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• W2040334711 abstract "Extensive data indicate that the transcription factor NFκB is activated by signals downstream of oncoproteins such as Ras or breakpoint cluster region (BCR)-ABL. Consistent with this, evidence has been presented that NFκB activity is required for Ras and BCR-ABL to transform cells. However, it remains unclear whether these oncoproteins activate a full spectrum of NFκB-dependent gene expression or whether they may augment or interfere with other stimuli that activate NFκB. The data presented here indicate that BCR-ABL expression in 32D myeloid cells or oncogenic Ras expression in murine fibroblasts blocks the ability of tumor necrosis factor (TNF) to activate NFκB. This suppression of NFκB is manifested by an inhibition of TNF-induced inhibitor of NFκB (IKK) activity and NFκB DNA binding potential but not by blocking TNF-induced nuclear accumulation of NFκB/p65. The inhibition of NFκB is not observed in oncogenic Raf-expressing cells and is not fully restored by the suppression of PI3-kinase or MEK pathways. Oncogenic Ras suppresses the ability of TNF to activate the expression of NFκB-dependent genes, such as iNOS (inducible nitric oxide synthase) and RANTES (regulated on activation normal T-cell expressed and secreted). These studies suggest that the ability of Ras and BCR-ABL to activate NFκB involves an uncharacterized pathway that does not involve classic IKK activity and that suppresses the TNF-induced IKK pathway through a Raf/MEK/Erk-independent mechanism. Extensive data indicate that the transcription factor NFκB is activated by signals downstream of oncoproteins such as Ras or breakpoint cluster region (BCR)-ABL. Consistent with this, evidence has been presented that NFκB activity is required for Ras and BCR-ABL to transform cells. However, it remains unclear whether these oncoproteins activate a full spectrum of NFκB-dependent gene expression or whether they may augment or interfere with other stimuli that activate NFκB. The data presented here indicate that BCR-ABL expression in 32D myeloid cells or oncogenic Ras expression in murine fibroblasts blocks the ability of tumor necrosis factor (TNF) to activate NFκB. This suppression of NFκB is manifested by an inhibition of TNF-induced inhibitor of NFκB (IKK) activity and NFκB DNA binding potential but not by blocking TNF-induced nuclear accumulation of NFκB/p65. The inhibition of NFκB is not observed in oncogenic Raf-expressing cells and is not fully restored by the suppression of PI3-kinase or MEK pathways. Oncogenic Ras suppresses the ability of TNF to activate the expression of NFκB-dependent genes, such as iNOS (inducible nitric oxide synthase) and RANTES (regulated on activation normal T-cell expressed and secreted). These studies suggest that the ability of Ras and BCR-ABL to activate NFκB involves an uncharacterized pathway that does not involve classic IKK activity and that suppresses the TNF-induced IKK pathway through a Raf/MEK/Erk-independent mechanism. Mechanisms controlling gene-specific transcription downstream of oncoprotein-dependent signaling are poorly understood. Although certain transcription factors, including Ets and NFκB proteins, have been shown to be required for transformation in response to oncoprotein expression (1Langer S.J. Bortner D.M. Roussel M.F. Sherr C.J. Ostrowski M.C. Mol. Cell. Biol. 1992; 12: 5355-5362Crossref PubMed Scopus (136) Google Scholar, 2Yang B.S. Hauser C.A. Henkel G. Colman M.S. Van Beveren C. Stacey K.J. Hume D.A. Maki R.A. Ostrowski M.C. Mol. Cell. Biol. 1996; 16: 538-547Crossref PubMed Scopus (318) Google Scholar, 3Finco T.S. Westwick J.K. Norris J.L. Beg A.A. Der C.J. Baldwin Jr., A.S. J. Biol. Chem. 1997; 272: 24113-24116Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar), it is unclear whether transforming proteins elicit a full transcription factor-dependent response or whether a limited set of transcription factor-regulated genes are induced to control transformation. In fact, based on the broad range of genes regulated by NFκB, it has been hypothesized that its role in oncogenic transformation would involve induction of a limited set of genes to prevent an effective immunological response against neoplastic cells (4Karin M. Cao Y. Greten F.R. Li Z.W. Nat. Rev. Cancer. 2002; 2: 301-310Crossref PubMed Scopus (2265) Google Scholar). Small GTPases of the Ras family are important signaling molecules in the regulation of a variety of cellular processes, including growth, differentiation, and survival (5Reuther G.W. Der C.J. Curr. Opin. Cell Biol. 2000; 12: 157-165Crossref PubMed Scopus (348) Google Scholar). Growth factors and other external stimuli lead to transient activation of Ras; however, mutations in Ras alleles, which occur in ∼30% of human tumors, result in a constitutively active protein (6Bos J.L. Cancer Res. 1989; 49: 4682-4689PubMed Google Scholar). Active, GTP-bound Ras signals to a variety of downstream effector pathways. The three most extensively characterized Ras effectors are Raf kinase, phosphatidylinositol 3-kinase (PI3-kinase), and RalGDS (7Campbell S.L. Khosravi-Far R. Rossman K.L. Clark G.J. Der C.J. Oncogene. 1998; 17: 1395-1413Crossref PubMed Scopus (920) Google Scholar, 8Joneson T. Bar-Sagi D. J. Mol. Med. 1997; 75: 587-593Crossref PubMed Scopus (146) Google Scholar). Activation of Ras and its downstream signal transduction cascades ultimately leads to activation of transcription factors involved in proliferation, differentiation, and apoptosis. Ras also functions to control downstream signaling of other oncoproteins, such as the fusion protein breakpoint cluster region (BCR) 1The abbreviations used are: BCR, breakpoint cluster region; IκB, inhibitor of κ B; IKK, inhibitor of IκB kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; TNF, tumor necrosis factor; MEF, mouse embryo fibroblast; IL, interleukin; EMSA, electrophoretic mobility shift assay; RANTES, regulated on activation normal T cell expressed and secreted; RLE, rat liver epithelial; iNOS, inducible nitric oxide synthase; PI, phosphatidylinositol.-ABL that is associated with acute lymphoblastic and chronic myelogenous leukemia (9Pendergast A.M. Quilliam L.A. Cripe L.D. Bassing C.H. Dai Z. Li N. Batzer A. Rabun K.M. Der C.J. Schlessinger J. Gishizy M.L. Cell. 1993; 75: 175-185Abstract Full Text PDF PubMed Scopus (594) Google Scholar, 10Cortez D. Stoica G. Pierce J.H. Pendergast A.M. Oncogene. 1996; 13: 2589-2594PubMed Google Scholar, 11Gishizky M.L. Cortez D. Pendergast A.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10889-10893Crossref PubMed Scopus (77) Google Scholar, 12Cortez D. Kadlec L. Pendergast A.M. Mol. Cell. Biol. 1995; 15: 5531-5541Crossref PubMed Scopus (268) Google Scholar, 13Goga A. McLaughlin J. Afar D.E. Saffran D.C. Witte O.N. Cell. 1995; 82: 981-988Abstract Full Text PDF PubMed Scopus (257) Google Scholar). Work from our laboratory and others has demonstrated that the transcription factor NFκB is activated by Ras (3Finco T.S. Westwick J.K. Norris J.L. Beg A.A. Der C.J. Baldwin Jr., A.S. J. Biol. Chem. 1997; 272: 24113-24116Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar, 14Norris J.L. Baldwin Jr., A.S. J. Biol. Chem. 1999; 274: 13841-13846Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 15Mayo M.W. Wang C.Y. Cogswell P.C. Rogers-Graham K.S. Lowe S.W. Der C.J. Baldwin Jr., A.S. Science. 1997; 278: 1812-1815Crossref PubMed Scopus (506) Google Scholar, 16Arsura M. Mercurio F. Oliver A.L. Thorgeirsson S.S. Sonenshein G.E. Mol. Cell. Biol. 2000; 20: 5381-5391Crossref PubMed Scopus (112) Google Scholar) and by BCR-ABL in a Ras-dependent manner (17Reuther J.Y. Reuther G.W. Cortez D. Pendergast A.M. Baldwin Jr., A.S. Genes Dev. 1998; 12: 968-981Crossref PubMed Scopus (353) Google Scholar). Inhibition of NFκB blocks Ras-induced transformation in vitro (3Finco T.S. Westwick J.K. Norris J.L. Beg A.A. Der C.J. Baldwin Jr., A.S. J. Biol. Chem. 1997; 272: 24113-24116Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar) and BCR-ABL-induced tumorigenesis (17Reuther J.Y. Reuther G.W. Cortez D. Pendergast A.M. Baldwin Jr., A.S. Genes Dev. 1998; 12: 968-981Crossref PubMed Scopus (353) Google Scholar). NFκB is a family of dimeric transcription factors involved in immune and inflammatory responses, cellular growth, differentiation, and apoptosis (18Ghosh S. Karin M. Cell. 2002; 109: S81-S96Abstract Full Text Full Text PDF PubMed Scopus (3300) Google Scholar). There are five mammalian NFκB family members: RelA/p65, RelB, c-Rel, p50/p105, and p52/p100. All members share homology in a 300-amino acid region called the Rel homology domain. The Rel homology domain is important for dimerization, nuclear translocation, DNA binding, and binding to the inhibitor of κ B (IκB) family of proteins (18Ghosh S. Karin M. Cell. 2002; 109: S81-S96Abstract Full Text Full Text PDF PubMed Scopus (3300) Google Scholar). In unstimulated cells, IκB proteins localize NFκB dimers in the cytoplasm by masking the nuclear localization sequence of NFκB. Activation of NFκB can occur through various stimuli, including cytokine stimulation, bacterial and viral infection, and oncogenic signals (18Ghosh S. Karin M. Cell. 2002; 109: S81-S96Abstract Full Text Full Text PDF PubMed Scopus (3300) Google Scholar). Receptor activation of TNFR1, IL-1R, and various Toll-like receptors initiates signal transduction cascades ultimately leading to activation of the IκB kinase (IKK) complex. The IKK complex is composed of three subunits, IKKα, IKKβ, and IKKγ (18Ghosh S. Karin M. Cell. 2002; 109: S81-S96Abstract Full Text Full Text PDF PubMed Scopus (3300) Google Scholar). Whereas IKKγ is a regulatory subunit, both IKKα and IKKβ have inducible catalytic activity. Upon activation, the IKK complex phosphorylates IκBα and IκBβ at specific serine residues, which targets IκB for ubiquitination and subsequent degradation by a proteasome-dependent pathway. Once IκB is degraded, the nuclear localization sequence of NFκB is unmasked, allowing nuclear accumulation, DNA binding, and transcriptional activation of target genes (18Ghosh S. Karin M. Cell. 2002; 109: S81-S96Abstract Full Text Full Text PDF PubMed Scopus (3300) Google Scholar). Although nuclear accumulation is an important step in NFκB activation, post-translational modifications on p65 are proposed to be necessary for the transcriptional competence of nuclear NFκB. For example, phosphorylation of p65 on serine 276 is required for stable interactions with the transcriptional coactivator CBP and to stimulate transcriptional activation of NFκB target genes (19Zhong H. SuYang H. Erdjument-Bromage H. Tempst P. Ghosh S. Cell. 1997; 89: 413-424Abstract Full Text Full Text PDF PubMed Scopus (727) Google Scholar, 20Zhong H. Voll R.E. Ghosh S. Mol. Cell. 1998; 1: 661-671Abstract Full Text Full Text PDF PubMed Scopus (1024) Google Scholar). Other sites of phosphorylation have also been described that may contribute to the inherent transcriptional activity of NFκB (21Wang D. Baldwin Jr., A.S. J. Biol. Chem. 1998; 273: 29411-29416Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar, 22Wang D. Westerheide S.D. Hanson J.L. Baldwin Jr., A.S. J. Biol. Chem. 2000; 275: 32592-32597Abstract Full Text Full Text PDF PubMed Scopus (373) Google Scholar, 23Sakurai H. Chiba H. Miyoshi H. Sugita T. Toriumi W. J. Biol. Chem. 1999; 274: 30353-30356Abstract Full Text Full Text PDF PubMed Scopus (711) Google Scholar). Evidence has also been presented that Akt, which functions downstream of PI3-kinase, can control the transcriptional activation function of the p65 NFκB subunit through a mechanism dependent on IKK function but in a manner which does promote enhanced DNA binding potential (24Madrid L.V. Mayo M.W. Reuther J.Y. Baldwin Jr., A.S. J. Biol. Chem. 2001; 276: 18934-18940Abstract Full Text Full Text PDF PubMed Scopus (700) Google Scholar, 25Sizemore N. Lerner N. Dombrowski N. Sakurai H. Stark G.R. J. Biol. Chem. 2002; 277: 3863-3869Abstract Full Text Full Text PDF PubMed Scopus (353) Google Scholar). In addition, a number of oncoproteins activate NFκB by increasing transcriptional activation function (26Mayo M.W. Norris J.L. Baldwin A.S. Methods Enzymol. 2001; 333: 73-87Crossref PubMed Scopus (61) Google Scholar). Consistent with this, we have shown that both oncogenic Ras and Raf activate an NFκB-dependent reporter gene in mouse fibroblasts without stimulating enhanced DNA binding of NFκB subunits (3Finco T.S. Westwick J.K. Norris J.L. Beg A.A. Der C.J. Baldwin Jr., A.S. J. Biol. Chem. 1997; 272: 24113-24116Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar, 14Norris J.L. Baldwin Jr., A.S. J. Biol. Chem. 1999; 274: 13841-13846Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). Similarly, previous work suggests that BCR-ABL activates an NFκB-dependent reporter but in a manner that did not promote strong DNA binding activity (17Reuther J.Y. Reuther G.W. Cortez D. Pendergast A.M. Baldwin Jr., A.S. Genes Dev. 1998; 12: 968-981Crossref PubMed Scopus (353) Google Scholar). Here we investigate the ability of BCR-ABL and Ras to modulate cytokine-induced NFκB-dependent responses. Our studies indicate that in murine myeloid cells and fibroblasts, BCR-ABL and Ras, respectively, strongly suppress TNF-induced NFκB activation by blocking both IKK activity and DNA binding. Inhibition of the PI3-kinase and MEK/extracellular signal-regulated kinase (Erk) pathways could not fully overcome the Ras-induced block on NFκB activation. Additionally, oncogenic Ras suppresses TNF-induced activation of the NFκB-dependent genes iNOS and RANTES. These studies suggest that the ability of Ras and BCR-ABL to activate NFκB involves an uncharacterized pathway that does not involve classic IKK activity and that suppresses the TNF-induced IKK pathway. Cell Culture and Reagents—Spontaneously immortalized mouse embryo fibroblasts (MEFs) were maintained in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum and penicillin-streptomycin. Cells stably expressing a constitutively active form of H-Ras, H-RasV12, or vector control were obtained by transfection of pZip-H-RasV12 or pZipneo with Polyfect (Qiagen). Cells expressing H-RasV12 were selected in medium containing 500 μg/ml G418. Surviving cells were pooled 2 weeks later. NIH 3T3 control and Raf-expressing cells were described previously (3Finco T.S. Westwick J.K. Norris J.L. Beg A.A. Der C.J. Baldwin Jr., A.S. J. Biol. Chem. 1997; 272: 24113-24116Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar). 32D myeloid cells were maintained in RPMI 1640 supplemented with 10% fetal bovine serum, 10% Wehi-conditioned medium, and penicillin-streptomycin. Generation of 32D cells expressing BCR-ABL fusion protein p185 was described previously (17Reuther J.Y. Reuther G.W. Cortez D. Pendergast A.M. Baldwin Jr., A.S. Genes Dev. 1998; 12: 968-981Crossref PubMed Scopus (353) Google Scholar). Cells were treated with 10 ng/ml TNF (Promega), 5 ng/ml IL-1β (Sigma), 20 μm PD98059 (Alexis Pharmaceuticals), or 10 μm LY294002 (Alexis Pharmaceuticals) where indicated. EMSAs and Western Blot Analysis—Preparation of nuclear and cytoplasmic extracts and subsequent electrophoretic mobility shift assays (EMSAs) were performed as previously described (27Haskill S. Beg A.A. Tompkins S.M. Morris J.S. Yurochko A.D. Sampson-Johannes A. Mondal K. Ralph P. Baldwin Jr., A.S. Cell. 1991; 65: 1281-1289Abstract Full Text PDF PubMed Scopus (586) Google Scholar). Briefly, nuclear extracts were prepared following TNF stimulation and incubated with an α-32P-labeled DNA probe containing an NFκB consensus from the major histocompatibility complex class I promoter. For supershifts, 1 μl of antibody (described below) was incubated with protein-DNA complexes for 10 min prior to loading gel. DNA-protein complexes were separated on a 5% non-denaturing polyacrylamide gel. The gel was dried, and DNA-protein complexes were visualized by autoradiography. Western blot analysis was performed by preparing nuclear, cytoplasmic, or whole cell extracts and separating proteins by SDS-PAGE. After transferring the separated proteins to nitrocellulose the blots were blocked in TBST with 5% milk and then incubated in primary antibody (IκBα, c-Rel, RelB, p50, and p52 (Santa Cruz Biotechnology), p65 (Rock-land), phospho-IκBα Ser-32/36, phospho-c-Jun Ser-73, and phospho-Akt Ser-473 (Cell Signaling), phospho-p42/p44 Thr-202/Tyr-204 (New England Biolabs)) for either 1–2 h or overnight. The membranes were then washed in TBST and incubated for 1 h in anti-mouse or anti-rabbit horseradish peroxidase-conjugated secondary antibody (Promega) and washed again. Protein bands were visualized with enhanced chemiluminescence detection (Amersham Biosciences). Kinase Assay—500 μg of whole cell extracts prepared from cells as previously described (28Mercurio F. Zhu H. Murray B.W. Shevchenko A. Bennett B.L. Li J. Young D.B. Barbosa M. Mann M. Manning A. Rao A. Science. 1997; 278: 860-866Crossref PubMed Scopus (1855) Google Scholar) was incubated with 25 μl of IKKγ antibody (Upstate) overnight at 4 °C. Protein A-Sepharose beads washed in whole cell extract buffer were added and incubated for 1–2 h at 4 °C. The beads were then washed three times in 1 ml of pulldown buffer and one time in assay dilution buffer (ADB). Next, the beads were incubated at 30 °C for 30 min in 25 μl of 1× ADB plus 4 μg of GST-IκBα, MgCl2, and ATP. 30 μl of 2× sample buffer was added, and the proteins were separated by SDS-PAGE. Separated proteins were transferred to nitrocellulose, and Western blotting for phospho-IκBα serine 32/36 (New England Biolabs) was performed. Luciferase Assays—Control and H-RasV12-transformed cells were plated at 2 × 104 cells/well in a 24-well plate. 24 h later 100 ng of pGl4-κB luciferase, 200 ng of β-actin LacZ, and 200 ng of empty vector or 100 ng of Gal4-luciferase, 50 ng of Gal4-p54, and 350 ng of empty vector DNA was transfected into the cells with Polyfect (Qiagen) according to the manufacturer's protocol. 24 h post-transfection the cells were harvested in 100 μl of M-PER buffer (PIERCE) and assayed for luciferase and β-galactosidase activity. All data are represented as luciferase units/β-galactosidase units. Ribonuclease Protection Assay—Ribonuclease protection assays were performed on a custom template according to the manufacturer's protocol (BD PharMingen). Briefly, 20 μg of total RNA, isolated with Trizol (Invitrogen), was hybridized overnight to biotinylated RNA probes. Unhybridized RNA and biotinylated probes were then digested with RNase A. The protected RNA complexes were then precipitated and resolved on a 4.75% polyacrylamide urea sequencing gel. The complexes were then transferred to a nitrocellulose membrane. After UV cross-linking, the membrane was incubated with streptavidin-horseradish peroxidase. Protected complexes were visualized by autoradiography after incubation with luminal/enhancer solution. BCR-ABL and H-RasV12 Expression Inhibits TNF–induced NFκB Activation—Previous reports indicate that oncogenic Ras and BCR-ABL can activate NFκB transactivation function independent of enhanced DNA binding. However, little is known about how oncoprotein-induced signaling may affect the ability of other stimuli to activate NFκB. To determine whether oncogenic Ras or BCR-ABL have an effect on TNF-induced NFκB activation, immortalized MEFs transformed with H-RasV12 or 32D cells expressing the p185 BCR-ABL fusion protein were examined. Transformed cells (H-Ras or BCR-ABL-expressing) and control cells (MEFs or 32D cells) were stimulated with TNF for 15, 30, or 60 min. Nuclear extracts were prepared, and NFκB DNA binding activity was determined by EMSA. As expected, TNF rapidly induces NFκB DNA binding activity by 15 min in control cells; however, this activation is greatly reduced in both H-RasV12-transformed MEFs (Fig. 1A) and BCR-ABL-expressing 32D cells (Fig. 1B). Super-shift analysis of the MEF control 30′ time point revealed that the NFκB DNA binding complex I contains p65 and p50 subunits, while complex II is shifted by the c-Rel antibody (Fig. 1A, right panel). BCR-ABL transformation is dependent on its kinase function and on its ability to utilize Ras-dependent downstream signaling (9Pendergast A.M. Quilliam L.A. Cripe L.D. Bassing C.H. Dai Z. Li N. Batzer A. Rabun K.M. Der C.J. Schlessinger J. Gishizy M.L. Cell. 1993; 75: 175-185Abstract Full Text PDF PubMed Scopus (594) Google Scholar, 10Cortez D. Stoica G. Pierce J.H. Pendergast A.M. Oncogene. 1996; 13: 2589-2594PubMed Google Scholar, 11Gishizky M.L. Cortez D. Pendergast A.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10889-10893Crossref PubMed Scopus (77) Google Scholar, 12Cortez D. Kadlec L. Pendergast A.M. Mol. Cell. Biol. 1995; 15: 5531-5541Crossref PubMed Scopus (268) Google Scholar, 13Goga A. McLaughlin J. Afar D.E. Saffran D.C. Witte O.N. Cell. 1995; 82: 981-988Abstract Full Text PDF PubMed Scopus (257) Google Scholar). We have previously published that BCR-ABL-induced NFκB activation is dependent on BCR-ABL-induced Ras activation (17Reuther J.Y. Reuther G.W. Cortez D. Pendergast A.M. Baldwin Jr., A.S. Genes Dev. 1998; 12: 968-981Crossref PubMed Scopus (353) Google Scholar). Therefore, in the remaining experiments, we have focused largely on the ability of oncogenic Ras to modulate TNF-induced NFκB activation. H-RasV12 Blocks TNF-induced IKK Activity—To determine the mechanism by which H-RasV12 inhibits TNF-induced NFκB activation, IκBα phosphorylation and degradation were examined. Control and Ras-transformed MEFs were treated with 10 ng/ml TNF for 5, 15, 30, 45, and 60 min, and whole cell extracts were prepared. These extracts were analyzed for the induction of phosphorylation and degradation of IκBα. As observed in Fig. 2A, Ras-transformed cells exhibit reduced levels of IκBα phosphorylation at serines 32 and 36 in response to TNF treatment. Consistent with this result, Ras-transformed MEFs exhibit significantly reduced IκBα degradation (Fig. 2A, middle panel). Similar results were also obtained in BCR-ABL-transformed myeloid cells (data not shown). To determine whether other downstream signaling components associated with TNF receptor activation are affected in Ras-transformed cells, we examined the phosphorylation status of c-Jun. In control MEFs, TNF-inducible phosphorylated c-Jun levels were detected. Although phosphorylated c-Jun levels are higher in Ras-transformed cells, TNF stimulation further increases phosphorylated c-Jun (Fig. 2A, lower panel). These data suggest the TNF-induced c-Jun NH2-terminal kinase signaling pathway is not affected in Ras-transformed MEFs. Because IκBα phosphorylation and subsequent degradation are controlled by IKK, the activity of IKK was examined in control and Ras-transformed MEFs. IKK assays were performed by treating MEFs with TNF for the indicated times, harvesting whole cell extracts, and immunoprecipitating the IKK complex with an antibody to IKKγ. The immunoprecipitated complex was then incubated with GST-IκBα as a substrate in an in vitro kinase assay. The proteins were separated by SDS-PAGE, and Western blot analysis for phospho-IκBα Ser-32/36 was performed. As expected, TNF rapidly activates the IKK complex in control MEFs, whereas no activation of the complex by TNF is observed in Ras-transformed MEFs (Fig. 2B). Interestingly, in Western blot analysis of whole cell extracts with antibodies to phospho-IκBα Ser-32/36 (Fig. 2A), minimal phosphorylation is detected in the H-RasV12-transformed MEFs, whereas the results from the kinase assay suggest that IKK activation was completely blocked (Fig. 2B). One possible explanation is that the IKK assay may not be as sensitive as protein immunoblotting for phosphorylated IκBα. Alternatively, it is possible that another IκB kinase activity, not associated with IKKγ, is activated by TNF and is able to phosphorylate IκB in vivo. Currently, it is proposed that IκBα degradation controls the ability of NFκB to accumulate in the nucleus following cytokine treatment. Therefore we examined whether the inhibition of NFκB in BCR-ABL- and Ras-transformed cells results in a defect in nuclear accumulation of NFκB subunits. Whereas TNF induces nuclear accumulation of p65 in 32D cells, nuclear levels of p65 are very high in unstimulated BCR-ABL-transformed 32D cells and remain high following TNF treatment (Fig. 2C, upper panel). Unexpectedly, the ability of TNF to stimulate nuclear accumulation of the p65 subunit appears unaffected in Ras-transformed cells, whereas nuclear accumulation of c-Rel is blocked (Fig. 2D, lower panel). Nuclear levels of RelB and p52 are unaffected by TNF treatment in control and Ras-transformed MEFs. Overall, these data indicate that IKK-directed IκBα phosphorylation is not required for nuclear accumulation of p65 but is required for optimal NFκB DNA binding activity and gene expression. Alternatively, expression of Ras may bypass this requirement. Notably, basal levels of nuclear NFκB p65 subunit are modestly elevated in Ras-transformed cells and even higher in BCR-ABL-transformed 32D cells, suggesting that in the absence of external stimuli, oncoprotein-induced signaling pathways promote the nuclear accumulation of the p65 subunit. Together these data indicate that oncogenic Ras and BCR-ABL do not inhibit TNF-induced nuclear translocation of p65; however, H-RasV12 does inhibit TNF-induced c-Rel nuclear translocation. H-RasV12 Does Not Inhibit IL-1β-induced NFκB Activation—A variety of different cytokines are known to activate NFκB. To determine whether oncogenic Ras inhibits NFκB activation by other cytokines, control and H-RasV12-transformed MEFs were treated with 5 ng/ml IL-1β for 5, 10, 15, or 30 min. Nuclear and cytoplasmic extracts were prepared and analyzed by EMSA and Western blot, respectively. EMSA analysis revealed no defect in IL-1β-induced NFκB DNA binding activity (Fig. 3, upper panel). Furthermore, IL-1β-induced IκBα phosphorylation is equivalent in control and H-RasV12-transformed MEFs (Fig. 3, lower panel). These data suggest that oncogenic Ras specifically inhibits TNF-induced NFκB activation but not IL-1β-induced NFκB activation. The signaling components involved in TNF and IL-1β receptor activation are well established. Although activation of both receptors results in NFκB activation, the signaling molecules that lead to IKK activation are different for these two pathways (29Mercurio F. Manning A.M. Oncogene. 1999; 18: 6163-6171Crossref PubMed Scopus (366) Google Scholar). The differences in these signaling pathways suggest that oncogenic Ras may inhibit a component of the TNF pathway that does not overlap with the IL-1β pathway. However, Western blot analysis for TNF signaling components (TNFR1, TRADD, TRAF2, RIP, MEKK3, IKKα, IKKβ, and IKKγ) shows no changes in protein levels between control and Ras-transformed MEFs (data not shown). Inhibition of TNF-induced NFκB Activation Requires Multiple Ras Effector Pathways—Ras proteins activate a variety of downstream effectors that ultimately lead to activation of transcription factors involved in cellular growth control. The most extensively studied effector pathways of Ras are controlled by PI3 kinase, Raf, and RalGEFs (7Campbell S.L. Khosravi-Far R. Rossman K.L. Clark G.J. Der C.J. Oncogene. 1998; 17: 1395-1413Crossref PubMed Scopus (920) Google Scholar, 8Joneson T. Bar-Sagi D. J. Mol. Med. 1997; 75: 587-593Crossref PubMed Scopus (146) Google Scholar). A recent study revealed that normal rat kidney cells expressing an inducible, constitutively active Raf-1 suppressed TNF- and IL-1β-induced NFκB activation (30Liu Q. Fan J. McMahon M. Prince A.M. Zhang P. Mol. Cell Biol. Res. Commun. 2001; 4: 381-389Crossref PubMed Scopus (8) Google Scholar), suggesting that Ras inhibition of TNF signaling could be because of the ability of Ras to activate the Raf-1 kinase pathway. To determine whether the effect observed in Ras-transformed murine fibroblasts is due solely to activated Raf, control and Raf-transformed 3T3 cells were treated with TNF, and NFκB DNA binding potential was analyzed by EMSA (Fig. 4). This experiment demonstrates that NFκB DNA binding is not impaired in Raf-transformed 3T3 cells. In fact, the expression of Raf appears to augment the ability of TNF to activate NFκB as measured by EMSA. These data show Ras activation of Raf is not sufficient to inhibit TNF-induced NFκB DNA binding. To further analyze Ras signaling pathways involved in NFκB inhibition, pharmacological inhibitors of the MEK/Erk and PI3 kinase pathways, PD98059 (PD) and LY294002 (LY), respectively, were used. Cytoplasmic and nuclear extracts of the treated control and Ras-transformed cells were prepared to determine whether the inhibitors have an effect on TNF-induced NFκB DNA binding potential. In control cells the inhibitors have no effect on NFκB DNA binding (Fig. 4, B and C, lanes 1–3). Furthermore, the PD and LY compounds alone or in combination (data not shown) have minimal effects on NFκB DNA binding potential in Ras-transformed fibroblasts (Fig. 4, B and C, lanes 10–12). Western blot analysis of cytoplasmic extracts for phospho-p42/p44 clearly demonstrates the effectiveness of the PD compound (Fig. 4B, lower panel). Immunoblotting for phospho-Akt demonstrates that the LY compound is capable of inhibiting TNF-induced phosphorylation of Akt in control MEFs and decreasing basal levels of phospho-Akt in Ras-transformed MEFs. The LY compound, however, does not completely block TNF-induced phosphorylation of Akt (Fig 4C, lower panel). A slight increase in TNF-induced NFκB DNA binding in Ras-transformed MEFs treated with the LY compound is observed, and further investigation is needed to determine whether complete inhibition of TNF-induced phosphorylation of Akt would further enhance TNF-indu" @default.
• W2040334711 created "2016-06-24" @default.
• W2040334711 creator A5015742165 @default.
• W2040334711 creator A5042676724 @default.
• W2040334711 creator A5059458097 @default.
• W2040334711 creator A5077791038 @default.
• W2040334711 date "2003-09-01" @default.
• W2040334711 modified "2023-09-27" @default.
• W2040334711 title "Oncoprotein Suppression of Tumor Necrosis Factor-induced NFκB Activation Is Independent of Raf-controlled Pathways" @default.
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