ERK1 and ERK2 mitogen-activated protein kinases affect Ras-dependent cell signaling differentially

Abstract

Background: The mitogen-activated protein (MAP) kinases p44ERK1 and p42ERK2 are crucial components of the regulatory machinery underlying normal and malignant cell proliferation. A currently accepted model maintains that ERK1 and ERK2 are regulated similarly and contribute to intracellular signaling by phosphorylating a largely common subset of substrates, both in the cytosol and in the nucleus.

Results: Here, we show that ablation of ERK1 in mouse embryo fibroblasts and NIH 3T3 cells by gene targeting and RNA interference results in an enhancement of ERK2-dependent signaling and in a significant growth advantage. By contrast, knockdown of ERK2 almost completely abolishes normal and Ras-dependent cell proliferation. Ectopic expression of ERK1 but not of ERK2 in NIH 3T3 cells inhibits oncogenic Ras-mediated proliferation and colony formation. These phenotypes are independent of the kinase activity of ERK1, as expression of a catalytically inactive form of ERK1 is equally effective. Finally, ectopic expression of ERK1 but not ERK2 is sufficient to attenuate Ras-dependent tumor formation in nude mice.

Conclusion: These results reveal an unexpected interplay between ERK1 and ERK2 in transducing Ras-dependent cell signaling and proliferation. Whereas ERK2 seems to have a positive role in controlling normal and Ras-dependent cell proliferation, ERK1 probably affects the overall signaling output of the cell by antagonizing ERK2 activity.

Figure 1

ERK1 ablation in mouse embryo fibroblasts results in enhancement of ERK2 activity and facilitates cell proliferation. (a) Wild-type (+) and ERK1-deficient (-) mouse embryonic fibroblasts (MEFs) were serum starved for 24 h and then stimulated with 20% serum for the indicated times. Western blotting was performed with both anti-ERK and anti-phospho-ERK antibodies. (b) Bands from (a) were quantified and the fold increase in phospho-ERK2 levels over total ERK2 levels calculated. (c) RNA from cells stimulated as in (a) was subjected to an RNase protection assay and probed for either c-fos or zif-268. A histone H4 probe was used as internal standard for normalization. (d) Wild-type (+) and ERK1-deficient (-) MEFs were seeded in triplicate in the presence of either 10% or 2.5% serum and cells were counted after the indicated times. Data are the mean ± standard error of the mean (SEM) of three independent experiments.

Figure 2

ERK-specific gene silencing unmasks differential roles for ERK1 and ERK2 in cell signaling and proliferation. (a) Schematic representation (top) of the proviral vector form used in shRNA-mediated RNA interference. ΔU3, R and U5 constitute a chimeric long terminal repeat (LTR) of the HIV-1 5' LTR with a deletion in U3 abolishing LTR mediated transcription; SD and SA, splice donor and acceptor sites; ψ encapsidation signal including the 5' portion of the gag gene (GA); RRE, Rev-response element; cPPT, central polypurine tract; shRNA, small hairpin RNA; H1, human H1 promoter; mPGK, mouse phosphoglycerate kinase promoter; Puro, puromycin-resistance gene; WPRE, woodchuck hepatitis virus post-transcription regulatory element. The western blot (bottom) shows expression levels of ERK proteins in wild-type MEFs transduced with equal amounts of lentiviral vectors carrying the indicated knock-down (KD) shRNA cassette or the corresponding control sequence (ctr). α-tubulin was used as a loading control. (b) Wild type (+), ERK1 KD or ERK2 KD MEFs were serum starved for 24 h and then stimulated with 20% serum for 5, 10, 30, 60 and 120 min. Western blots were analyzed with anti-phospho-ERK and anti-ERK antibodies, as in Figure 1. (c) Bands from (b) were quantified and fold increases in phospho-ERK2 or phospho-ERK1 levels over total ERK2 or total ERK1 levels calculated. Mean ± SEM of three experiments is indicated. (d) Growth curve of wild-type, ERK1 and ERK2 KD fibroblasts and their corresponding controls, seeded in triplicate in the presence of 10% serum and 2 μg/ml puromycin and counted after the indicated times. The data are the mean of three independent experiments ± SEM.

Figure 3

ERK-specific gene silencing in NIH 3T3 cells differentially affects MEK-ERK interactions. (a) ERK1- and ERK2-specific NIH 3T3 clones with stable shRNA expression only (left) or also co-transfected with H-Ras^Q61L (right) were isolated and checked for ERK expression levels by western blot analysis, as in Figure 1. Two clones (I and II) for each transfection are shown. (b) Lysates from wild-type NIH 3T3 control, ERK1 KD and ERK2 KD clones growing in 10% serum were incubated with anti-MEK-1/2 polyclonal antibody. Immune complexes (IP) were resolved in SDS-PAGE and western blotted (WB) with polyclonal anti-ERK1 (sc-94, top) and anti-ERK2 (sc-153, bottom) antibodies. (c) Bands from (b) were quantified and the relative fold increase in ERK1 and ERK2 levels in the knockdown samples over the corresponding wild-type controls were calculated (only samples probed with anti-ERK antibody sc-94 are indicated). Data are representative of three independent experiments, expressed as mean ± SEM.

Figure 4

ERK1 knockdown in NIH 3T3 cells facilitates growth in colony formation assays, whereas ERK2 knockdown shows inhibitory effects. NIH 3T3 cells were transfected as indicated with the specific shRNA construct (KD) against ERK1 or ERK2 or the corresponding control sequence (ctr), all cloned into the pSUPER_Puro vector; cells were transfected either with shRNA alone or also with an oncogenic form of H-Ras (Ras^Q61L), and colony formation was scored after 10 days. (a) Representative plates; (b) graph of the number of colonies formed (the result of four independent experiments, expressed as mean ± SEM). Asterisks indicate a statistically significant genotype effect calculated from a post-hoc comparison in one-way ANOVA (Scheffe's test: control versus ERK1 KD; control versus ERK2 KD; Ras^Q61L versus Ras^Q61L-ERK1 KD; Ras^Q61L versus Ras^Q61L-ERK2 KD); single asterisk, p < 0.01; double asterisk, p < 0.0001.

Figure 5

Overexpression of ERK1 attenuates Ras-dependent cell growth in NIH 3T3 cells. (a) NIH 3T3 cells were stably transfected with different plasmids bearing hemagglutinin (HA) epitope-tagged ERK1, ERK1^K72R, ERK2 or p38 or Myc epitope-tagged Ras^Q61L, all in the vector pMEX. Stable transfectants were generated and expression of the transgene monitored by western blotting. Clones were also serum starved and stimulated with 20% serum for 10 min and extracts were probed with either anti-ERK or anti-phospho-ERK antibodies. (b) Three independent NIH 3T3 clones per plasmid from (a) were plated in 10% serum and their growth was monitored for 5 days, as in Figure 1d. The data are the mean ± SEM of three independent experiments. (c) Expression of double transfectants was determined as in (a). (d) Clones from (c) were monitored for cell growth as in (b). Data are expressed as mean ± SEM of three independent experiments.

Figure 6

Ectopic expression of ERK1 in NIH 3T3 cells inhibits Ras-mediated colony formation. (a,b) NIH 3T3 cells were transfected as indicated and colony formation was scored after 10 days. Graphs represent quantitations of six independent experiments, expressed as mean ± SEM. Double asterisk indicates a genotype effect that is statistically significant (p < 0.0001), calculated from a post-hoc comparison in one-way ANOVA (Scheffe's test: Ras^Q61L-ERK1 versus Ras^Q61L-pMex; Ras^Q61L-ERK1^K72R versus Ras^Q61L-pMex; Ras^Q61L-ERK2^K52R versus Ras^Q61L-pMex). (c) A representative plate for each clone from (a,b) is shown.

Figure 7

ERK1 expression inhibits Ras-dependent tumor formation in nude mice. (a) NIH 3T3 clones were transfected as indicated and expression of the relevant transgenes assessed by western blotting. (b) Growth of tumors in injected nude mice was monitored over 6 days starting from day 4 after injection, by determining the skin area covered by the tumor mass (mm²). The data are expressed as mean ± SEM of two independent experiments (ten animals per clone). (c) Representative tumors after sacrifice at day 10 are shown. (d) Mean weight (± SEM) of the different tumor samples is indicated. Asterisk indicates a genotype effect significant at p < 0.001, calculated using a post-hoc comparison in one-way ANOVA (Scheffe's test: Ras^Q61L-ERK1 versus Ras^Q61L-pMex; Ras^Q61L-ERK1^K72R versus Ras^Q61L-pMex).

Figure 8

ERK2 activation is significantly reduced in tumors overexpressing ERK1. (a) Two individual tumors for each clone were explanted from treated mice and subjected to western blot analysis to determine ERK2 activation and transgene expression. (b) Mean ± SEM of the data in (a) are plotted as indicated.