Stimulus-induced uncoupling of extracellular signal-regulated kinase phosphorylation from nuclear localization is dependent on docking domain interactions.
Many stimuli activate the extracellular signal-regulated kinase (ERK) by phosphorylation on the TEY motif. Activated ERK characteristically accumulates in the nucleus, but the underlying mechanisms involved are unclear. Using automated microscopy to explore ERK regulation in single intact cells, we find that, when protein kinase C or epidermal growth factor receptors are activated, a substantial fraction of the ERK nuclear localization response is uncoupled from TEY phosphorylation. This phosphorylation-unattributable nuclear localization response occurs in the presence of inhibitors of tyrosine phosphatases and protein synthesis. It was also evident with a catalytically inactive ERK2-GFP 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 was, however, reduced by MEK inhibition and by mutations preventing either TEY phosphorylation or D (docking)-domain-dependent ERK binding (D319N). Thus, we show that MEK-catalysed ERK phosphorylation is necessary but not sufficient for the full nuclear localization response: there is an additional phosphorylation-unattributable component of the response that does not reflect induced expression of nuclear anchors and is independent of ERK catalytic activity or DEF-domain binding. It is, however, dependent upon D-domain binding, highlighting distinct roles of ERK motifs during nuclear targeting.
Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath, UK BA2 7AY.
Christopher J
CJ
Caunt
Craig A
CA
McArdle
D002467:Cell Nucleus / Q000187:drug effects / Q000201:enzymology*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000737:chemistry* / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D020935:MAP Kinase Signaling System / Q000187:drug effects; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D050505:Mutant Proteins / Q000378:metabolism; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding / Q000187:drug effects; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship
D050505:Mutant Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D015240:Phorbol 12,13-Dibutyrate; D048049:Extracellular Signal-Regulated MAP Kinases; D020929:Mitogen-Activated Protein Kinase Kinases