title: The role of c-Jun N-terminal kinase, p38, and extracellular signal-regulated kinase in insulin-induced Thr69 and Thr71 phosphorylation of activating transcription factor 2.
abstract: The stimulation of cells with physiological concentrations of insulin induces a variety of responses, e.g. an increase in glucose uptake, induction of glycogen and protein synthesis, and gene expression. One of the determinants regulating insulin-mediated gene expression may be activating transcription factor 2 (ATF2). Insulin activates ATF2 by phosphorylation of Thr69 and Thr71 via a two-step mechanism, in which ATF2-Thr71 phosphorylation precedes the induction of ATF2-Thr69+71 phosphorylation by several minutes. We previously found that in c-Jun N-terminal kinase (JNK)-/- fibroblasts, cooperation of the ERK1/2 and p38 pathways is required for two-step ATF2-Thr69+71 phosphorylation in response to growth factors. Because JNK is also capable of phosphorylating ATF2, we assessed the involvement of JNK, ERK1/2 and p38 in the insulin-induced two-step ATF2 phosphorylation in JNK-expressing A14 fibroblasts and 3T3L1-adipocytes. The induction of ATF2-Thr71 phosphorylation was sensitive to MAPK kinase (MEK) 1/2-inhibition with U0126, and this phosphorylation coincided with nuclear translocation of phosphorylated ERK1/2. Use of the JNK inhibitor SP600125 or expression of dominant-negative JNK-activator SAPK kinase (SEK1) prevented the induction of ATF2-Thr69+71, but not ATF2-Thr71 phosphorylation by insulin. ATF2-dependent transcription was also sensitive to SP-treatment. Abrogation of p38 activation with SB203580 or expression of dominant-negative MKK6 had no inhibitory effect on these events. In agreement with this, the onset of ATF2-Thr69+71 phosphorylation coincided with the nuclear translocation of phosphorylated JNK. Finally, in vitro kinase assays using nuclear extracts indicated that ERK1/2 preceded JNK translocation. We conclude that sequential activation and nuclear appearance of ERK1/2 and JNK, rather than p38, underlies the two-step insulin-induced ATF2 phosphorylation in JNK-expressing cells.
authors.affiliation: Department of Molecular Cell Biology, Section Signal Transduction and Ageing, Leiden University Medical Centre, The Netherlands.
authors.forename: Bart
authors.initials: B
authors.lastname: Baan
authors.forename: Hans
authors.initials: H
authors.lastname: van Dam
authors.forename: Gerard C M
authors.initials: GC
authors.lastname: van der Zon
authors.forename: J Antonie
authors.initials: JA
authors.lastname: Maassen
authors.forename: D Margriet
authors.initials: DM
authors.lastname: Ouwens
mesh_terms: D041721:3T3-L1 Cells; D051698:Activating Transcription Factor 2 / Q000378:metabolism*; D021581:Active Transport, Cell Nucleus; D000818:Animals; D004791:Enzyme Inhibitors / Q000494:pharmacology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000502:physiology*; D006801:Humans; D007328:Insulin / Q000494:pharmacology*; D048031:JNK Mitogen-Activated Protein Kinases / Q000502:physiology*; D051379:Mice; D008954:Models, Biological; D041681:NIH 3T3 Cells; D010766:Phosphorylation / Q000187:drug effects*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D021381:Protein Transport; D013997:Time Factors; D014162:Transfection; D048051:p38 Mitogen-Activated Protein Kinases / Q000502:physiology*
chemical_list: D051698:Activating Transcription Factor 2; C497106:Atf2 protein, mouse; D004791:Enzyme Inhibitors; D007328:Insulin; D017346:Protein Serine-Threonine Kinases; D048049:Extracellular Signal-Regulated MAP Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases