Original Article

Oncogene (2007) 26, 4774–4796; doi:10.1038/sj.onc.1210271; published online 12 February 2007

Ras effector pathways modulate scatter factor-stimulated NF-kappaB signaling and protection against DNA damage

S Fan1, Q Meng1, J J Laterra2 and E M Rosen1

  1. 1Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University, Washington, DC, USA
  2. 2Department of Neurology, The Kennedy Krieger Institute and Johns Hopkins University School of Medicine, Baltimore, MD, USA

Correspondence: Dr EM Rosen, Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University, 3970 Reservoir Road, NW, Box 571469, Washington, DC 20057-1469, USA. E-mail: emr36@georgetown.edu

Received 27 April 2006; Revised 30 October 2006; Accepted 14 November 2006; Published online 12 February 2007.

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Abstract

Scatter factor (SF) (hepatocyte growth factor) is a pleiotrophic cytokine that accumulates within tumors in vivo and protects tumor cells against cytotoxicity and apoptosis due to DNA damaging agents in vitro. Previous studies have established that SF-mediated cell protection involves antiapoptotic signaling from its receptor (c-Met) to PI3 kinase right arrow c-Akt right arrow Pak1 (p21-activated kinase -1) right arrow NF-kappaB (nuclear factor-kappa B). Here, we found that Ras proteins (H-Ras and R-Ras) enhance SF-mediated activation of NF-kappaB and protection of DU-145 and MDCK (Madin–Darby canine kidney) cells against the topoisomerase IIalpha inhibitor adriamycin. Studies of Ras effector loop mutants and their downstream effectors suggest that Ras/PI3 kinase and Ras/Raf1 pathways contribute to SF stimulation of NF-kappaB signaling and cell protection. Further studies revealed that Raf1 positively regulates the ability of SF to stimulate NF-kappaB activity and cell protection. The ability of Raf1 to stimulate NF-kappaB activity was not due to the classical Raf1 right arrow MEK1/2 right arrow ERK1/2 pathway. However, we found that a MEK3/6 right arrow p38 pathway contributes to SF-mediated activation of NF-kappaB. In contrast, RalA, a target of the Ras/RalGDS pathway negatively regulated the ability of SF to stimulate NF-kappaB activity and cell protection. Ras, Raf1 and RalA modulate SF stimulation of NF-kappaB activity, in part, by regulating IkappaB kinase (IKK)-beta kinase activity. These findings suggest that Ras/Raf1/RalA pathways may converge to modulate NF-kappaB activation and SF-mediated survival signaling at the IKK complex and/or a kinase upstream of this complex.

Keywords:

scatter factor, Ras, RalA, Raf1, protection, adriamycin

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Introduction

Scatter factor (SF) (also called hepatocyte growth factor (HGF)) stimulates invasion, proliferation, differentiation, angiogenesis and tumorigenesis in different cell types under different circumstances. The actions are mediated through its receptor, the c-Met tyrosine kinase (Bottaro et al., 1991). Although SF can stimulate apoptosis in hepatocarcinoma cells (Matteucci et al., 2003), in most cell types and contexts, SF is cytoprotective because of to its ability to stimulate antiapoptotic signaling and enhance cell survival (Frisch and Francis, 1994; Bardelli et al., 1996). SF was found to protect epithelial, carcinoma and glioma cells against DNA damaging agents, including adriamycin (ADR, a DNA topoisomerase IIalpha inhibitor), ionizing radiation, cis-platinum (a DNA cross-linking agent), and other agents (Fan et al., 1998, 2000, 2001; Bowers et al., 2000). These findings may be significant because expression of SF and c-Met are upregulated in various tumor types (e.g., breast cancers, gliomas and bladder cancers) (Jin et al., 1997; Rosen et al., 1997). Thus, the accumulation of SF and c-Met might contribute to chemoresistance and radioresistance of the tumors.

Studies of the mechanisms of SF protection against DNA damaging agents have identified some of the signal transduction pathway components that mediate cell protection. This pathway involves signaling through phosphatidylinositol-3-kinase (PI3K) and activation of c-Akt, a serine/threonine protein kinase (Bowers et al., 2000; Fan et al., 2000, 2001). The p21-associated kinase-1 (Pak1) acts downstream of c-Akt to promote cell survival; and nuclear factor-kappa B (NF-kappaB) and several of its target genes are downstream of these kinases in the SF-protection pathway (Fan et al., 2001, 2005). In addition to these survival-promoting proteins, the multi-substrate adapter Grb2-associated binder-1 (Gab1) and the tumor suppressor phosphatase and tensin homolog (PTEN) function upstream of c-Akt to inhibit cell protection by SF (Fan et al., 2001).

Previous studies have identified three effector signaling pathways through which oncogenic Ras signaling can stimulate tumorigenesis and other activities. These include pathways involving PI3K, Raf1 and RalGDS (Ral guanine nucleotide dissociation stimulator) (Chung et al., 1993; White et al., 1995; Yang et al., 1998; Peyssonnaux et al., 2000; McFarlin et al., 2003). Specific mutations of the 'effector loop' of the Ras protein can preserve some interactions with effector proteins, whereas disrupting others (de Vos et al., 1988; Milburn et al., 1990), thus enabling studies of one or another signaling pathway downstream of Ras. We have investigated the roles of Ras and several pathways downstream of Ras in mediating the ability of SF to stimulate NF-kappaB transcriptional activity and protection against ADR. These studies identify a stimulatory role for Raf1 and an inhibitory role for the small GTPase RalA in modulating SF-stimulated NF-kappaB activity and cell protection. They have also identified roles for several mitogen-activated protein (MAP) kinases in these processes.

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Results

Stimulation of NF-kappaB signaling by Ras proteins

We tested the effects of wild-type (wt) and mutant Ras proteins on NF-kappaB signaling using an NF-kappaB-Luc reporter to measure NF-kappaB activity. DU-145 prostate cancer or Madin–Darby canine kidney (MDCK) epithelial cells were transfected with the indicated vectors, treatedplusminusSF (100 ng/ml times 24 h) and harvested for luciferase assays. Typically, SF gave a 50- to 60-fold increase in NF-kappaB-Luc activity; and the empty pcDNA3 vector had no effect on activity. In combination with SF, an activated H-Ras codon 12 mutant (V12Ras) expression vector yielded a 6.3- to 8-fold higher NF-kappaB activity than SF alone (P<0.001, two-tailed t-test) (Figure 1a). Without SF, V12Ras gave only about 2.2- to 2.5-fold higher activity, suggesting that the effect of Ras alone is relatively modest. A farnesylation-defective mutant V12Ras that is unable to activate downstream effectors (V12S186Ras (Table 1)) gave little or no stimulation of NF-kappaB activity, in the absence or presence of SF.

Figure 1.
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Ability of Ras mutants to modulate SF stimulation of NF-kappaB transcriptional activity. (a) Effect of series of Ras mutant proteins on basal and SF-stimulated NF-kappaB-Luc activity. Subconfluent proliferating DU-145 cells or MDCK cells in 2 cm2 wells were co-transfected overnight with the indicated Ras vector and NF-kappaB-Luc reporter using Lipofectamine (0.25 mug plasmid DNA per vector). The wells were washed, postincubated in fresh culture mediumplusminusSF (100 ng/ml) for 24 h and harvested for luciferase assays. Luciferase values are expressed relative to control cells (no vector, 0 SF) and are meansplusminuss.e.m.s of four replicate wells. The data shown are representative of at least two independent experiments. (b) Effect of additional Ras proteins on basal and SF-stimulated NF-kappaB-Luc activity. Assays were performed exactly as described above for panel a, except using a different set of Ras vectors.

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Effector loop mutations alter the ability of Ras to activate the Raf1 vs PI3K vs RalGDS signaling pathways and preferentially activate one pathway but not others. A mutant that activates PI3K but not Raf1 or RalGDS (V12C40Ras) gave slightly more enhancement of SF-stimulated NF-kappaB activity (8.3- to 9-fold) (P<0.001) than V12Ras; whereas a mutant that selectively activates RalGDS (V12G37Ras) showed reduced ability to enhance SF-induced NF-kappaB activity (3.9- to 4.4-fold, P<0.001) (Figure 1a). Mutants that selectively activate Raf1 (V12S35Ras and V12E38Ras) (McFarlin et al., 2003) enhanced SF-induced NF-kappaB activity (P<0.001) to a somewhat lesser extent (3.9- to 6-fold) than V12Ras. All mutants gave less stimulation of NF-kappaB in the absence of SF (1.5- to 2.2-fold) than in its presence.

We further found that both wt-H-Ras and wt-R-Ras (the related Ras viral oncogene homolog) enhanced SF-stimulated NF-kappaB activity (P<0.001), by 5.5- to 5.8-fold and 3.8- to 5.2-fold, respectively (Figure 1b). An activated R-Ras mutant (R-RasV38) gave a similar or slightly higher increase in SF-stimulated NF-kappaB activity (P<0.001) than wt-R-Ras. In contrast, dominant-negative (DN) H-Ras (RasN17) and R-Ras (R-RasN43) reduced SF-stimulated NF-kappaB activity to 18–32% of the control values (P<0.001). The stimulation of NF-kappaB activity by wt-H-Ras, wt-R-Ras and R-RasV38 was much smaller in the absence of SF (1.4- to 1.9-fold). RasN17 and RasN43 reduced the basal NF-kappaB activity to about 25–50% of control (P<0.001); and the pcDNA3 and pEXV vectors did not alter NF-kappaB activity. Cell transfection efficiencies were congruent80%, similar to the efficiency numbers found in previous transient transfection studies (Fan et al., 2001, 2005).

Ability of mutant Ras proteins to modulate cell protection by SF

To test the effect of Ras proteins on cell protection, DU-145 cells were transfected with different Ras mutant vectors and then assayed for their ability to protect cells against DNA damage caused by a 2 h exposure to the ADR following treatmentplusminusSF (100 ng/ml) for 48 h. Cell viability was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. In untransfected or empty vector-transfected cells (Figure 2a and g), SF protected the cells at all ADR doses tested (P<0.001). V12C40Ras gave the largest increase in cell viability in the presence of SF (Figure 2d), followed by V12Ras (Figure 2b). For comparison, the dashed lines show results of empty vector transfections. Both Ras vectors also increased cell survival in the absence of SF, but the increases were smaller than those owing to SF. The farnesylation-defective mutant V12S186Ras yielded little or no changes in cell viability with or without SF (Figure 2f), suggesting that farnesylation is required for modulation of cell survival. V12S35Ras, V12G37Ras and V12E38Ras enhanced cell survival (Figure 2c, e and h, respectively), but to a smaller degree than V12C40Ras or V12Ras. The increases in survival were larger with than without SF. Finally, the DN RasN17 abrogated cell protection (Figure 2i), whereas wt-H-Ras enhanced protection by SF (Figure 2j). These results are consistent with the NF-kappaB assays.

Figure 2.
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Effect of Ras proteins on SF protection against ADR. Proliferating DU-145 cells in six-well dishes were transfected overnight with the indicated Ras vector (5 mug of plasmid DNA per well), washed and allowed to recover for several hours. The cells were then harvested, plated into 96-well dishes, allowed to attach overnight, incubatedplusminusSF (100 ng/ml times 48 h), exposed to different doses of ADR (2 h at 37°C) and postincubated for 48 h in fresh drug-free medium. The cells were then analysed for cell viability using MTT assays. Cell viability values are expressed relative to non-ADR-treated control cells and represent meansplusminuss.e.m.s of 10-replicate wells. The results shown are representative of at least two independent experiments. The Ras vectors tested were: empty pcDNA3 vector (a), V12Ras (b), V12S35Ras (c), V12C40Ras (d), V12G37Ras (e), V12S186Ras (f), empty pSVE vector (g), V12E38Ras (h), RasN17 (i) and wt-H-Ras (j). The dashed lines represent empty vector-transfected cells.

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Raf1 enhances SF stimulation of NF-kappaB signaling and cell survival

Activated Ras interacts with the serine/threonine kinase Raf1 and causes activation of MEK1/2 (MKK1/2) and ERK1/2 (Moodie et al., 1993; Vojtek et al., 1993). We tested the effects of wild-type and DN Raf1 vectors on stimulation of NF-kappaB-Luc activity by SF. Here, wt-Raf1 enhanced SF-stimulated NF-kappaB-Luc activity by about fourfold (P<0.001); whereas in the absence of SF, wt-Raf1 gave a smaller (<2-fold) stimulation of NF-kappaB activity (Figure 3a). DN-Raf1 reduced SF-stimulated NF-kappaB activity by >90% (P<0.001). We performed a similar study in the presence of V12Ras, to maximally activate Ras signaling. V12Ras stimulated NF-kappaB-Luc activity by 2.30- to 2.5-fold in the absence of SF and enhanced the SF-stimulated NF-kappaB activity by 4- to 4.5-fold (P<0.001) (Figure 3b). In contrast, DN-Raf1 reduced the SF-stimulated NF-kappaB activity by over 90% and it reduced (SF+RasV12)-stimulated NF-kappaB activity by over 95% (P<0.001). The empty pcDNA3 vector did not affect NF-kappaB activity. Finally, wt-Raf1 enhanced, whereas DN-Raf1 blocked SF-stimulated cell survival (P<0.001) (Figure 3c). The effects of wt-Raf1 on survival were smaller without SF than with SF, indicating that exogenous Raf1 enhances protection by SF. DN-Raf1 had little effect on cell survival in the absence of SF, but attenuated the SF-mediated protection, suggesting that endogenous Raf1 contributes to SF protection against ADR. Expression of the wt-Raf1 and DN-Raf1 proteins in DU-145 cells is shown in Figure 3d. These results suggest that Raf1 signaling is required for SF stimulation of NF-kappaB activity and protection against ADR.

Figure 3.
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Raf1 enhances SF-mediated stimulation of NF-kappaB transcriptional activity and cell survival. (a) Effect of wtRaf1 and DN-Raf1 on NF-kappaB-Luc activity in DU-145 and MDCK cells. NF-kappaB activity was determined as above (Figure 1a), except using Raf1 rather than Ras expression vectors. (b) Effect of wtRaf1 and DN-Raf1 on (SF+RasV12) stimulation of NF-kappaB-Luc activity. Assays were performed as described above for panel a. (c) Raf1 enhances protection against ADR by SF. Cell protection assays were performed as described in Figure 2 legend, except using Raf1 instead of Ras vectors. The dashed lines represent empty pcDNA3 vector-transfected cells. (d) Western blot showing expression of wtRaf1 and DN-Raf1. DU-145 cells were transfected overnight with the indicated vector (15 mug plasmid DNA per 100 mm dish), washed and postincubated for 24 h to allow gene expression. The cells were then harvested and Western blotted to detect Raf1 or actin (control for loading and transfer).

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RalA inhibits SF stimulation of NF-kappaB signaling and cell survival

In NF-kappaB-Luc reporter assays, a wt-RalA vector reduced the basal activity and inhibited SF stimulation of NF-kappaB activity by about two-thirds (P<0.001) (Figure 4a). Conversely, a DN-RalA- enhanced SF-stimulated NF-kappaB activity (P<0.001), but had a modest effect on the basal NF-kappaB activity. RasV12 stimulated the SF-induced NF-kappaB activity by 3.5- to 4-fold; and wt-RalA attenuated the (RasV12+SF)-stimulated NF-kappaB activity by >5-fold (P<0.001). In contrast, DN-RalA enhanced the (RasV12+SF)-stimulated NF-kappaB activity by nearly twofold (P<0.001). Without SF, the empty pcDNA3 vector and various expression vectors yielded modest or no changes in NF-kappaB activity. Consistent with its ability to inhibit NF-kappaB signaling, wt-RalA abrogated the SF protection of DU-145 cells against ADR (Figure 4b). The expression of wt-RalA and DN-RalA was confirmed by Western blotting (Figure 4c).

Figure 4.
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RalA inhibits SF-mediated stimulation of NF-kappaB signaling and cell survival. (a) Effect of wtRalA on NF-kappaB-Luc activity. Proliferating cells in 2 cm2 wells were transiently co-transfected with indicated vectors overnight, washed, incubated in fresh medium without or with SF (100 ng/ml) for 24 h and harvested for luciferase assays. The luciferase values are expressed relative to control-treated cells (no vector, 0 SF) and are meansplusminuss.e.m.s of four replicate wells. (b) RalA inhibits SF protection against ADR. Cell protection assays were performed as described in Figure 2 legend, except that RalA vectors were used instead of Ras vectors. The dashed lines represent empty pcDNA3 vector-transfected cells. (c) Western blot showing expression of RalA and DN-RalA. Proliferating DU-145 cells were transfected overnight with the indicated vector (15 mug plasmid DNA per 100 mm dish), washed and postincubated for 24 h to allow gene expression. The cells were then harvested and Western blotted for RalA or actin. (d) RalA interacts with c-Met receptor in a SF-independent manner. DU-145 cells in 100 mm dishes were incubated in serum-free mediumplusminusSF (100 ng/ml) for 20 min; and whole-cell lysates were subjected to IP using an anti-c-Met antibody (see Materials and methods section). Precipitated proteins were analysed by SDS–PAGE and blotted using antibodies for total c-Met, phospho-c-Met (tyrosine-1349), RalA, Raf1 and Ras. (e) Effect of RalB on SF stimulation of NF-kappaB-Luc activity. Assays were performed as described for panel a, except that a wild-type RalB expression vector was used instead of RalA.

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To test if RalA associates with the SF receptor, DU-145 cells were treatedplusminusSF for 20 min; and c-Met was immunoprecipitated (IP'd). Western blotting showed an increase of tyrosine-phosphorylated c-Met following SF treatment (Figure 4d). In addition, RalA, Raf1 and Ras were found in the c-Met immunoprecipitations (IPs), with no obvious differences owing to SF treatment (but see below). Finally, we tested the ability of wt-RalB to modulate the SF stimulation of NF-kappaB activity. wt-RalB caused a reduction in SF stimulated NF-kappaB activity, but to a slightly lesser extent than RalA (Figure 4e). These findings suggest that the Ral GTPases inhibit SF-mediated NF-kappaB signaling and cell protection.

Ability of Ras, Raf1 and RalA to modulate SF stimulation of IKK-beta kinase activity

NF-kappaB activity is inhibited by IkappaB proteins which maintain p65RelA within the cytoplasm. These proteins (IkappaB-alpha and IkappaB-beta) are regulated by serine phosphorylations, which mark them for ubiquitin-mediated degradation. A key mediator of IkappaB phosphorylation is the IkappaB kinase (IKK) complex (Woronicz et al., 1997; Hu and Wang, 1998; May et al., 2000), which contains IKK-alpha, IKK-beta and IKK-italic gamma (NEMO). One measure of IKK activation is phosphorylation on serine of the IKK-alpha/beta proteins. First, we tested the effect of SF and V12Ras on IkappaB-alpha degradation. In both DU-145 and MDCK cells, SF treatment for 20 min caused a large reduction of IkappaB-alpha protein; whereas V12Ras alone caused only a modest reduction of IkappaB-alpha protein (Figure 5a). The combination (V12Ras+SF) caused loss of detectable IkappaB-alpha protein, suggesting that V12Ras enhances the ability of SF to induce IkappaB-alpha degradation.

Figure 5.
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Impact of Ras, Raf1 and RalA on SF-mediated stimulation of IKK-beta kinase activity. (a) Ability of SF and V12Ras to induce degradation of IkappaB-alpha. Proliferating cells in 100 mm dishes were transfected with V12Ras (15 mug per dish) or sham-treated for 24 h. The cells were then treatedplusminusSF (100 ng/ml) in serum-free DMEM for 20 min, harvested and subjected to Western blotting for IkappaB-alpha or actin. (b and c) Ability of Ras mutants to modulate phosphorylation of IKK complex. Proliferating DU-145 cells were transfected with the indicated Ras vectors and then treated with SF (100 ng/ml) (b) or without SF (c) for 20 min. The cells were harvested and subjected to Western blotting for Ras, phospho-IKK-alpha/beta, total IKK-alpha, total IKK-beta or actin. (d) Ability of Ras mutant proteins to modulate SF stimulation of IKK-beta activity. DU-145 cells were transfected with different Ras vectors and treatedplusminusSF, as described above. Cell lysates were prepared; and IKK-beta kinase activity was determined using a commercial assay kit (see Materials and methods section). The IKK-beta kinase activity values are meansplusminuss.e.m.s of five replicate wells expressed as a percentage of the control value (no SF, mock transfection). (e) Ability of additional Ras mutant proteins to modulate SF stimulation of IKK-beta activity. Assays were performed as described in panel d. (f) Effect of wtRaf1 and DN-Raf1 on IKK-beta kinase activity in DU-145 cells. Assays were performed as described in panel d, except that Raf1 vectors were used instead of Ras vectors. (g) Effect of wtRalA and DN-RalA on IKK-beta kinase activity in DU-145 cells. Assays were performed as described in panel d, except that RalA vectors were used instead of Ras vectors.

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To test the effect of Ras mutants on IKK phosphorylation, DU-145 cells were transfected with different Ras vectors, treated with SF for 20 min, and Western blotted for phosphorylated and total IKK proteins. Each mutant Ras protein was expressed (Figure 5b). SF increased the phosphorylation on IKK-alpha/beta; and with SF present, V12Ras and the effector loop mutants (but not the farnesylation-defective mutant V12S186Ras) further enhanced the phosphorylation of IKK-alpha/beta, compared with the pcDNA3 vector control (Figure 5b). Several experiments gave similar results, with some experiments showing no basal phosphorylation of IKK-alpha/beta in control cells in the absence of SF. As controls, there were no consistent differences in total IKK-alpha, total IKK-beta or actin protein levels. In the absence of SF, there was no detectable phosphorylation of IKK-alpha/beta in control or V12S186 Ras-transfected cells (Figure 5c). Phosphorylation on IKK-alpha/beta was detected in the other V12Ras-transfected cells, suggesting that the phosphorylation can be mediated by activated Ras proteins in the absence of SF.

We tested the effect of Ras, Raf1 and RalA proteins on IKK-beta kinase activity in DU-145 cells using an assay kit that measures the ability of the sample to phosphorylate a biotinylated IkappaB-alpha substrate peptide (see Materials and methods). Here, SF-stimulated kinase activity by about fivefold; and the activated Ras proteins V12Ras, V12C40Ras, V12S35Ras, V12G37Ras and V12E38Ras- enhanced SF-stimulated kinase activity (P<0.001) by five- to sevenfold (Figure 5d). V12S186Ras and the empty pcDNA3 vector did not alter IKK activity. In the absence of SF, the active RasV12 mutants showed modest (less than or equal to1.6-fold) stimulation of IKK-beta kinase activity. wt-R-Ras, the activated mutant R-RasV38 and wt-H-Ras- enhanced SF-stimulated IKK-beta activity by five- to sixfold (P<0.001) (Figure 5e). In contrast, the DN R-RasN43 and RasN17 proteins blocked SF-stimulated IKK-beta activity (P<0.001), suggesting that endogenous Ras contributes to SF stimulation of IKK-beta activity.

In further studies, wt-Raf1 enhanced the SF-stimulated IKK-beta kinase activity by 4.8-fold (P<0.001); but it had a modest effect on the kinase activity in the absence of SF (about twofold stimulation) (Figure 5f). In contrast, DN-Raf1 abrogated SF stimulation of IKK-beta kinase activity (P<0.001). Finally, wt-RalA inhibited the SF-stimulated kinase activity (P<0.001); whereas DN-RalA enhanced SF-stimulated kinase activity by another fourfold (P<0.001) (Figure 5g). These findings suggest that Ras and Raf1 positively regulate, whereas RalA negatively regulates the ability of SF to stimulate IKK-beta kinase activity. Thus, the results of the IKK-beta activity assays correlated well with the NF-kappaB-transcriptional assays.

Role of MAP kinases in SF stimulation of NF-kappaB activity

We tested the role of various MAP kinase pathways in the SF stimulation of NF-kappaB activity, using expression vectors for wt, constitutively active (ca) and DN MAP kinase kinase proteins. MKK1 and MKK2 activate the classic extracellular signal-regulated kinases (ERK1 and ERK2) (Zheng and Guan, 1993). Neither wt-MKK1 nor DN-MKK1 nor wt-MKK2 nor DN-MKK2 had any significant effect on NF-kappaB-Luc activity in DU-145 cells without or with SF present, even though the proteins were well expressed (data not shown). MKK4 is a dual-specificity kinase that activates the stress-activated kinases c-jun NH2-terminal kinase and p38 (Hog1), but not ERK1/2 and is unresponsive to Raf1 (Lin et al., 1995). wt-MKK4 caused a modest increase in SF-stimulated NF-kappaB activity (congruent20%); whereas DN-MKK4 caused a modest decrease in activity (congruent20%) (data not shown). MKK5 is an upstream activator of ERK5 (MAPK7) (Mody et al., 2003). Neither wt-MKK5 nor DN-MKK5 altered the basal or SF-stimulated NF-kappaB activity (data not shown).

In contrast, MKK3 and MKK6 (both of which activate p38 (Derijard et al., 1995; Wang et al., 1997)) strongly regulated NF-kappaB activity (Figure 6a and b). The wt and ca mutant MKK3 and MKK6 proteins stimulated basal NF-kappaB-Luc activity by about three- to fourfold (P<0.001) and enhanced the SF-stimulated NF-kappaB-Luc activity by 3.5- to 5-fold (P<0.001). Conversely, DN-MKK3 and DN-MKK6 reduced SF-stimulated NF-kappaB activity (P<0.001) to about 15% of control. A selective inhibitor of p38 kinase activity, SB202190 (10 muM), reduced SF-stimulated NF-kappaB activity by 50–70% and abrogated the ability of MKK3 and MKK6 to stimulate NF-kappaB activity in the presence or absence of SF (Figure 6c and d). To confirm a role for the endogenous MKK3 and MKK6 proteins in NF-kappaB signaling, DU-145 cells were treated with MKK3 small interfering RNA (siRNA), MKK6-siRNA or control-siRNA (50 nM times 48 h) before testing for SF-stimulated NF-kappaB activity. The MKK3 and MKK6 siRNAs reduced the SF-stimulated NF-kappaB-Luc activity to congruent30–40% of that of cells treated with control-siRNA or no siRNA (P<0.01) (Figure 6e). These findings suggest that in DU-145 cells, NF-kappaB activity is regulated by an MKK3/6 right arrow p38 pathway, but not by the Raf1 right arrow MEK1/2 right arrow ERK1/2 pathway.

Figure 6.
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Role of MAP kinase pathways in SF stimulation of NF-kappaB transcriptional activity. DU-145 cells were co-transfected overnight with the NF-kappaB-Luc reporter and the indicated expression vectors for wild-type or mutant MKK (mitogen-activated protein kinase kinase). The cells were then washed, treatedplusminusSF (100 ng/ml times 24 h) and assayed for luciferase activity. Data are shown for MKK3 (a) and MKK6 (b). (c and d) Assays were carried out as above using MKK3 (c) and MKK6 (d) vectors in the absence or presence of the selective p38 inhibitor SB202190 (10 muM). Note: wt, wild-type; DN, dominant negative; ca, constitutively active. (e) the cells were pretreated with MKK3-siRNA or MKK6-siRNA, control-siRNA (50 nM times 48 h) or no siRNA; transfected with the NF-kappaB-Luc reporter; treatedplusminusSF (100 ng/ml times 24 h); and assayed for luciferase activity. (f) DU-145 cells were transfected with V12Ras vs empty pcDNA3 vector, pre-incubatedplusminusSB202190 (50 muM), treatedplusminusSF (100 ng/ml times 20 min) and then assayed for IKK-beta kinase activity. (g) Ability of the MKK3 and MKK6 siRNAs to knock down the endogenous MKK3 and MKK6 proteins, respectively, after a 48 h treatment with 50 nM of gene-specific or control siRNAs.

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We tested the role of p38 in mediating the enhanced SF-stimulated IKK-beta kinase activity because of V12Ras in DU-145 cells, using the p38 inhibitor SB202190. Here, we used a higher concentration of SB202190 (50 muM) than in the NF-kappaB-Luc assay (10 muM), to ensure maximal inhibition of p38. As before, SF caused a fivefold increase in IKK-beta activity; and (SF+V12Ras) caused another fivefold increase beyond that owing to SF alone or (SF+pcDNA3) (Figure 6f). SB202190 abrogated the increase in IKK-beta activity owing to SF alone and the further increase in IKK-beta activity owing to V12Ras. Thus, IKK-beta activity for (SF+V12 Ras+SB202190) was slightly less than that for SF alone. The ability of the siRNAs to knock down MKK3 and MKK6 proteins is shown in Figure 6g. These findings suggest that the ability of V12Ras to enhance the SF-stimulated IKK-beta activity in DU-145 cells is dependent upon p38.

Ras mutants stimulate c-Akt activity

We showed that c-Akt signaling is required for SF stimulation of NF-kappaB activity and protection against ADR (Fan et al., 2001, 2005). Here, we tested the ability of Ras mutant proteins to activate c-Akt, indicated by phosphorylation on serine-473 and threonine-309. A 20 min exposure to SF-stimulated Akt phosphorylation on both residues in empty vector-transfected cells (Figure 7a). V12Ras and all V12Ras effector loop mutants (but not the farnesylation-defective mutant V12S186Ras) enhanced Akt phosphorylation in SF-stimulated cells; whereas there were no significant changes in total Akt or actin protein. V12Ras and the effector loop mutants (but not V12S186Ras) also induced Akt phosphorylation in the absence of SF (Figure 7b). There were no consistently observed differences among these mutants (except V12S186Ras) in the ability to activate Akt.

Figure 7.
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Ability of Ras mutants to stimulate c-Akt activity. (a and b) Ability of mutant Ras proteins to stimulate Akt phosphorylation. DU-145 cells were transfected with the indicated vectors and then treated with SF (100 ng/ml) (a) or without SF (b) in serum-free DMEM for 20 min. Western blotting of whole-cell lysates was carried out using antibodies against phospho-Akt (serine-473 or threonine-309), total Akt or actin. (c) Interaction of effector loop mutant Ras proteins with effector proteins. DU-145 cells were transfected with the indicated pCMV-Tag2 Ras mutant expression vector, washed, postincubated for 24 h to allow gene expression and harvested. Aliquots of cell lysates (800 mug of total cell protein) were IP'd using an anti-FLAG antibody or non-immune IgG (negative control) (see Materials and methods section). The IPs were Western blotted using an anti-FLAG antibody (to detect transfected Ras) or antibodies against the Ras effectors p110alpha, Raf1 and RalGDS. A lane corresponding to unprecipitated cell lysate (50 mug) from untransfected control cells is also shown. (d) DN-Akt blocks (SF+RasV12)-mediated stimulation of NF-kappaB transcriptional activity. Assays were performed as described in Figure 1a legend. (e) PTEN blocks (SF+V12Ras)-mediated stimulation of NF-kappaB activity. Assays were performed as described in Figure 1a legend.

Full figure and legend (174K)

Thus, V12Ras mutant proteins that selectively activate three effector pathways (PI3K, Raf1 or RalGDS) each enhanced SF-stimulated Akt phosphorylation, IKK-beta kinase activity, NF-kappaB activity and cell survival. The finding that a Ras mutant that selectively activates RalGDS (V12G37Ras) has these activities was surprising, because RalA and RalB, two major targets of RalGDS, inhibited the same SF-stimulated activities. We tested our Ras effector loop mutants to confirm that they behave as expected. DU-145 cells were transfected with different mutants or with V12Ras itself and tested for association of the Ras proteins with p110alpha (catalytic subunit of PI3K), Raf1 and RalGDS. For this purpose, we constructed FLAG-tagged Ras mutant expression vectors to facilitate IP of the Ras proteins. By IP-Western blotting, V12Ras associated with all three effector proteins; whereas V12C40Ras associated only with p110alpha. V12S35Ras and V12E38Ras interacted only with Raf1; and V12G37Ras interacted only with RalGDS (Figure 7c). Thus, the mutant Ras proteins behave as expected.

Finally, we studied the role of c-Akt in regulating the ability of V12Ras to enhance SF-stimulated NF-kappaB activity. In the absence of V12Ras, a kinase-dead DN-Akt vector blocked the SF-stimulated NF-kappaB activity (P<0.001) (Figure 7d). As before, V12Ras further enhanced the SF-stimulated NF-kappaB activity by five- to sixfold. However, DN-Akt nearly abrogated the (SF+V12Ras)-stimulated NF-kappaB activity (P<0.001). DN-Akt also inhibited the basal NF-kappaB activity in the absence of SF (P<0.001); and wt-PTEN (a negative regulator of c-Akt activity), but not a mutant phosphatase-defective PTEN, inhibited basal, SF-stimulated and (SF+V12Ras)-stimulated NF-kappaB activity (P<0.001) (Figure 7e). These findings suggest that c-Akt is required for basal and SF/Ras-stimulated NF-kappaB activity in DU-145 and MDCK cells.

Effects of Raf1 and RalA knock down on NF-kappaB and IKK-beta activity

Consistent with DN-Raf1 experiments, treatment of DU-145 and MDCK cells with Raf1-siRNA (but not a control-siRNA) reduced the basal and SF-stimulated NF-kappaB-Luc activity (P<0.001) (Figure 8a). And like DN-RalA, RalA-siRNA increased the basal and SF-stimulated NF-kappaB-Luc activity cell types (P<0.001) (Figure 8b). The extent of the increase owing to RalA-siRNA was greater in the presence (>3-fold) than in the absence (twofold) of SF. Similar to the transcriptional assays, Raf1-siRNA (but not control-siRNA) strongly reduced the SF-stimulated (and basal) IKK-beta kinase activity (P<0.001) (Figure 8c); and RalA-siRNA enhanced basal and SF-stimulated IKK-beta kinase activity (P<0.001) (Figure 8d). Finally, we tested the effects of Raf1 or RalA on IKK-beta kinase activity in cells treated with (SF+V12Ras). SF alone gave a fivefold increase in kinase activity and (SF+V12Ras) gave a further fivefold increase beyond that of SF alone (Figure 8e). DN-Raf1 abrogated the increased IKK-beta kinase activity owing to (SF+V12Ras); whereas wt-RalA reduced the kinase activity in the presence of (SF+V12Ras) to 55% of that found in the absence of wt-RalA (P<0.001). The ability of the siRNAs to knock down Raf1 and RalA protein is shown in Figure 8f. Thus, DN-Raf1 was more effective than wt-RalA in modulating the stimulatory effect of the combination of (SF+V12Ras).

Figure 8.
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Effects of knock down of Raf1 and RalA on NF-kappaB and IKK-beta activity. (a) Effect of Raf1-siRNA on NF-kappaB-transcriptional activity. Cells were pretreated with Raf1-siRNA vs control-siRNA (50 nM) vs no siRNA (vehicle only) for 48 h. The cells were then transfected with the NF-kappaB-Luc reporter, treatedplusminusSF (100 ng/ml) for 24 h and harvested for luciferase assays. The relative luciferase values are meansplusminuss.e.m.s of four replicate wells. (b) Effect of RalA-siRNA on NF-kappaB activity. Experiments were performed as described above, except using RalA-siRNA rather than Raf1-siRNA. (c) Effect of Raf1-siRNA on IKK-beta kinase activity. DU-145 cells were pre-treated Raf1-siRNA vs control-siRNA (50 nM) vs no siRNA (vehicle only) for 48 h, then treatedplusminusSF (100 ng/ml) for 20 min and harvested for IKK-beta kinase activity assays. The values shown are meansplusminuss.e.m.s of five-replicate wells, expressed relative to untreated control cells (=100%). (d) Effect of RalA-siRNA on IKK-beta kinase activity. Experiments were performed as described in panel c, except using RalA-siRNA rather than Raf1-siRNA. (e) Effects of Raf1 and RalA on IKK-beta kinase activity stimulated by (SF+V12 Ras). DU-145 cells were transfected with the indicated expression vectors; treatedplusminusSF (100 ng/ml) for 20 min and assayed for IKK-beta kinase activity as above. (f) Knock down of Raf1 and RalA protein levels by the siRNAs. Cells were treated with the indicated siRNAs (50 nM times 48 h) followed by Western blotting.

Full figure and legend (170K)

Raf1 and IKK phosphorylation in the c-Met complex

We tested the effect of SF and V12Ras on the phosphorylation (a measure of activation) of Raf1. DU-145 cells were transfected with V12Ras vs pcDNA3 vector; treatedplusminusSF (20 min) and subjected to IP-Western blotting using an antiRaf1 antibody. The total levels of Raf1 and c-Met protein associated with Raf1 were unaffected by these treatments; but the phosphorylated c-Met associated with Raf1 was increased in V12Ras-transfected cells compared to pcDNA3-transfected cells (Figure 9a). SF increased the phospho-Raf1 (serine-43) levels in both pcDNA3 and V12Ras-transfected cells; but the SF-induced increase in phospho-Raf1 was greater in V12Ras-transfected cells. V12Ras had little effect on Raf1 phosphorylation without SF; but with SF present, V12Ras increased the amount of phospho-Met associated with Raf1. In a similar experiment using an anti-c-Met antibody for IP, SF increased the levels of phospho-Met; and V12Ras further enhanced SF-induced c-Met phosphorylation (Figure 9b). There were no changes in the total Raf1, but there was more phospho-Raf1 associated with c-Met in cells treated with (SF+V12Ras) as compared with (SF+pcDNA3) (Figure 9b). These findings suggest that activated c-Met may mediate SF-induced Raf1 phosphorylation and that V12Ras enhances SF-induced Raf1 phosphorylation in the signaling complex.

Figure 9.
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Effects of SF and V12Ras on Raf1 phosphorylation. (a and b) Raf1 and c-Met IPs. DU-145 cells were transfected with V12Ras or empty pcDNA3 vector (15 mug of plasmid DNA per 100 mm dish), postincubated for 24 h to allow gene expression and treatedplusminusSF (100 ng/ml) for 20 min. The cells were harvested; and equal aliquots of total cell protein (500 mug) were IP'd using antibodies against total Raf1 (a) or total c-Met (b) The IPs were Western blotted for total c-Met, phospho-c-Met (tyrosine-1349), total Raf1, phospho-Raf1 (serine-43), RalA, phospho-IKK-alpha/beta and total IKK-alpha/beta. (c and d) Effects of Raf1 (c) and RalA (d) siRNAs on IKK phosphorylation. DU-145 cells were transfected with the indicated siRNA (50 nM times 48 h), treatedplusminusSF (100 ng/ml times 48 h) and then Western blotted to detect Raf1 or RalA, phospho-IKK-alpha/beta, total IKK-alpha/beta and actin.

Full figure and legend (98K)

In the same experiments, RalA associated with both Raf1 and c-Met, in a SF- and V12Ras-independent manner. IKK-alpha and IKK-beta also associated with Raf1 and c-Met. Phospho-IKK-alpha/beta associated with Raf1 and c-Met in a SF- and V12Ras-dependent manner: SF increased the amount of phospho-IKK-alpha/beta in Raf1 and c-Met IPs; whereas V12Ras further increased the phospho-IKK-alpha/beta levels in these IPs in SF-treated cells. Finally, we tested the effect of Raf1-siRNA (Figure 9c) or RalA-siRNA (Figure 9d) on the ability of SF to induce IKK-alpha/beta phosphorylation. As expected, SF stimulated IKK-alpha/beta phosphorylation, but did not significantly alter total IKK-alpha, IKK-beta or actin levels. Knock down of Raf1 blocked the SF-induced IKK-alpha/beta phosphorylation; whereas knock down of RalA enhanced the SF-stimulated phosphorylation. These findings suggest that endogenous Raf1 and RalA, each of which associates with c-Met, modulate the ability of SF to induce IKK-alpha/beta phosphorylation.

V12Ras enhances SF-induced p65 nuclear translocation and NF-kappaB target gene expression

We had shown that SF causes rapid translocation of the p65 subunit of NF-kappaB from cytoplasm to nucleus (Fan et al., 2005). Here, we tested the effect of V12Ras on nuclear translocation of p65 after a brief (20 min) exposure to SF, by immunofluorescence microscopy. In basal conditions, cells transfected with V12Ras or pcDNA3 vector showed a predominantly cytoplasmic distribution of p65 (Figure 10a). In response to SF, p65 become mostly localized in the nucleus. Consistent with the ability of V12Ras to stimulate NF-kappaB transcriptional activity, V12Ras-transfected cells showed higher levels of immunoreactive p65 in the nucleus than pcDNA3-transfected cells. The ability of SF to cause 'scattering' (cell dispersion and a fibroblastic appearance) of DU-145 cells is illustrated in the phase contrast micrographs in Figure 10a. We have also shown that SF stimulates the expression of several known NF-kappaB target genes (cIAP1, cIAP2 and TRAF2) in an NF-kappaB-dependent manner (Fan et al., 2005). Here, we tested whether V12Ras could enhance the expression of several of these genes in SF-treated cells. The ability of SF to induce increased protein levels of cIAP2 and TRAF2 was greater in V12Ras-transfected cells than in pcDNA3-transfected cells (Figure 10b). As expected, the total Ras levels were significantly greater in the V12Ras-transfected cells. In contrast, there were no differences in the levels of c-Met or actin. These findings are consistent with the above studies showing that V12Ras enhances the SF-stimulated NF-kappaB transcriptional activity.

Figure 10.
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Effect of V12Ras on SF-induced p65 translocation and NF-kappaB target gene expression. (a) Effect of V12Ras on SF-induced nuclear translocation of p65. DU-145 cells cultured on glass coverslips in six-well dishes were transfected with V12Ras or empty pcDNA3 vector, washed and allowed to recover from transfection. The cells were then treatedplusminusSF (100 ng/ml) for 20 min and processed for immunofluorescence microscopy to detect the p65 subunit of NF-kappaB (see Materials and methods). (b) Effect of V12Ras on SF-induced NF-kappaB target gene expression. DU-145 cells were transfected with V12Ras or empty pcDNA3 vector, washed, allowed to recover from transfection and treatedplusminusSF (100 ng/ml) for 24 h. The cells were then harvested; and whole-vcell lysates were Western blotted to detect cIAP2, TRAF2 and actin.

Full figure and legend (132K)

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Discussion

These studies identify a role for p21Ras and several of its downstream effectors (Raf1 and RalA) in modulating the ability of SF to stimulate IKK-beta kinase activity, NF-kappaB transcriptional activity and protection against ADR-induced DNA damage. In particular, Ras and Raf1 signaling enhanced SF stimulation of IKK-beta activity, NF-kappaB activity and cell protection, whereas the RalA inhibited these activities. The ability of activated Ras (RasV12) to enhance SF-stimulated NF-kappaB signaling was dependent upon signaling downstream of c-Akt and Raf1; and it was negatively regulated by RalA. In addition, SF-stimulated NF-kappaB signaling was dependent upon endogenous Ras, Raf1 and c-Akt and was negatively regulated by endogenous RalA. Finally, although Raf1 was required for SF-stimulated NF-kappaB activation (and cell protection), the Raf1 right arrow MEK1/2 right arrow ERK1/2 pathway was dispensable. We do not yet know the mechanism through which Raf1 mediates the activation of IKK-beta and NF-kappaB. However, in a different context, it was observed that Raf1 can stimulate IKK-beta activity by a mechanism that involves the kinase MEKK1 (MAP3K1), but is independent of the ERK1/2 pathway (Baumann et al., 2000).

Consistent with the ability of Ras to enhance SF-stimulated IKK-beta kinase and NF-kappaB activity, the activated mutant V12Ras also enhanced the nuclear translocation of the p65RelA subunit of NF-kappaB caused by SF as well as the SF-induced increases in the expression of two known NF-kappaB target genes, cIAP2 and TRAF2. Previously, we showed that SF induces expression of these genes in an NF-kappaB-dependent manner and that they contribute to the SF-mediated protection against ADR (Fan et al., 2005).

The V12C40Ras mutant, which selectively binds p110alpha (Rodriguez-Viciana et al., 1997) was slightly more active than V12Ras in enhancing the SF-stimulated IKK-beta kinase activity, NF-kappaB activity and protection against ADR. This finding is consistent with our previous studies indicating a role for PI3K/c-Akt signaling in SF-mediated cell protection (Bowers et al., 2000; Fan et al., 2000, 2001, 2005), and it suggests that Ras acts, in part, upstream of PI3K in the cell protection pathway. The increased activity of V12C40Ras relative to V12Ras is consistent with an inhibitory component of Ras signaling owing to another pathway downstream of Ras (e.g., RalA) that is active in V12Ras but not in V12C40Ras. Alternatively, the V12C40Ras pathway may sequester signaling proteins from another SF signaling pathway that negatively regulates cell protection (Fan et al., 2001).

Two Ras effector mutants that selectively bind Raf1 (V12S35 Ras and V12E38Ras) enhanced SF-stimulated IKK-beta activity, NF-kappaB activity and cell survival. These findings, coupled to the finding that wtRaf1 enhanced the kinase, NF-kappaB and survival-stimulating activity of SF, suggest that Ras/Raf1 signaling contributes to these activities. The ability of DN-Raf1 or Raf1-siRNA to these activities suggests that Raf1 is required for the SF survival pathway in the cell types studied. And the ability of DN-Raf1 to block (SF+V12Ras)-stimulated NF-kappaB activity is consistent with the idea that Raf1 acts downstream of Ras in this pathway. Although Raf1 contributes to SF-stimulated NF-kappaB activity, neither wtMEK1/2 nor DN-MEK1/2 mutants altered the SF-stimulated NF-kappaB activity, suggesting that the ability of Raf1 to enhance the SF-stimulated NF-kappaB activity is not owing to the Raf1/MEK/ERK pathway. In contrast, the ability of DN-Raf1 to block and wt-Raf1 to enhance SF-stimulated IKK-beta kinase activity suggest that Raf1 targets the IKK complex or a kinase upstream of IKK.

Our previous studies indicate that c-Akt signaling is required for SF-mediated NF-kappaB activation and cell protection (Bowers et al., 2000; Fan et al., 2000, 2001, 2005). The present study revealed that DN-Akt also blocks the ability of RasV12 to enhance the SF-stimulated NF-kappaB activity, suggesting that both Raf1 and c-Akt act downstream of Ras in the SF survival pathway. The findings are consistent with a model in which Raf1 and c-Akt act in a direct pathway leading to NF-kappaB activation, although these proteins are not known to participate in the same phosphorylation cascade. Alternatively, they also support a model in which Raf1 and c-Akt signaling impinge on the NF-kappaB pathway (e.g., at the level of IKK, a kinase upstream of IKK, and/or NF-kappaB itself) to synergistically activate this pathway. Here, inactivation of either Raf1 or c-Akt is sufficient to block SF-induced NF-kappaB activation and cell survival. The finding that wtRaf1 enhances SF-stimulated IKK-beta activity and NF-kappaB activity by three- to fivefold is consistent with this model, because it shows that the pathway through which Raf1 stimulates SF-inducible NF-kappaB activity can be further activated. Similarly, we previously reported that exogenous c-Akt enhances SF-stimulated NF-kappaB activity by 2.5- to 3-fold, suggesting that the pathway through which endogenous c-Akt enhances SF-stimulated NF-kappaB activity is not maximally activated and can be further stimulated.

V12G37Ras, a mutant that selectively activates the guanine nucleotide dissociation stimulator RalGDS (Albright et al., 1993; Hofer et al., 1994), enhanced SF-stimulated IKK-beta activity and NF-kappaB activity, but to a lesser extent than V12Ras. Studies utilizing DN-RalA and RalA-siRNA suggest that RalA negatively regulates SF stimulation of IKK-beta and NF-kappaB activity and SF protection against ADR and inhibits V12Ras-mediated enhancement of the SF stimulation of IKK-beta and NF-kappaB activity. Our findings suggest that the endogenous protein RalA inhibits the SF survival pathway at the level of the IKK complex and NF-kappaB transcriptional activity. In addition, RalB, another target of RalGDS, inhibited the SF-stimulated NF-kappaB activity. We verified that all Ras effector loop mutant proteins tested were expressed and behaved as expected (e.g., V12G37Ras interacted with RalGDS but not p110alpha or Raf1). The finding that V12G37Ras enhances SF-mediated NF-kappaB survival signaling deserves comment, as two major targets of RalGDS, the RalA and RalB GTPases, blocked SF-stimulated NF-kappaB signaling.

Two possible explanations are that: (1) the V12G37Ras/RalGDS pathway activates other downstream components besides RalA and RalB; or (2) V12G37Ras (and other Ras effector mutants) activate an NF-kappaB stimulatory pathway independent of RalGDS, Raf1 and p110alpha/PI3K. Possibility (2) is consistent with the finding that each V12Ras effector mutant enhanced the SF-stimulated c-Akt phosphorylation and caused c-Akt phosphorylation in the absence of SF. The finding that a farnesylation-defective mutant (V12S186Ras) did not stimulate IKK-beta activity, NF-kappaB activity or cell protection suggests that this alternative NF-kappaB stimulatory pathway requires farnesylation of the Ras protein. Ras can stimulate other effectors than p110alpha, Raf1 and RalGDS, including RIN1, AF6, NORE1, PLC-alt epsilon and other RalGDS family members (RGL, RLF and RGL2) (Shao et al., 1999; Herrmann, 2003; Wohlgemuth et al., 2005). Whether these or other potential Ras effectors contribute to regulation of NF-kappaB signaling downstream of Ras remains to be discovered.

A role for RalA in an NF-kappaB-survival pathway is novel, as the role of RalA in cell survival or apoptosis is not well characterized. The pathways downstream of RalA that mediate inhibition of NF-kappaB signaling and cell protection by SF remain to be identified. RalA signaling is not understood in great depth, but one target of RalA is phospholipase D1 (PLD1), a protein implicated in vesicle trafficking (Luo et al., 1998). Emerging evidence suggests that the exocyst, a protein complex involved in the interaction of secretory vesicles with the plasma membrane, is a target for activated RalA and that RalA stimulates exocytosis (Camonis and White, 2005). PLD1 may contribute to RalA-mediated exocytosis (Vitale et al., 2005). Whether PLD1 or exocyst proteins also participate in the regulation of NF-kappaB signaling is conjectural at this time. RalBP1 (RLIP76) is a RalA-interacting protein implicated in the transport of glutathione conjugates and in mediating drug resistance (Cantor et al., 1995; Stuckler et al., 2005), but the role of RalA in these processes is unclear.

Although MAP kinase kinases 1–5 had little or no effect on NF-kappaB signaling, we found that DN-MKK3 and DN-MKK6 inhibited SF stimulation of NF-kappaB activity and wild-type or active mutant MKK3 and MKK6 enhanced SF-stimulated NF-kappaB activity. Knock down of MKKs 3 or 6 blocked SF-induced NF-kappaB activity, suggesting roles for endogenous MKK3/6 in SF-mediated NF-kappaB signaling, although we did not tested whether these kinases contribute to cell survival. The stress-activated kinase p38 is a substrate for MKK3 and MKK6; and several studies suggest that p38 can stimulate NF-kappaB signaling (Nick et al., 1999; Shuto et al., 2001; Costantini et al., 2005). Here, we found that a selective inhibitor of p38 (SB202190) inhibited SF-, MKK3/6- and (SF+V12Ras)-stimulated NF-kappaB activity, suggesting that an MKK3/6 right arrow p38 pathway contributes to SF-stimulated NF-kappaB activity. Interestingly, in addition to regulating IKK-beta kinase activity (demonstrated herein), p38 has been reported to mediate the phosphorylation and activation the p65RelA subunit of NF-kappaB in another context, whether directly or indirectly (Rahman et al., 2004).

Using IP-Western blotting, we showed that Ras, Raf1, RalA, IKK-alpha and IKK-beta each associated with the liganded or unliganded c-Met receptor. We further demonstrated an association of c-Met, RalA, IKK-alpha and IKK-beta with Raf1. Within these signaling complexes, SF induced and V12Ras further enhanced the phosphorylation (activation) of Raf1 and IKK-alpha/beta. In studies using siRNAs to knock down endogenous protein levels, we found that Raf1 is required for the SF-induced phosphorylation of IKK-alpha/beta and that endogenous RalA suppresses IKK-alpha/beta phosphorylation. Taken together, these findings suggest a physiological connection between Raf1, RalA and the IKK complex that can modulate the activation of IKK-alpha/beta and NF-kappaB signaling. Although we did not test the activation state of the Ras and RalA GTPases, it was previously demonstrated that ligation of c-Met is required for Ras activation (conversion to the GTP-bound state) (Graziani et al., 1993); and it is, thus, likely that c-Met ligation is also required for RalA activation within the signaling complex. Whether c-Met can transmit a survival signal in the absence of SF is unclear; but it is interesting that the multisubstrate docking site of the c-Met receptor (1349YVHVxxxYVNV) – which mediates the association of c-Met with multiple signaling proteins (e.g., Grb2, Shc, Gab1 and p110) and is required for much of its signaling activity – is not required for transduction of a Ras signal or cell scattering (Tulasne et al., 1999). It was suggested that GDP-bound Ras may have a role in signal transduction (Stewart and Guan, 2000). These unconventional receptor-dependent Ras signaling mechanisms are poorly understood.

Interestingly, although the Ras/Raf1 signaling pathways stimulate IKK-beta activity, NF-kappaB activity and cell survival both in the presence and absence of SF, and to mediate cell protection in carcinoma and epithelial cell lines, the stimulatory effects of Ras and Raf1 are significantly greater in the presence of SF than in its absence. These findings suggest the possibility that the SF and Ras/Raf1 are acting through parallel pathways that interact (cross-talk) with each other. SF may activate more steps than Ras/Raf1, accounting for the greater effects of SF alone than of Ras/Raf1 alone.

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Materials and methods

Cell lines and culture

DU-145 human prostate cancer cells and MDCK epithelial cells were obtained from the American Type Culture Collection (Manassas, VA, USA). The cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% fetal calf serum, non-essential amino acids (100 mM), L-glutamine (5 mM), streptomycin (100 mug/ml) and penicillin (100 U/ml) (all purchased from BioWhittaker, Walkersville, MD, USA). The cells were grown at 37°C in a humidified atmosphere of 95% air and 5% CO2 and were subcultured weekly, using trypsin.

Reagents

Recombinant human two-chain SF was a gift from Dr Ralph Schwall, Department of Endocrine Research, Genentech Inc. (South San Francisco, CA, USA). ADR (doxorubicin hydrochloride) and MTT were obtained from the Sigma Chemical Co. (St Louis, MO, USA). The p38 inhibitor SB202190 was purchased from CalBiochem (La Jolla, CA, USA). The antibodies used in this study are described below.

Expression vectors

The expression vectors utilized in this study are described in Table 1.

Transient transfections

Subconfluent proliferating cells were transfected overnight with the vector of interest or with the empty pcDNA3 vector (Invitrogen, Carlsbad, CA, USA) (15 mug of plasmid DNA per 100 mm dish or 5 mug per well in six-well dishes) using Lipofectamine (Life Technologies, Gaithersburg, MD, USA) and washed to remove the excess vector and Lipofectamine. To determine the transfection efficiency, cultures were co-transfected with plasmid pRSV-beta-gal (Promega Corporation, Madison, WI, USA) to allow staining with 5-bromo-4-choloro-3indolyl-beta-D-galactoside reagent and visualization of transfected (blue-staining) cells.

siRNA treatments

Validated siRNAs for human Raf1 (ID no. 42858), RalA (ID no. 120429) and a negative control siRNA were purchased from Ambion (Austin, TX, USA) (see Ambion siRNA catalog for details). MKK3-siRNA (catalog no. PA-11386) and MKK6-siRNA (catalog no. PA-11388) were purchased from Dharmacon Inc. (Lafayette, CO, USA). For siRNA transfections, proliferating cells at about 30–50% of confluence were treated with the indicated siRNA using OligofectAMINE (Invitrogen, Carlsbad, CA, USA) according to manufacturer's instructions. Cells were treated with 50 nM of siRNA for 48 h; and the efficacy of the siRNA treatment was verified by Western blotting.

Measurement of NF-kappaB transcriptional activity

NF-kappaB activity was measured as described earlier (Fan et al., 2005). The NF-kappaB-Luc reporter plasmid (NF-kappaB-Luc) (Stratagene, La Jolla, CA, USA) is composed of five copies of the NF-kappaB enhancer element upstream of a TATA box and the luciferase gene. Briefly, subconfluent proliferating cells in 2 cm2 wells were transfected overnight with 0.25 mug of NF-kappaB-Luc reporter and 0.25 mug of each indicated vector, using Lipofectamine. The cells were then washed to remove Lipofectamine and excess vectors, allowed to recover for several hours, treatedplusminusSF (100 ng/ml) for 24 h and harvested for measurement of luciferase activity, using the Dual Luciferase Reporter Assay System (Promega, Piscataway, NJ, USA). Transfection efficiency was determined using the Galacto-Star Mammalian Reporter Gene Assay System (Applied Biosystems, Foster City, CA, USA). Luciferase values were meansplusminuss.e.m.s of quadruplicate wells. Each experiment was performed at least twice to assure that the findings were reproducible.

ADR treatment

Subconfluent proliferating cells in six-well dishes were transfected overnight with the indicated vector(s) (5 mug of plasmid DNA per well), washed and allowed to recover for several hours in fresh culture medium. The cells were then harvested, inoculated into 96-well dishes in complete culture medium, allowed to attach overnight, washed, incubatedplusminusSF (100 ng/ml times 48 h) in serum-free medium, exposed to different doses of ADR (2 h at 37°C) and postincubated for 48 h in fresh drug-free complete culture medium (plusminusSF). The cells were then analysed for cell viability using MTT assays. For each assay condition, cell viability was determined in 10 replicate wells. At least two independent experiments were performed for each vector tested.

MTT cell viability assay

This assay is based on the ability of viable mitochondria to convert MTT, a soluble tetrazolium salt, into an insoluble formazan precipitate, which is dissolved in dimethylsulfoxide and quantitated by spectrophotometry (Alley et al., 1988). After ADR treatment, cells in 96-well dishes were tested for MTT dye conversion. The cell viability was calculated as the amount of MTT dye conversion relative to sham-treated control cells.

Measurement of IKK-beta kinase activity

After cell transfections and SF treatment of DU-145 cells as described in the figure legends, IKK-beta kinase activity was measured using the HTScan IKK-beta Kinase Assay Kit (Cell Signaling Technology Inc., Danvers, MA, USA). This assay measures the ability of the cell lysate being tested to phosphorylate a biotinylated IkappaB-alpha (Ser32) substrate peptide. A phospho-IkappaB-alpha (Ser32/36)- specific mouse monoclonal antibody is used to detect the phosphorylated form of the substrate peptide. As a positive control and for standardization of the assay, the kit provides an active IKK-beta kinase in the form of a glutathione S-transferase fusion protein. Each experimental condition was tested in five replicate wells, and the values of IKK-beta kinase activity were expressed relative to the untransfected, nonSF treated control. Each experiment was performed at least twice to assure reproducibility of the results.

Immunoprecipitation

Ras IP
 

To facilitate this experiment, the V12Ras, V12C40Ras, V12S35Ras, V12G37Ras and V12E38 Ras cDNAs were cloned into the pCMV-Tag2B mammalian expression vector (Invitrogen) (to allow expression of the protein with an N-terminal FLAG epitope tag) using the BamH1 and XhoI cloning sites and sequenced. DU-145 cells were transfected overnight with the different FLAG-Ras vectors (10 mug plasmid DNA per 100 mm dish), washed and postincubated for 24 h to allow gene expression. The cells were then harvested by scraping; and total cell lysates were prepared in Tris-buffered saline (TBS) buffer (20 mM Tris–HCl pH 7.5, 137 mM NaCl and 0.5% NP-40) containing Complete Protease Inhibitor Cocktail (Roche, Indianapolis, IN, USA). The lysates were passed through a 22 G needle to shear the DNA and centrifuged at 100 000 g at 4°C for 30 min. The supernatant protein was quantified; and aliquots of protein (700 mug) were incubated with anti-FLAG monoclonal M2 (2.5 mug) agarose beads (Sigma, St Louis, MO, USA) at 4°C for 4 h. A control IP was performed using the same quantity of non-immune mouse IgG (catalog no. 7056, Cell Signaling Technology). The beads were then washed three times with TBS buffer. The precipitated proteins were solubilized in 2% sodium dodecyl sulfate (SDS) buffer at 45°C for 1 h and analysed by Western blotting.

Raf1 and c-Met Ips
 

Following transfection with V12Ras or empty pcDNA3 vector and treatment with SF (as described in the figure legends), DU-145 cells were scraped into 1 ml of lysis buffer (20 mM Tris–HCl (pH 7.5), 150 mM NaCl, 10% glycerol, 1% NP-40 and protease inhibitor cocktail set I (Calbiochem, San Diego, CA, USA)) per 100 mm dish. The lysed cells were incubated for 30 min at 0°C, centrifuged at 17 000g for 15 min at 4°C, and the supernatants were collected and protein concentrations determined with the Bio-Rad protein assay, using bovine serum albumin (BSA) as a standard. The IP antibodies were as follows: anti-Raf1 rabbit polyclonal antibody (Ab32025) or anti-c-Met rabbit polyclonal antibody (Ab14570) from Abcam (Cambridge, MA, USA). For IPs, 500 mug of protein was pre-cleared for 1 h by addition of Bio-Mag beads (Qiagen, Valencia, CA, USA). The pre-cleared lysates were incubated with IP antibody (5 mug) plus Bio-Mag beads at 4°C overnight. The beads were washed four times with 0.5 ml of lysis buffer; and 1 times loading buffer (25 mM Tris–HCl (pH 6.5), 5% glycerol, 1% SDS, 1% 2-mercaptoethanol and 0.05% bromphenol blue) was added. The samples were boiled for 3 min, analysed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE)); and Western blotted as described below.

Western blotting

For straight Western blotting, cells were collected using trypsin, washed twice with phosphate-buffered saline (PBS) and pelleted by centrifugation. The pellet was resuspended in radioimmunoprecipitation assay buffer (1 times PBS, 1% NP-40, 0.5% sodium desoxycholate and 0.1% SDS), placed on ice for 30 min and spun for 15 min at 14 000 r.p.m. at 4°C to remove the insoluble material. Western blotting was performed as described earlier (Fan et al., 2005). Briefly, aliquots of whole cell protein (50 mug per lane) or IP'd proteins (see above) were electrophoresed in 4–20% SDS–polyacrylamide gradient gels and transferred to nitrocellulose membranes. The membranes were blotted using the primary antibodies listed below and then blotted with the appropriate secondary antibodies (Santa Cruz Biotech, Santa Cruz, CA, USA, 1:1000 dilution). The blotted proteins were visualized using the enhanced chemiluminescence system (Amersham Pharmacia Biotech, Piscataway, NY, USA), with colored markers (Bio-Rad Laboratoriess, Hercules, CA, USA) as molecular size standards.

The primary antibodies were: phospho-Akt (serine-473) (np. 9271S, Cell Signaling Technology Inc., Beverly, MA, USA; 1:500); phospho-Akt (threonine-309) (monoclonal 244F9, Cell Signaling Technology); total Akt (no. 9271, Cell Signaling Technology, 1:500); c-Met (H-190, Santa Cruz, 1:1000); phospho-c-Met (tyrosine-1349) (Biosource International, Camarillo, CA, USA); FLAG (rabbit anti-FLAG, Sigma, 1:2000); IkappaB-alpha (C-15, Santa Cruz, 1:500); IKK-alpha (rabbit anti-human IKK-alpha, Sigma, 1:1000); IKK-beta (monoclonal 62AT216, Abgent Inc., San Diego, CA, USA; 1:500); phospho-IKK-alpha/beta (sc-21661, Santa Cruz, 1:500); p110alpha (sc-8010, Santa Cruz, 1:300); MKK3 (no. 9232, rabbit polyclonal, Cell Signaling Technology, 1:1000); MKK6 (no. 9264, rabbit polyclonal, Cell Signaling Technology, 1:1000); Raf1 (C-20, Santa Cruz, 1:1000 or no. 1560–1, Epitomics Inc., Burlingame, CA, USA, 1:500); phospho-Raf1 (pS259, Epitomics, 1:500); RalA (mouse monoclonal, BD Transduction Laboratories, San Diego, CA, USA, 1:100 or R23520, rabbit polyclonal, 1:400, Signal Transduction Laboratories, Lexington, KY, USA); RalGDS (C-19, Santa Cruz, 1:500); Ras (R4025, Sigma, 1:1000); and actin (goat polyclonal, Santa Cruz, 1:1000).

Immunofluorescence microscopy to detect p65 (RelA) subcellular translocation

After transfection and treatment with SF (see text and figure legends), DU-145 cells cultured on glass coverslips in six-well plastic dishes were fixed with 4% paraformaldehyde (Electron Microscopy Sciences, Fort Washington, PA, USA) in PBS for 10 min at 25°C. The cells were then permeabilized using Tween-20 (in PBS) for 10 min at 25°C; incubated in PBS containing 1% BSA for 1 h at 25°C to block nonspecific staining; and incubated overnight at 4°C with rabbit anti-human p65 polyclonal antibody (1:100 dilution) (Santa Cruz). The cells were then washed and incubated with Cy5-conjugated goat anti-rabbit IgG antibody (Sigma) (1:100 in blocking buffer) for 1 h at 37°C and washed five times with PBS. The coverslips were mounted on glass slides with Mowiol 4-88 (Hoechst Celanese, Somerville, NJ, USA). A laser with a wavelength of 638 nm was utilized for excitation of Cy5 fluorescence. Fluorescence images were captured with a Nikon Eclipse E800 confocal laser fluorescence microscope with an objective lens (times 40) (Diagnostic Instruments, Sterling Heights, MI, USA). The images were collected through the specimens every 3 s in the vertical plane and overlaid to generate focus composite images. The images were exported and displayed images were exported to Adobe Photoshop (Adobe, San Jose, CA, USA).

Statistical analyses

Where appropriate, statistical comparisons were made using the two-tailed Student's t-test.

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Acknowledgements

Sources of support: This work is supported, in part, by United States Public Health Service Grants RO1-ES09169 (EMR) and RO1-NS43987 (JJL/EMR).