Heterodimerization with different Jun proteins controls c-Fos intranuclear dynamics and distribution

Abstract

The c-Fos proto-oncogenic transcription factor defines a multigene family controlling many processes both at the cell and the whole organism level. To bind to its target AP-1/12-O-tetradecanoylphorbol-13-acetate-responsive element or cAMP-responsive element DNA sequences in gene promoters and exert its transcriptional part, c-Fos must heterodimerize with other bZip proteins, its best studied partners being the Jun proteins (c-Jun, JunB, and JunD). c-Fos expression is regulated at many transcriptional and post-transcriptional levels, yet little is known on how its localization is dynamically regulated in the cell. Here we have investigated its intranuclear mobility using fluorescence recovery after photobleaching, genetic, and biochemical approaches. Whereas monomeric c-Fos is highly mobile and distributed evenly with nucleolar exclusion in the nucleus, heterodimerization with c-Jun entails intranuclear redistribution and dramatic reduction in mobility of c-Fos caused by predominant association with the nuclear matrix independently of any binding to AP-1/12-O-tetradecanoylphorbol-13-acetate-responsive element or cAMP-responsive element sequences. In contrast to c-Jun, dimerization with JunB does not detectably affect c-Fos mobility. However, dimerization with JunB affects intranuclear distribution with significant differences in the localization of c-Fos.c-Jun and c-Fos.JunB dimers. Moreover, c-Jun and JunB exert comparable effects on another Fos family member, Fra-1. Thus, we report a novel regulation, i.e. differentially regulated intranuclear mobility and distribution of Fos proteins by their Jun partners, and suggest the existence of intranuclear storage sites for latent c-Fos.c-Jun AP-1 complexes. This may affect the numerous physiopathological functions these transcription factors control.

FIGURE 1.

Monomeric c-Fos is highly mobile in the nucleus. A, comparison of transfected EGFP-c-Fos and endogenous c-Fos expressions. HeLa cells were either stimulated for 1 h with 20% serum (left panel) or transfected for 16 h with the EGFP-c-Fos-encoding plasmid (right panel). After cell fixation and permeabilization, c-Fos and EGFP-c-Fos were stained with a rabbit anti-c-Fos antiserum followed by an Alexa 647-labeled anti-rabbit antiserum. EGFP-c-Fos-expressing cells emitting a far red signal close to that of cells expressing endogenous c-Fos were selected and observed in the green channel to set up the green fluorescence intensity range usable in FRAP experiments. B, FRAP experiment. Asynchronously growing HeLa cells were transfected with expression plasmids encoding EGFP-c-Fos or EGFP, and FRAP experiments were carried out as described under “Materials and Methods.” A typical experiment with EGFP-c-Fos is presented. Arrows indicate the bleached area, before, during, and after the bleach. C, FRAP data. Typical fluorescence recovery curves are presented for both EGFP- and EFGP-c-Fos-expressing cells. <t½> and F_mob30 calculated from 20 FRAP experiments are given. They include standard deviations. D, immunoblotting analysis of cell fractions. Cells transfected as in A were lysed in the presence of Triton X-100, and soluble (S), wash (W), and nonsoluble (NS) fractions were prepared and analyzed by immunoblotting as described under “Materials and Methods.” Phax and topoisomerase I (Topo 1) were used as internal controls of soluble and nonsoluble proteins, respectively.

FIGURE 2.

Reduced intranuclear mobility of c-Fos in the presence of c-Jun. A, alteration of intranuclear distribution of EGFP-c-Fos in the presence of c-Jun. HeLa cells were transfected with EGFP-c-Fos in the absence or in the presence of a 2-fold excess of c-Jun expression plasmid and analyzed by confocal microscopy on living cells. B, F_mob30 of EGFP-c-Fos in the presence of varying amounts of c-Jun. HeLa cells were co-transfected with c-Jun and EGFP-c-Fos expression plasmids in the indicated ratios. FRAP experiments were performed as in Fig. 1A. F_mob30 values were calculated 30 s after the end of the bleach from 20 experiments for each condition and presented as histograms. The error bars indicate standard deviations. C, FRAP experiment. Asynchronously growing HeLa cells were transfected with expression plasmids encoding EGFP with or without a 2-fold excess of c-Jun vector. Each curve corresponds to the averages of 20 FRAP experiments. D and E, cell fractionation experiments. Fractionation experiments were carried out as in Fig. 1C using HeLa cells co-transfected with expression plasmids for (i) EGFP-c-Fos in the absence or in the presence of a 2-fold excess of c-Jun expression plasmid (D) or (ii) c-Fos in the absence or in the presence of a 2-fold excess of c-Jun expression plasmid (E). S, soluble; W, wash; NS, nonsoluble.

FIGURE 3.

Role of heterodimerization with c-Jun and binding to genomic AP-1 DNA sequences on c-Fos intranuclear dynamics. A, structures of wild type and mutant c-Fos and c-Jun proteins. B, F_mob30 of wild type and mutant EGFP-c-Fos proteins in the presence of wild type and mutant c-Jun. Co-transfections were conducted in the presence of 2-fold more c-Jun expression plasmids than vectors for either wild type or mutant EGFP-c-Fos. FRAP experiments were conducted as in Fig. 1B. F_mob30 are the averages of 6–20 experiments. The given values include standard deviations. C, localization of wild type and mutant EGFP-c-Fos in the presence of wild type c-Jun. Transfection conditions were as in B. Confocal microscopy was conducted on living cells. D, dimerization of EGFP-c-Fos mutants with c-Jun. Transfection conditions were as in B. Co-immunoprecipitations were performed with the anti-FLAG antibody, because c-Jun constructs contained a FLAG tag at the C terminus, and immunoblotting experiments were conducted with the indicated antibodies as described under “Materials and Methods.” E, cell fractionation experiments. Transfection conditions were as in B, and cell fractionations were conducted and analyzed as in Fig. 1C. T, total cell extract; SN, supernatant; S, soluble; W, wash; NS, nonsoluble; IP, immunoprecipitation.

FIGURE 4.

Effect of JunB on c-Fos intranuclear distribution and mobility. A, heterodimerization of JunB-FLAG with EGFP-c-Fos. The expression plasmid for EGFP-c-Fos was transfected in HeLa cells in the presence of a 2-fold excess of JunB-FLAG construct. Co-immunoprecipitations were conducted with the anti-FLAG antibody as in Fig. 3D. The antibodies used in immunoblotting analyses are indicated. B, intranuclear distribution of EGFP-c-Fos in the presence of JunB. EGFP-c-Fos localization in the presence of JunB-FLAG was assessed by confocal microscopy on living cells. The JunB versus EGFP-c-Fos expression plasmid ratio was of 2. C, FRAP experiments. FRAP experiments were conducted in HeLa cells transfected with expression plasmids for either EGFP-c-Fos or EGFP-c-Fos + JunB. In the latter case, the plasmid ratio was 2. The curves correspond to the averages of 20 FRAP experiments in each case. D, mobility of EGFP-c-Fos in the presence of different amounts of JunB. HeLa cells were transfected in the presence of different ratios of JunB versus EGFP-c-Fos plasmids as indicated. F_mob30 were calculated from 15–20 FRAP experiments in each case. E, cell fractionation experiments. Cell fractionation experiments of cells transfected with plasmids encoding JunB and EGFP-c-Fos in a ratio of 2 were conducted and analyzed as in Fig. 1D. T, total cell extract; SN, supernatant; IP, immunoprecipitation; S, soluble; W, wash; NS, nonsoluble.

FIGURE 5.

Alteration of Fra-1 intranuclear distribution and mobility by c-Jun and JunB. A, dimerization of EGFP-Fra-1 with c-Jun and JunB. Heterodimerization assays were conducted as in Fig. 3D using HeLa cells transfected with plasmids for either c-Jun-FLAG + EGFP-Fra-1 or JunB-FLAG + EGFP-Fra-1 in a ratio of 2. B, intracellular localization of EGFP-Fra-1 in the presence of c-Jun and JunB in a 2-fold excess. Intracellular localization was assessed on living cells by confocal microscopy analysis of HeLa cells transfected as in A. C, FRAP experiments. FRAP experiments were conducted in HeLa cells transfected with expression plasmids for either EGFP-Fra1 or EGFP-Fra1 and a 2-fold excess of JunB- or c-Jun plasmid. The curves correspond to the averages of 10–20 FRAP experiments. D, F_mob30 of EGFP-Fra-1 in the presence of c-Jun and JunB. Transfection conditions were as in A. F_mob30 were calculated from more than 10 FRAP experiments. E, fractionation experiments. Fractionation and analyses of HeLa cells transfected as in A were conducted as in Fig. 1C. T, total cell extract; SN, supernatant; IP, immunoprecipitation; S, soluble; W, wash; NS, nonsoluble.

FIGURE 6.

Cell and subnuclear fractionation. HeLa cells were (i) transfected with the EGFP-c-Fos vector in the absence or in the presence of an equivalent vector for either c-Jun or JunB in a 1:2 ratio or (ii) simply stimulated with 20% serum for 1 h. They were then fractionated as described under “Materials and Methods.” Equivalent amounts of each fraction were loaded in each lane for immunoblotting analysis. The names of the various proteins assayed are indicated in the figure. T, total cell extract; S1, Triton X-100-soluble fraction; W, wash; S2, soluble fraction after MNase digestion; S3, soluble fraction after 2 m NaCl treatment; P, nuclear matrix fraction.

FIGURE 7.

Microscopic analysis of c-Fos and EGFP-c-Fos intranuclear localization in fractionated cells. These experiments were carried out in parallel with those presented in Fig. 6 with HeLa cells seeded and grown on coverslips. Endogenous c-Fos was detected using sequentially the sc52 rabbit anti-c-Fos antibody and an Alexa 488-labeled anti-rabbit secondary antibody. The nuclei were stained with Hoechst 33342. All of the pictures in the EGFP channel were acquired using the same time exposure.