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 kinase (MAPKKK) homologues have been reported. However, there have been no reports of protein-protein interactions between these kinases or molecular analysis of MAPK cascades in higher plants. To analyze a possible MAPK cascade 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, MAPKK, and MAPKKK homologues from Arabidopsis. In two-hybrid screening using ATMEKK1 as bait, we isolated a novel MAPKK homologue, ATMKK2, a MAPK homologue, ATMPK4, and an unknown protein. ATMKK2 has high sequence similarity with MEK1 (a MAPKK) in Arabidopsis. Based on yeast two-hybrid analysis, we detected protein-protein interactions between ATMEKK1 and ATMKK2/MEK1 (MAPKKs), between ATMKK2/MEK1 and ATMPK4 (a MAPK), and between ATMPK4 and ATMEKK1. ATMPK4 and ATMKK2/MEK1 interacted with two distinct regions of ATMEKK1, the N-terminal regulatory domain and the C-terminal kinase domain, respectively. Coexpression of ATMEKK1 increased the ability of two closely related MAPKKs, ATMKK2 and MEK1, to complement a growth defect of the yeast pbs2 mutant. Coexpression of ATMPK4 and MEK1 complemented a growth defect of the yeast mpk1 and bck1 mutants. By contrast, other combinations of MAPKs and MAPKKs did not suppress these yeast mutations. These results suggest that ATMEKK1, ATMKK2/MEK1, and ATMPK4 may constitute a MAP kinase cascade.
Ichimura
K
K
Laboratory of Plant Molecular Biology, Institute of Physical and Chemical Research (RIKEN), Tsukuba Life Science Center, Japan.
Mizoguchi
T
T
Irie
K
K
Morris
P
P
Giraudat
J
J
Matsumoto
K
K
Shinozaki
K
K
D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics*; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000302:isolation & purification; D005656:Fungal Proteins / Q000302:isolation & purification / Q000378:metabolism*; D005816:Genetic Complementation Test; D048370:MAP Kinase Kinase 2; D020930:MAP Kinase Kinase Kinases; D020929:Mitogen-Activated Protein Kinase Kinases; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017346:Protein Serine-Threonine Kinases / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D017434:Protein Structure, Tertiary; D011505:Protein-Tyrosine Kinases / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D013912:Threonine / Q000235:genetics
D018076:DNA, Complementary; D005656:Fungal Proteins; D013912:Threonine; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D020930:MAP Kinase Kinase Kinases; D048370:MAP Kinase Kinase 2; D020929:Mitogen-Activated Protein Kinase Kinases; D000409:Alanine