Abstract
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.
Publication types
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Research Support, N.I.H., Extramural
MeSH terms
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Amino Acid Sequence
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Animals
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Bacterial Proteins / genetics
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Bacterial Proteins / metabolism
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Cells, Cultured
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Dimerization
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Enzyme Activation
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Fluorescence Resonance Energy Transfer
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Green Fluorescent Proteins / genetics
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Green Fluorescent Proteins / metabolism
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Luminescent Proteins / genetics
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Luminescent Proteins / metabolism
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MAP Kinase Kinase 5 / drug effects
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MAP Kinase Kinase 5 / genetics
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MAP Kinase Kinase 5 / metabolism*
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MAP Kinase Kinase Kinase 2 / genetics
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MAP Kinase Kinase Kinase 2 / metabolism
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Mitogen-Activated Protein Kinase 7 / drug effects
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Mitogen-Activated Protein Kinase 7 / genetics
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Mitogen-Activated Protein Kinase 7 / metabolism*
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Molecular Sequence Data
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Multiprotein Complexes
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Mutation
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Protein Interaction Mapping
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Protein Structure, Tertiary
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Recombinant Proteins / genetics
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Recombinant Proteins / metabolism
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Signal Transduction*
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Sorbitol / pharmacology
Substances
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Bacterial Proteins
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Cyan Fluorescent Protein
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Luminescent Proteins
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Multiprotein Complexes
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Recombinant Proteins
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yellow fluorescent protein, Bacteria
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Green Fluorescent Proteins
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Sorbitol
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Mitogen-Activated Protein Kinase 7
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MAP Kinase Kinase Kinase 2
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MAP Kinase Kinase 5
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MAP2K5 protein, human