title: ERK phosphorylation and nuclear accumulation: insights from single-cell imaging.
abstract: Many stimuli mediate activation and nuclear translocation of ERK (extracellular-signal-regulated kinase) by phosphorylation on the TEY (Thr-Glu-Tyr) motif. This is necessary to initiate transcriptional programmes controlling cellular responses, but the mechanisms that govern ERK nuclear targeting are unclear. Single-cell imaging approaches have done much to increase our understanding of input-output relationships in the ERK cascade, but few studies have addressed how the range of ERK phosphorylation responses observed in cell populations influences subcellular localization. Using automated microscopy to explore ERK regulation in single adherent cells, we find that nuclear localization responses increase in proportion to stimulus level, but not the level of TEY phosphorylation. This phosphorylation-unattributable nuclear localization response occurs in the presence of tyrosine phosphatase and protein synthesis inhibitors. It is also seen with a catalytically inactive ERK2-GFP (green fluorescent protein) mutant, and with a mutant incapable of binding the DEF (docking site for ERK, F/Y-X-F/Y-P) domains found in many ERK-binding partners. It is, however, reduced by MEK (mitogen-activated protein kinase/ERK kinase) inhibition and by mutations preventing TEY phosphorylation or in the ERK common docking region. We therefore show that TEY phosphorylation of ERK is necessary, but not sufficient, for the full nuclear accumulation response and that this 'phosphorylation-unattributable' component of stimulus-mediated ERK nuclear localization requires association with partner proteins via the common docking motif.
authors.affiliation: Department of Biology and Biochemistry, University of Bath, Bath BA2 7AY, UK. c.caunt@bath.ac.uk
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Caunt
authors.forename: Craig A
authors.initials: CA
authors.lastname: McArdle
mesh_terms: D021581:Active Transport, Cell Nucleus; D020816:Amino Acid Motifs; D002467:Cell Nucleus / Q000378:metabolism; D006801:Humans; D008856:Microscopy, Fluorescence; D019950:Mitogen-Activated Protein Kinase 1 / Q000737:chemistry / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D021381:Protein Transport; D059010:Single-Cell Analysis
chemical_list: D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3