinase cascade in Arabidopsis

In plants, a number of MAP kinase (MAPK), MAPK kinase (MAPKK), and MAPKK ki nase (MAPKKK) homologues have been reported. However, there have been no re ports of protein-protein interactions between these kinases or molecular an alysis of MAPK cascades in higher plants, To analyze a possible MAPK cascad e in Arabidopsis thaliana, we took two molecular approaches. One is the two -hybrid screening of ATMEKK1 (a MAPKKK)-interacting proteins; the other is an analysis of physical and functional interactions among isolated MAPK, MA PKK, and MAPKKK homologues from Arabidopsis. In two-hybrid screening using ATMEKK1 as bait, we isolated a novel MAPKK homologue, ATMKK2, a MAPK homolo gue, ATMPK4, and an unknown protein. ATMKK2 has high sequence similarity wi th MEK1 (a MAPKK) in Arabidopsis. Based on yeast two-hybrid analysis, we de tected protein-protein interactions between ATMEKK1 and ATMKK2/MEK1 (MAPKKs ), between ATMKK2/MEK1 and ATMPK4 (a MAPK), and between ATMPK4 and ATMEKK1. ATMPK4 and ATMKK2/1MEK1 interacted with two distinct regions of ATMEKK1, t he N-terminal regulatory domain and the C-terminal kinase domain, respectiv ely. Coexpression of ATMEKK1 increased the ability of two closely related M APKKs, ATMKK2 and MEK1, to complement a growth defect of the yeast pbs2 mut ant. Coexpression of ATMPK4 and MEK1 complemented a growth defect of the ye ast mpk1 and bck1 mutants. By contrast, other combinations of MAPKs and MAP KKs did not suppress these yeast mutations. These results suggest that ATME KK1, ATMKK2/MEK1, and ATMPK4 may constitute a MAP kinase cascade. (C) 1998 Academic Press.