Pathway- and expression level-dependent effects of oncogenic N-Ras: p27(Kip1) mislocalization by the Ral-GEF pathway and Erk-mediated interference with Smad signaling

Abstract

Overactivation of Ras pathways contributes to oncogenesis and metastasis of epithelial cells in several ways, including interference with cell cycle regulation via the CDK inhibitor p27(Kip1) (p27) and disruption of transforming growth factor beta (TGF-beta) anti-proliferative activity. Here, we show that at high expression levels, constitutively active N-Ras induces cytoplasmic mislocalization of murine and human p27 via the Ral-GEF pathway and disrupts TGF-beta-mediated Smad nuclear translocation by activation of the Mek/Erk pathway. While human p27 could also be mislocalized via the phosphatidylinositol 3-kinase/Akt pathway, only Ral-GEF activation was effective for murine p27, which lacks the Thr157 Akt phosphorylation site of human p27. This establishes a novel role for the Ral-GEF pathway in regulating p27 localization. Interference with either Smad translocation or p27 nuclear localization was sufficient to disrupt TGF-beta growth inhibition. Moreover, expression of activated N-Ras or specific effector loop mutants at lower levels using retroviral vectors induced p27 mislocalization but did not inhibit Smad2/3 translocation, indicating that the effects on p27 localization occur at lower levels of activated Ras. These findings have important implications for the contribution of activated Ras to oncogenesis and for the conversion of TGF-beta from an inhibitory to a metastatic factor in some epithelial tumors.

FIG. 1.

Activation of the Ral-GEF pathway by N-Ras^61K/37G induces mislocalization of both murine and human p27. Mv1Lu cells (A to C) or HEK-293T cells (D) were cotransfected (see Materials and Methods) with (A and B) murine GFP-p27 in pEGFP and an excess (sixfold) of empty pcDNA3 vector (Ctrl), N-Ras^61K, or one of its effector domain mutants in pcDNA3. (C) Murine GFP-p27 was replaced by human HA-p27 in pcDNA3. (D) pEGFP (transfection marker) replaced murine GFP-p27. After 24 h, the cells were fixed with 4% paraformaldehyde. For immunofluorescent labeling (C and D), the paraformaldehyde-fixed cells were permeabilized with 0.2% Triton X-100 prior to being immunostained. Fluorescence images wererecorded by a charge-coupled device camera. Bar, 20 μm. (A) Typical images of murine GFP-p27 localization. (B) Quantification of murine GFP-p27 localization. (C) Quantification of human HA-p27 localization. To label HA-p27, the cells were incubated successively with (i) rabbit anti-HA (2 μg/ml), (ii) biotinylated GαR IgG (5 μg/ml), and (iii) Cy3-streptavidin (1.2 μg/ml). (D) Quantification of the localization of endogenous human p27. Endogenous p27 in HEK-293T cells was labeled by successive incubations with (i) rabbit anti-p27 (1.25 μg/ml), (ii) biotinylated GαR IgG (5 μg/ml), and (iii) Cy3-streptavidin (1.2 μg/ml). The bars are means plus standard errors of the mean of five to seven samples in each case, scoring 100 transfected cells per sample for nuclear and cytoplasmic localization of p27. The asterisks indicate significant differences from the control (*, P < 0.003; **, P < 10⁻⁶; Student's t test). Mainly nuclear localization is evident for the control and for the Mek/Erk-activating mutant N-Ras^61K/35S. N-Ras^61K/37G, which activates Ral-GEF but not the Mek/Erk or the PI3K pathway, was as effective as constitutively active N-Ras^61K in mislocalizing both murine and human p27. In contrast, the PI3K-activating mutant N-Ras^61K/40C was highly effective in mislocalizing human HA-p27 but had a much weaker effect on the localization of murine GFP-p27. Control experiments (not shown) on Mv1Lu cells transfected with dominant-negative H-Ras^17N in pRSV (54) or N-Ras^17N in pEGFP showed no mislocalization of murine p27 and even a slight increase (to 86 to 92%) in the percentage of cells with nuclear p27.

FIG. 2.

Impairment of TGF-β-induced Smad2/3 nuclear translocation by N-Ras^61K is mediated via the Mek/Erk pathway. Mv1Lu cells were cotransfected with murine GFP-p27 and N-Ras^61K mutants in pcDNA3 as in Fig. 1. In the control (Ctrl), empty pcDNA3 replaced the N-Ras construct. After 24 h, the cells were incubated without (A) or with (B) TGF-β1 (100 pM; 20 min; 37°C), fixed/permeabilized, and processed for immunofluorescence (see Materials and Methods). They were labeled successively with (i) rabbit anti-Smad3, reactive with both Smad3 and Smad2 (5 μg/ml); (ii) biotinylated goat anti-rabbit IgG (5 μg/ml); and (iii) Cy3-streptavidin (1.2 μg/ml). Bar, 20 μm. The white arrows in the Smad2/3 images indicate the transfected cells (which also show GFP-p27 fluorescence). As shown in the arrow-marked cells in panel B, in the absence of TGF-β, Smad2/3 was mainly cytoplasmic. TGF-β failed to induce Smad2/3 nuclear translocation in cells transfected with constitutively active N-Ras^61K and the N-Ras^61K/35S mutant but not with the other mutants. (C) Quantification of Smad2/3 localization in the entire transfected-cell population. Transfected cells were identified by GFP-p27 fluorescence, and Smad2/3 nuclear localization was scored in the GFP-expressing cells. (D) More pronounced effects are observedin cells displaying cytoplasmic p27 localization. Only transfected cells exhibiting cytoplasmic GFP-p27 were scored. The results shown are with TGF-β1; in the absence of ligand, the results were similar to those in panel C (not shown). In both panels C and D, the percentages of cells with predominantly nuclear Smad2/3 were derived by scoring 100 cells per sample in four or five samples. The bars are means plus standard errors of the mean. The asterisks indicate a significant difference from the control in the presence of TGF-β (P < 0.0003, except for N-Ras^61K/35S in panel D, which yielded a P value of <0.0008).

FIG. 3.

Ral^28N blocks p27 cytoplasmic mislocalization by N-Ras^61K, while Mek inhibition restores TGF-β-mediated Smad2/3 nuclear translocation. (A and B) Pharmacological inhibitors are effective in blocking growth factor stimulation in Mv1Lu cells. After 24 h in serum-free medium, cells were incubated with LY294002 (20 μM) or U0126 (50 μM) for 12 h. They were then stimulated (100 ng/ml; 5 min) with PDGF (A) or EGF (B), lysed, and subjected to 12.5% SDS-PAGE, loading 100 μg protein/lane for Akt and 60 μg/lane for Erk analysis. Electrotransfer was followed by immunoblotting with anti-Akt or anti-Erk antibodies to determine the total levels of these proteins or with antibodies specific to phospho-Akt (P-Akt) and phospho-Erk (P-Erk) to determine the activated forms. Visualization was by ECL. PDGF induced a 3.5- ± 0.8-fold increase in P-Akt, and EGF induced a 7.0- ± 1.2-fold increase in P-Erk (n = 3 in both cases); both effects were totally blocked by the respective inhibitors LY294002 and U0126. (C) Interference of inhibitors with N-Ras^61K effects on p27 and Smad2/3 localization. Mv1Lu cells were cotransfected with murine GFP-p27 together with an excess (sixfold) of empty pcDNA3 vector (Ctrl) or N-Ras^61K in pcDNA3. To assess the effects of dominant-negative Ral^28N (DN Ral), cells were triply transfected with GFP-p27 together with both N-Ras^61K and Ral^28N at DNA ratios of 1:6:6, respectively. After 24 h, the cells were incubated for 16 h with LY294002 (20 μM) or U0126 (50 μM); control samples and cells transfected with Ral^28N were left untreated. The cells were then incubated with TGF-β1 (100 pM; 20 min; 37°C), fixed/permeabilized, and labeled for Smad2/3 as in Fig. 2. Typical images of GFP-p27 (green) and Smad2/3 (Cy3; red) are shown. In the Smad2/3 images (bottom row), arrows indicate the transfected cells, identified by GFP-p27 fluorescence (top row). Bar, 20 μm. (D and E) Quantification of p27 and Smad2/3 localization. Cells were scored for nuclear versus cytoplasmic localization of GFP-p27 as in Fig. 1. The Smad2/3 localization results shown were obtained by counting cells exhibiting cytoplasmic GFP-p27, as described in the legend to Fig. 2D. Qualitatively similar, albeit somewhat smaller, effects of N-Ras^61K on Smad2/3 nuclear translocation in response to TGF-β were observed in the entire population of transfected cells (not shown). The bars are means plus standard errors of the mean of three or four samples in each case, scoring 100 cells per sample.The asterisks indicate a significant difference (Student's t test) from the control (*, P < 10⁻⁴; **, P < 2 × 10⁻⁵).

FIG. 4.

Rlf-CAAX induces p27 mislocalization but does not affect Smad2/3 nuclear translocation. Mv1Lu cells were cotransfected with murine GFP-p27 (in pEGFP) together with a sixfold excess of HA-Rlf-CAAX (in pBABE-puro) or empty pBABE-puro vector (Ctrl). After 24 h, the cells were incubated with TGF-β1 (100 pM; 20 min; 37°C), fixed/permeabilized, and labeled for Smad2/3 as in Fig. 2. (A) Typical images of GFP-p27 and Smad2/3. Smad2/3 is endogenously expressed in all the cells, while Rlf-CAAX and GFP-p27 are expressed only in the transfected cells, identified by GFP-p27 fluorescence (marked by arrows in the Smad2/3 images). Bar, 20 μm. (B and C) Quantification of p27 and Smad2/3 localization. The percentage of cells with nuclear Smad2/3 was scored in cells with cytoplasmic p27 localization, as in Fig. 2D. Similar effects (not shown) were obtained for Smad2/3 in the entire population of transfected cells. In the absence of TGF-β, only 4 to 6% of the cells exhibited nuclear Smad2/3 labeling, both in mock-transfected and in Rlf-CAAX-transfected cells. The bars are means plus standard errors of the mean of three or four samples (100 cells per sample). The asterisks indicate a significant difference from the control (P < 1.5 × 10⁻⁶).

FIG. 5.

Growth arrest by TGF-β is disrupted by N-Ras mutants that mediate either p27 mislocalization or impairment of Smad2/3 nuclear translocation. Mv1Lu cells were cotransfected with GFP (pEGFP vector, as a marker for transfected cells) and an excess (sixfold) of empty pcDNA3 vector (Ctrl), N-Ras^61K, or one of the effector domain mutants in pcDNA3. After 24 h, the cells were incubated without (A) or with (B) TGF-β1 (10 pM; 24 h; 37°C). They were then subjected to BrdU incorporation and immunostaining (resulting in Cy3-labeled BrdU), as detailed in Materials and Methods. The arrows in the BrdU images (red) indicate transfected cells, identified by GFP fluorescence (green). Bar, 20 μm. (A and B) Typical images of BrdU incorporation. (C) Quantification of BrdU incorporation in Mv1Lu cells transiently expressing Ras effector loop mutants. Transfected cells (identified by GFP expression) were scored for nuclear BrdU labeling. The bars are means plus standard errors of the mean of four samples in each case, scoring 100 cells per sample. Inhibition of BrdU incorporation by TGF-β1 was highly significant (marked by asterisks) in the control cells (P < 2 × 10⁻⁶) and in cells expressing the N-Ras^61K/40C mutant (P < 0.005) but was completely lost in cells expressing N-Ras^61K, N-Ras^61K/37G, or N-Ras^61K/35S (P > 0.1; comparing pairs of samples without and with TGF-β).

FIG. 6.

Different dependences of p27 mislocalization and Smad2/3 nuclear translocation on the expression level of N-Ras^61K. (A) Ras expression levels in Mv1Lu cells transfected with N-Ras^61K in pcDNA3 and in retroviral vectors. Mv1Lu cells were transfected with empty vector [pcDNA3 or one of the retroviral vectors pMX-IRES-GFP1.1 or pZIP-NeoSV(X)1; Ctrl], with N-Ras^61K in pcDNA3, or with N-Ras^61K in one of the retroviral vectors (designated pMX or pZIP); a small amount of pEGFP (one-sixth) was included. The cells were lysed 24 h later. Transfection efficiencies were similar (∼40% expressing cells), as evaluated from the percent cells expressing GFP (by fluorescence microscopy) prior to lysis. Aliquots of the lysates (30 μg protein) were subjected to SDS-PAGE, followed by immunoblotting with pan-anti-Ras antibody (left), to determine total Ras. To determine the level of GTP-bound activated Ras, aliquots (500 μg protein) were subjected to the GST-RBD pull-down assay (see Materials and Methods), followed by SDS-PAGE and immunoblotting with pan-anti-Ras. The immunoblots were visualized by ECL. The data shown are representative of three independent experiments. Transfection with N-Ras^61K in pcDNA3 or in a retroviral vector resulted in 5.0- ± 0.2-fold and 2.1- ± 0.2-fold increases in activated Ras, respectively. Similar results (right) were obtained when blotting was performed with anti-N-Ras antibodies. (B) Typical images of p27 (GFP; green) and Smad2/3 (Cy3; red) localization. Mv1Lu cells were cotransfected with GFP-p27 in pEGFP, together with a sixfold excess of either empty vector (pcDNA3 or pMX; Ctrl), N-Ras^61K in pcDNA3, or N-Ras^61K in the pMX retroviral vector. N-Ras^61K in the pZIP retroviral vector yielded results similar to those obtained with the pMX vector (Fig. 7A). After 24 h, the cells were incubated with TGF-β1 (100 pM; 20 min; 37°C), fixed/permeabilized, and subjected to labeling of Smad2/3 as in Fig. 2. In the Smad2/3 images, arrows indicate the transfected cells, identified by GFP-p27 fluorescence. Bar, 20 μm. (C and D) Quantification of p27 and Smad2/3 localization. The percentage of cells with nuclear Smad2/3 was scored in cells with cytoplasmic p27 localization, as described in Fig. 2D. Similar, although somewhat smaller, effects were found for Smad2/3 in the entire population of transfected cells (data not shown). The bars are means plus standard errors of the mean of three or four samples (100 cells per sample) in each case. The asterisks indicate a significant difference from the control (P < 1.5 × 10⁻⁶ in panel C; P < 3 × 10⁻⁴ in panel D).

FIG. 7.

Selective impairment of p27 localization and TGF-β-mediated growth arrest, but not Smad nuclear translocation, by low levels of N-Ras^61K effector loop mutants. Mv1Lu cells were cotransfected with GFP-p27 (A and B) or GFP (C) in pEGFP, together with an excess of one of the N-Ras^61K effector loop mutants [in pZIP-NeoSV(X)1] as in Fig. 6B to D. The bars are means plus standard errors of the mean of three or four samples in each case, scoring 100 cells per sample. (A and B) Quantification of p27 and Smad2/3 localization. Twenty-four hours posttransfection, cells were incubated with 100 pM TGF-β1 (20 min) and assayed for p27 and Smad2/3 localization as in Fig. 6B to D. The asterisks indicate significant differences from the control (P < 2 × 10⁻⁴). (C) TGF-β-mediated inhibition of BrdU incorporation. Twenty-four hours posttransfection, cells were incubated with TGF-β1 (10 pM; 24 h), followed by a BrdU incorporation assay as in Fig. 5. Inhibition of BrdU nuclear incorporation by TGF-β1 was significant (asterisks) in the control (cotransfection with an empty pZIP vector) and in cells expressing N-Ras^61K/40C or N-Ras^61K/35S (P < 0.005), but was completely lost (P > 0.2) upon expression of N-Ras^61K or N-Ras^61K/37G.