A network of immediate early gene products propagates subtle differences in mitogen-activated protein kinase signal amplitude and duration

Abstract

The strength and duration of mitogen-activated protein kinase (MAPK) signaling have been shown to regulate cell fate in different cell types. In this study, a general mechanism is described that explains how subtle differences in signaling kinetics are translated into a specific biological outcome. In fibroblasts, the expression of immediate early gene (IEG)-encoded Fos, Jun, Myc, and early growth response gene 1 (Egr-1) transcription factors is significantly extended by sustained extracellular signal-regulated kinase 1 and 2 (ERK1 and -2) signaling. Several of these proteins contain functional docking site for ERK, FXFP (DEF) domains that serve to locally concentrate the active kinase, thus showing that they can function as ERK sensors. Sustained ERK signaling regulates the posttranslational modifications of these IEG-encoded sensors, which contributes to their sustained expression during the G(1)-S transition. DEF domain-containing sensors can also interpret the small changes in ERK signal strength that arise from less than a threefold reduction in agonist concentration. As a result, downstream target gene expression and cell cycle progression are significantly changed.

FIG. 1.

DEF domain-dependent regulation of Fra proteins. (A) Alignment of the c-Fos, Fra-1, and Fra-2 COOH termini showing amino acid identity (boldface letters), ERK and RSK priming phosphorylation sites (underlined), and DEF domains (box). The sequences are from rat (c-Fos) and mouse (Fra-1 and Fra-2) proteins, and the numbers indicate amino acid positions. (B) NIH 3T3 cells were transfected with the indicated Fos alleles, serum deprived, and treated with EGF (50 ng/ml) for 5 min. Where indicated, cells were pretreated with UO126 (5 μM) for 30 min before treatment with EGF for 5 min. Cell extracts were then subjected to immunoblotting with anti-c-Fos, anti-phospho-Thr325 c-Fos, and anti-ERK antibodies. (C and D) NIH 3T3 cells were transfected with the indicated DEF domain mutant Fra-2 and Fra-1 alleles and treated as described for panel B. Immunoblotting was performed with the anti-Fra-2, anti-Fra-1, and anti-ERK antibodies.

FIG. 2.

DEF domain-mediated regulation of c-Myc phosphorylation. (A) The illustration details the putative DEF domain (FPYP), NH₂-terminal phosphorylation site Thr58 and Ser62, Myc box II (MbII), basic DNA binding domain (B), and helix-loop-helix (HLH)-leucine zipper (zip) domains. (B) NIH 3T3 cells were transfected with c-Myc or control vector and treated as described in the legend to Fig. 1B. c-Myc was immunoprecipitated (IP) from cell lysates, and each sample was analyzed with anti-c-Myc or anti-phospho-specific Ser62 (pS62) antibodies. Western blot analysis of ERK activation in the cell lysate was measured with the anti-phospho-ERK1/2 antibody. IgG, immunoglobulin G. (C) Quiescent cells expressing the indicated c-Myc proteins were treated with EGF for 5 min and lysed, and c-Myc immunoprecipitations were performed. Western blot analysis of immunoprecipitates used anti-c-Myc or anti-phospho-Ser62 antibodies, and analysis of cell lysate used anti-c-Myc or anti-ERK antibodies. The data shown are representative of three experiments.

FIG. 3.

Global induction of IEGs. (A) Quiescent Swiss 3T3 cells were treated with EGF (25 ng/ml) or PDGF-BB (20 ng/ml) for the indicated times and lysed. Western blotting was used to visualize the expression of Fos family proteins and c-Jun, as well as ERK activation kinetics (lower panel). (B) Cells were treated as described for panel A, and lysates were analyzed with anti-Egr-1, anti-JunB, or anti-ERK antibodies. (C) The induction of c-Myc was analyzed by initially immunoprecipitating c-Myc from extracts followed by Western analysis. The activation of ERK was analyzed with an anti-phospho-ERK1/2 (αpERK) antibody. All data shown are representative of at least four experiments.

FIG. 4.

Sustained expression of IEG products requires the ERK pathway. Swiss 3T3 cells were induced with PDGF for 90 min and then treated with UO126 (5 μM) or DMSO (0.1%) for a further 120 min. (A) The levels of Egr-1, Fra-2, c-Jun, and JunB and the ERK activation kinetics were assayed by Western blotting. (B) After initially treating the cells with PDGF for 90 (lanes 2 to 10) or 120 min (lanes 11 to 13), DMSO or UO126 was added to cells. c-Myc was immunoprecipitated from cell lysates and subjected to Western analysis. (C) Swiss 3T3 cells were induced for 5 h with PDGF and then treated with UO126 for 20 or 30 min before cell lysis. Control cells (−) were treated with DMSO for 30 min. α pERK, anti-phospho-ERK. (D) Cells induced with PDGF for 5 h were incubated in the presence and absence (DMSO) of UO126 for a further 7 h. Cell extracts were prepared at the indicated times and subjected to Western analysis to detect levels of Fra-1 and the activation kinetics of ERK. The data shown are representative of three experiments.

FIG. 5.

Incremental decreases in ERK signal strength and duration have large effects on IEG product phosphorylation and sustained expression. (A) Quiescent Swiss 3T3 cells were treated with different amounts of PDGF for the indicated times and lysed. The levels of endogenous c-Fos and Egr-1 and the activation kinetics of ERK were measured by Western blotting.α pERK, anti-phospho-ERK. (B) Quiescent Swiss 3T3 cells were treated with different doses of PDGF and lysed. The phosphorylation of Thr325 in c-Fos and total levels of c-Fos were measured by Western blotting. (C) ERK1 was immunoprecipitated from PDGF-treated cells, and its kinase activity was quantitated with an immune complex kinase assay. The data shown are the mean ± standard error from three independent experiments. (D and E) Cells were treated as described for panel A, and the levels of Fra-2 and Fra-1 were analyzed by Western analysis. The data are representative of three to four experiments.

FIG. 6.

ERK signal strength affects AP-1 target gene expression and S-phase entry. (A) Quiescent Swiss 3T3 cells were treated with PDGF (10 or 4 ng/ml), and cyclin D1 levels in cell extracts were measured with an anti-cyclin D antibody. (B) Quiescent Swiss 3T3 cells cultured on glass coverslips were treated with PDGF, and BrdU incorporation was assayed as described in Materials and Methods. The data shown are representative of three independent experiments. DAPI, 4′,6′-diamidino-2-phenylindole. (C) Quiescent Swiss 3T3 cells were induced with PDGF for 11.75 h, treated with BrdU (20 μM) for 15 min, and then fixed. The number of BrdU-positive cells in each treatment group was quantitated by FACS analysis as described in Materials and Methods. The data shown are the mean ± standard error from triplicate determinations and are representative of three independent experiments.