PB1 domain-dependent signaling complex is required for extracellular signal-regulated kinase 5 activation.
MEKK2, MEK5, and extracellular signal-regulated kinase 5 (ERK5) are members of a three-kinase cascade for the activation of ERK5. MEK5 is the only MAP2K to express a PB1 domain, and we have shown that it heterodimerizes with the PB1 domain of MEKK2. Here we demonstrate the MEK5 PB1 domain is a scaffold that also binds ERK5, functionally forming a MEKK2-MEK5-ERK5 complex. Reconstitution assays and CFP/YFP imaging (fluorescence resonance energy transfer [FRET]) measuring YFP-MEKK2/CFP-MEK5 and CFP-MEK5/YFP-ERK5 interactions define distinct MEK5 PB1 domain binding sites for MEKK2 and ERK5, with a C-terminal extension of the PB1 domain contributing to ERK5 binding. Stimulus-dependent CFP/YFP FRET in combination with mutational analysis was used to define MEK5 PB1 domain residues critical for the interaction of MEKK2/MEK5 and MEK5/ERK5 required for activation of the ERK5 pathway in living cells. Fusion of the MEK5 PB1 domain to the N terminus of MEK1 confers ERK5 regulation by a MAP2K normally regulating only ERK1/2. The MEK5 PB1 domain confers stringent MAP3K regulation of ERK5 relative to more promiscuous MAP3K control of ERK1/2, JNK, and p38.
Nakamura
Kazuhiro
K
Department of Pharmacology, CB#7365, 1108 Mary Ellen Jones Building, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7365, USA.
Uhlik
Mark T
MT
Johnson
Nancy L
NL
Hahn
Klaus M
KM
Johnson
Gary L
GL
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D019281:Dimerization; D004789:Enzyme Activation; D031541:Fluorescence Resonance Energy Transfer; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D048671:MAP Kinase Kinase 5 / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D048730:MAP Kinase Kinase Kinase 2 / Q000235:genetics / Q000378:metabolism; D048054:Mitogen-Activated Protein Kinase 7 / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes; D009154:Mutation; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D013012:Sorbitol / Q000494:pharmacology
D001426:Bacterial Proteins; C485182:Cyan Fluorescent Protein; D008164:Luminescent Proteins; D046912:Multiprotein Complexes; D011994:Recombinant Proteins; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins; D013012:Sorbitol; D048054:Mitogen-Activated Protein Kinase 7; D048730:MAP Kinase Kinase Kinase 2; D048671:MAP Kinase Kinase 5; C482563:MAP2K5 protein, human