Ribosomal subunit kinase-2 is required for growth factor-stimulated transcription of the c-Fos gene

Abstract

Ribosomal subunit kinases (Rsk) have been implicated in the regulation of transcription by phosphorylating and thereby activating numerous transcription factors, such as c-Fos, cAMP responsive element binding protein (CREB), and nuclear receptors. Here we describe the generation and characterization of immortalized embryonic fibroblast cell lines from mice in which the Rsk-2 gene was disrupted by homologous recombinant gene targeting. Rsk-2-deficient (knockout or KO) cell lines have no detectable Rsk-2 protein, whereas Rsk-1 expression is unaltered as compared with cell lines derived from wild-type control mice. KO cells exhibit a major reduction in platelet-derived growth factor (PDGF) and insulin-like growth factor (IGF)-1-stimulated expression of the immediate-early gene c-Fos. This results primarily from a reduced transcriptional activation of the ternary complex factor Elk-1 and reduced activation of the serum response factor. The reduced Elk-1 activation in KO cells occurs despite normal activation of the mitogen-activated protein kinase pathway and normal PDGF- and IGF-1-stimulated Elk-1 phosphorylation. By contrast, PDGF- and IGF-1-stimulated phosphorylation and transcriptional activation of CREB is unaltered in KO cells. Thus Rsk-2 is required for growth factor-stimulated expression of c-Fos and transcriptional activation of Elk-1 and the serum response factor, but not for activation of CREB or the mitogen-activated protein kinase pathway in response to PDGF and IGF-1 stimulation.

Figure 1

Rsk-2 is absent in 3T3 cell lines from KO embryos. (A) PCR genotyping to identify male embryos (Top) and the presence of the neomycin-resistance gene in the targeted Rsk-2 allele (Middle). Western blot analyses using Rsk-2-specific antibody on cell extracts from primary mouse embryonic fibroblasts (Bottom). (B) Western blot analysis on extracts from immortalized 3T3 cells of control embryo no. 7 (WT) and KO embryo no. 6 (KO) with Rsk-1-specific (Upper) and Rsk-2-specific antibodies (Lower).

Figure 2

PDGF- and IGF-1-stimulated activation of p70S6-kinase, Akt, and MAPK is unaltered in KO cells. Western blot analyses were performed on cell extracts from control (WT) and KO cells, which had been stimulated with PGDF or IGF-1 for the indicated times (min). Immunoblotting was performed with antibodies against phosphorylated p70S6-kinase (A, Top and Middle), phosphorylated Akt (B, Top and Middle), and phosphorylated MAPK (C, Top and Middle) or with corresponding antibodies, detecting these proteins independent of activation (A–C, Bottom)

Figure 3

Growth factor-stimulated expression of c-Fos is reduced in KO cells. (A) Northern blot analyses with c-Fos and GAPDH probes were performed on RNA extracted from WT or KO cells stimulated for the indicated times (min) with PDGF. The autoradiography after incubation with the c-Fos probe (Top). The integrity of RNA on the ethidium bromide-stained gel (Middle), and the autoradiography after hybridization with the GAPDH probe (Bottom). (B) Quantification was performed by densitometric scanning of the specific c-Fos signal/intensity of the GAPDH signal. Data are the mean ± SEM of three independent experiments expressed as percentage of maximum intensity in WT cells (*, P < 0.05; **, P < 0.01). (C) Schematic representation of the c-Fos promoter-luciferase constructs. WT c-Fos contains base pairs −734 to +43 of the human c-Fos gene cloned into promoterless luciferase gene (pGL2basic). In the construct δTCF-c-Fos, the binding site for TCF (base pair −320 to −328) was mutated (12). WT cells (black bars) and KO cells (gray bars) were cotransfected with the reporter plasmid WT c-Fos (black bars with white stippling) or the mutant reporter (gray bars with white stippling). After overnight culture in serum-free medium, cells were treated with PDGF or IGF-1 for 3 hr, and cell extracts were subsequently assayed for luciferase and β-galactosidase activity. Data represent the stimulation of luciferase activity/β-galactosidase activity in PDGF- and IGF-1-stimulated vs. untreated cells. Data are the mean of four separate experiments ± SEM.

Figure 4

PDGF- and IGF-1-stimulated transcriptional activity of Elk-1 is reduced in the presence of unaltered Elk-1 phosphorylation. (A) Western blot analyses were performed with an anti-Elk-1 antibody (Bottom) or an phospho-specific anti-Elk-1 antibody (Top and Middle) on cell extracts of PDGF- (Top and Bottom) or IGF-1-treated (Middle) cells. (B) Schematic representation of the heterologous Elk-1 reporter system. (C) WT and KO cells were cotransfected with the described reporter plasmid, Gal-4–Elk-1 and the plasmid PEF–β-galactosidase (PEF–β-gal) expressing β-galactosidase under control of the elongation factor promoter. After culture in serum-free medium for 36 hr, cells were left untreated (white bars) or treated with PDGF (black bars) or IGF-1 (gray bars) for 4 hr before lysis. Data represent the stimulation of luciferase activity/β-galactosidase activity in stimulated vs. untreated cells. Data are the mean of four independent experiments ± SEM. (*, P < 0.05; **, P < 0.01). (D) Cotransfections were performed with the described Elk-1-reporter in the presence of pcDNA or a MEK-1-expression plasmid (black bars) (**, P < 0.01). (E) KO cells were transfected with 5XUAS-luciferase and Gal-4–ElkC together with the expression plasmid pcDNA (white bar), RSV-WTRsk (black bar), or RSV-DNRsk (gray bar) expressing wild-type Rsk (WTRsk) or a dominant–negative mutant of Rsk-2 (DNRsk). Data presented are the mean of four experiments ± SEM expressed as stimulation compared with pcDNA-transfected cells (**, P < 0.01).

Figure 5

Transcriptional activation of a heterologous SRF in response to PDGF and MEK-1 stimulation is reduced in KO cells. (A) Schematic representation of the heterologous SRF-reporter system with a fusion protein of the Gal-4–DNA-binding domain (amino acids 1–147 of Gal-4) and human SRF (amino acids 10–301 of SRF). (B) WT and KO cells were cotransfected with the described reporter plasmid. After overnight culture in serum-free medium, cells were left untreated or treated with PDGF for 3 hr before lysis. Data represent the stimulation of luciferase activity/β-galactosidase activity in stimulated vs. untreated cells. Data are the mean of four independent experiments ± SEM. (C) Cotransfections were performed as described for B with the exception that PDGF stimulation was replaced by cotransfection of an MEK-1 expression plasmid (**, P < 0.01).

Figure 6

PDGF- and IGF-1-stimulated phosphorylation and activation of CREB are unaltered in the absence of Rsk-2. (A) Western blot analysis using an anti-CREB antibody (Bottom) shows comparable cellular content of CREB in WT and KO cells. Western blot with an Ser-133 phospho-specific anti-CREB antibody reveals normal phosphorylation of CREB in KO cells in response to PDGF (Top) and IGF-1 stimulation (Middle). (B) Schematic representation of the heterologous CREB reporter system. (C) WT and KO cells were cotransfected with the described reporter plasmid. After serum depletion for 36 hr, cells were left untreated (open bars) or treated with PDGF (black bars) or IGF-1 (gray bars) for 4 hr before lysis. Data represent the stimulation of luciferase activity/β-galactosidase activity in stimulated vs. untreated cells.