title: Live Cell Imaging of ERK and MEK: simple binding equilibrium explains the regulated nucleocytoplasmic distribution of ERK.
abstract: In response to epidermal growth factor (EGF), the mitogen-activated protein kinase ERK2 translocates into the nucleus. To probe the mechanisms regulating the subcellular localization of ERK2, we used live cell imaging to examine the interaction between MEK1 and ERK2. Fluorescence resonance energy transfer (FRET) studies show that MEK1 and ERK2 directly interact and demonstrate that this interaction in the cytoplasm is largely responsible for cytoplasmic retention of ERK2. Stimulation with EGF caused loss of FRET as ERK separated from MEK and moved into the nucleus. FRET was recovered as ERK returned to the cytosol, indicating ERK reassociation with MEK in the cytoplasm. The EGF-induced transit of ERK through the nucleus was complete within 20 min, and there was no significant movement of MEK into the nucleus. Fluorescence recovery after photobleaching experiments was used to assess the rate of movement of MEK and ERK. The steady-state rate of ERK entry into the nucleus in resting cells was energy-independent and greater than the rate of ERK entry upon EGF stimulation. This suggests that the rate constant for ERK transport across the nuclear membrane is not limiting nuclear entry. Thus, we suggest that the movement of ERK into and out of the nucleus in response to agonist occurs primarily by diffusion and is controlled by interactions with binding partners in the cytosol and nucleus. No evidence of ERK dimerization was detected by FRET methods; the kinetics for nucleocytoplasmic transport were unaffected by mutations in the ERK putative dimerization domain.
authors.affiliation: Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri 63110, USA. rburack@path.wustl.edu
authors.forename: W Richard
authors.initials: WR
authors.lastname: Burack
authors.forename: Andrey S
authors.initials: AS
authors.lastname: Shaw
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001426:Bacterial Proteins / Q000493:pharmacokinetics; D001692:Biological Transport / Q000187:drug effects / Q000502:physiology; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D003593:Cytoplasm / Q000201:enzymology; D019281:Dimerization; D004815:Epidermal Growth Factor / Q000494:pharmacology; D036681:Fluorescence Recovery After Photobleaching; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D007668:Kidney / Q000166:cytology; D008164:Luminescent Proteins / Q000493:pharmacokinetics; D048369:MAP Kinase Kinase 1 / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009685:Nuclear Envelope / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D008164:Luminescent Proteins; C054562:yellow fluorescent protein, Bacteria; D004815:Epidermal Growth Factor; D000255:Adenosine Triphosphate; D019950:Mitogen-Activated Protein Kinase 1; D048369:MAP Kinase Kinase 1