Abstract

Nuclear factor-B (NF-B) is constitutively activated in diverse human malignancies by mechanisms that are not understood^{1, 2}. The MUC1 oncoprotein is aberrantly overexpressed by most human carcinomas and, similarly to NF-B, blocks apoptosis and induces transformation^{3, 4, 5, 6}. This study demonstrates that overexpression of MUC1 in human carcinoma cells is associated with constitutive activation of NF-B p65. We show that MUC1 interacts with the high-molecular-weight IB kinase (IKK) complex in vivo and that the MUC1 cytoplasmic domain binds directly to IKK and IKK. Interaction of MUC1 with both IKK and IKK is necessary for IKK activation, resulting in phosphorylation and degradation of IB. Studies in non-malignant epithelial cells show that MUC1 is recruited to the TNF-R1 complex and interacts with IKK–IKK in response to TNF stimulation. TNF-induced recruitment of MUC1 is dependent on TRADD and TRAF2, but not the death-domain kinase RIP1. In addition, MUC1-mediated activation of IKK is dependent on TAK1 and TAB2. These findings indicate that MUC1 is important for physiological activation of IKK and that overexpression of MUC1, as found in human cancers, confers sustained induction of the IKK–NF-B p65 pathway.

Introduction

Nuclear localization of NF-B p65 was studied in HCT116 colon cancer and HeLa cervical cancer cells that stably express either an empty vector or MUC1 (ref. 4, also see Supplementary Information, Fig. S1a). Levels of nuclear NF-B p65 were lower in vector cells than in cells expressing MUC1 (Fig. 1a). Human ZR-75-1 and MCF-7 breast cancer cells that express endogenous MUC1 were stably transfected to express either an empty vector or a MUC1 siRNA⁴ (Supplementary Information, Fig. S1a). Silencing of MUC1 in ZR-75-1 (ref. 4) and MCF-7 cells⁷ decreased nuclear NF-B p65 (Fig. 1b). MUC1 expression was also associated with a decrease in cytosolic NF-B p65 levels in HeLa and ZR-75-1 cells (Supplementary Information, Fig. S1b). To determine whether MUC1 is associated with activation of the NF-B p65 transcription function, HeLa and ZR-75-1 cells were transfected with a construct containing a NF-B-binding site upstream of the luciferase reporter (pNF-B-Luc). MUC1 expression was associated with activation of pNF-B-Luc (Fig. 1c). In contrast, MUC1 had no effect on activation of a pNF-B-Luc construct that was mutated at the NF-B p65 binding site (Fig. 1c). In addition, expression of Bcl-x_L, a gene activated by NF-B, was higher in cells expressing MUC1 (Fig. 1d). To determine whether MUC1 affects IB phosphorylation (as phosphorylated IB is targeted for ubiquitination and proteosomal degradation) cytosolic lysates were immunoblotted with an anti-phospho-IB antibody. Indeed, phospho-IB levels were significantly higher in cells expressing MUC1 (Fig. 1e). Assessment of IB stability indicated that MUC1 expression increases degradation of IB (Fig. 1f). The half-lives of IB in the absence and presence of MUC1, were 6.7 0.5 h and 3.8 0.3 h (mean s.d., n = 3), respectively. Similar results were obtained in ZR-75-1 cells (data not shown), indicating that MUC1-induced increases in phosphorylation of IB are associated with increases in IB degradation. Targeting of NF-B p65 to the nucleus activates IB gene transcription in an inducible, autoregulatory pathway that replenishes IB levels^{1, 2}. Consistent with this autoregulatory loop, RT-PCR analysis demonstrated that MUC1-induced increases in nuclear NF-B p65 are associated with upregulation of IB mRNA levels (Fig. 1g). These findings indicate that MUC1 contributes to IB degradation, resulting in activation of NF-B p65.

Figure 1: MUC1 targets NF-B p65 to the nucleus by inducing phosphorylation and degradation of IB.

(a) and (b) Nuclear lysates from the indicated cells were subjected to immunoblotting with anti-p65, anti-lamin B and anti-IB antibodies. Whole cell lysate (WCL) prepared from HCT116-vector cells was used as a control for anti-IB reactivity. Immunoblot analysis of the nuclear lysates with antibodies against nuclear lamin B and cytosolic IB confirmed equal loading of the lanes and lack of cytoplasmic contamination. (c) The indicated cells were transfected with a pNF-B-Luc reporter plasmid or a mutant at the NF-B binding site and, as a control, the SV40-Renilla-Luc plasmid. Luciferase activity was measured at 48 h after transfection. The results are expressed as the fold activation (mean s.d., of three separate experiments) compared with that obtained in HeLa-vector (left) or ZR-75-1-MUC1 siRNA (right) cells (each assigned a value of 1). (d) Whole cell lysates from the indicated cells were immunoblotted with anti-Bcl-xL and anti--actin antibodies. (e) Cytosolic lysates from the indicated cells were immunoblotted with anti-phospho-IB, anti-IB and anti--actin antibodies. (f) HeLa-vector and HeLa-MUC1 cells were pulsed with ³⁵S-methionine and chased for the indicated times. Anti-IB immunoprecipitates from equal amounts of lysate were subjected to SDS–PAGE and autoradiography (upper panels). Intensity of the IB signals was determined by scanning densitometry and is expressed as the percentage IB remaining compared with that obtained at 0 h (lower panels). Similar results were obtained in two separate experiments. (g) IB and -actin mRNA levels were determined for the indicated cells by quantitative RT-PCR. Full scans of the gels in a, b, e and f are shown in Supplementary Fig. S6-1.

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The presence of IKK in a complex with IKK is necessary and sufficient for phosphorylation of IB in the classical NF-B pathway. IKK binds directly to IKK and is required for IKK activation. Analysis of anti-IKK immunoprecipitates from ZR-75-1 and MCF-7 cells showed that MUC1 carboxy-terminal subunit (MUC1-C) associates with IKK (Fig. 2a). In vitro studies with purified GST–IKK and the MUC1 cytoplasmic domain (MUC1-CD) demonstrated that these proteins interact directly with each other (Fig. 2b). This interaction was confirmed in experiments with purified GST–MUC1-CD and IKK (Fig. 2c, lower left). Studies with MUC1-CD amino acid fragments 1–45 and 46–72 demonstrated that MUC1-CD(1–45) confers binding to IKK (Fig. 2c, left). Studies with IKK(1–458) and IKK(458–756) further demonstrated that MUC1-CD binds directly to the IKK-amino-terminal region (Fig. 2c, lower right). The IKK-carboxy-terminal region associates with the N-terminal region of IKK. Consistent with the formation of IKK–IKK complexes and binding of MUC1 to IKK, we found that MUC1-C co-precipitates with IKK (Fig. 2d). In vitro studies with GST–IKK demonstrated that MUC1-CD binds to purified IKK (Fig. 2e). In contrast to the interaction with IKK, MUC1-CD(46–72) but not MUC1-CD(1–45) binds to IKK (Fig. 2f) and at the C-terminal region of IKK (Fig. 2g). To further assess binding of MUC1-C to IKK and IKK in vivo, MUC1-C was immunodepleted from HeLa-MUC1 cell lysates by precipitation with increasing amounts of an anti-MUC1-C antibody (Supplementary Information, Fig. S1c, left). As a control, the lysates were incubated with a non-immune IgG (Supplementary Information, Fig. S1c, right). Immunoblot analysis of the lysates demonstrated that depletion of MUC1-C is associated with decreases in IKK and IKK (Supplementary Fig. S1c). These findings indicate that MUC1 binds directly to IKK and IKK, and potentially to both proteins in IKK–IKK complexes (Supplementary Information, Fig. S1d).

Figure 2: MUC1-CD binds directly to IKK and IKK.

(a) Lysates from the indicated cells were subjected to immunoprecipitation with a control IgG or anti-IKK antibody. The precipitates were immunoblotted with the indicated antibodies. (b) GST and GST–IKK bound to glutathione–agarose beads were incubated with purified MUC1-CD. The precipitates and input were immunoblotted with an anti-MUC1-C antibody. Input of the GST and GST–IKK proteins was assessed by Coomassie blue staining. (c) Amino-acid sequence of MUC1-CD (upper panel). GST and the indicated GST–MUC1-CD fusion proteins bound to glutathione beads were incubated with purified IKK. The precipitates and input were immunoblotted with an anti-IKK antibody (lower left). GST and the indicated GST–IKK fusion proteins bound to glutathione beads were incubated with MUC1-CD. The precipitates and input were immunoblotted with an anti-MUC1-C antibody (lower right). Input of GST and GST– fusion proteins was assessed by Coomassie blue staining. (d) Lysates from the indicated cells were subjected to immunoprecipitation with a control IgG or an anti-IKK antibody. The precipitates were immunoblotted with the indicated antibodies. (e) GST and GST–IKK bound to glutathione beads were incubated with purified MUC1-CD. The precipitates and input were immunoblotted with an anti-MUC1-C antibody. (f) GST and the indicated GST–MUC1-CD fusion proteins bound to glutathione beads were incubated with purified IKK. The precipitates and input were immunoblotted with an anti-IKK antibody. Input of the GST and GST– fusion proteins is shown in Fig. 2c, left. (g) GST and the indicated GST–IKK fusion proteins bound to glutathione beads were incubated with MUC1-CD. The precipitates and input were immunoblotted with an anti-MUC1-C antibody. Full scans of the gels in a, b, c, e and f and g are shown in Supplementary Fig. S6-2.

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To determine whether MUC1-C associates with the IKK complex, lysates from HeLa-vector and HeLa-MUC1 cells were subjected to gel filtration chromatography followed by immunoblotting of the fractions with anti-IKK and anti-IKK antibodies. Consistent with previous findings⁸, analysis of HeLa-vector cells showed that IKK and IKK are detectable in a prominent pool with a relative molecular mass (M_r) of 700,000 (fractions 10–12) and also in a pool of 300 K (fractions 14 and 15; Fig. 3a). Analysis of HeLa-MUC1 cells showed that IKK and IKK are detectable in fractions 10–12 but not in fractions 14 and 15 (Fig. 3a). MUC1-C was detected mainly in fraction 10, consistent with binding to the large 700-K complex (Fig. 3a). The 700-K IKK–IKK complexes isolated from HeLa-MUC1, but not HeLa-vector cells, exhibited constitutive activation (Fig. 3a). The HeLa-MUC1 lysates were also immunoprecipitated with an anti-MUC1-C antibody and the precipitates were released by adding MUC1-C peptide before gel filtration chromatography. Immunoblot analysis of the fractions confirmed that MUC1-C associates with the large IKK–IKK complexes (Supplementary Information, Fig. S2a). MUC1 expression was associated with increased binding of IKK to IKK (Fig. 3b). Moreover, incubation of purified IKK with IKK in vitro demonstrated that MUC1-CD increases the interaction between IKK and IKK (Fig. 3c). MUC1-CD(1–45) binds to IKK and MUC1-CD(46–72), which contains a serine-rich SAGNGGSSLS motif (SRM; amino acids 50–59), binds to IKK (Fig. 2). Mutation of the SRM to AAGNGGAAAA (mSRM) had no effect on the interaction between MUC1-CD and IKK (Supplementary Information, Fig. S2b), but attenuated binding to IKK (Supplementary Information, Fig. S2c). MUC1-CD(mSRM) was substantially less effective than MUC1-CD in inducing the association of IKK and IKK (Fig. 3c), indicating that this response is dependent on binding of both IKKs to MUC1-CD. Phosphorylation of IKK on Ser 181 in the activation loop, perhaps by a trans-autophosphorylation mechanism, is required for induction of IKK activity. Immunoblot analysis with an anti-phospho-IKK-Ser 181 antibody showed that IKK is phosphorylated on Ser 181 by a MUC1-dependent mechanism (Fig. 3d). Analysis of anti-IKK precipitates for phosphorylation of IB further demonstrated that MUC1 stimulates the IKK kinase function (Fig. 3e). Incubation of IKK and IKK with ATP in vitro resulted in phosphorylation of IKK on Ser 181 (Fig. 3f). The extent of IKK phosphorylation was increased significantly by adding MUC1-CD to the reaction (Fig. 3f). However, this effect of MUC1-CD was attenuated by mutation of the SRM (Fig. 3f).

Figure 3: MUC1 activates the IKK–IKK complex.

(a) HeLa-vector and HeLa-MUC1 cell lysates were separated on a Sephacryl S-200 HR column. The indicated fractions were analysed by immunoblotting with the indicated antibodies and for phosphorylation of GST–IB in kinase assays (KAs). (b) Anti-IKK immunoprecipitates from the indicated cells were immunoblotted with anti-IKK and anti-IKK antibodies. (c) GST or GST–IKK bound to glutathione beads was incubated with IKK in the absence and presence of MUC1-CD or MUC1-CD(mSRM). The precipitates were immunoblotted with an anti-IKK antibody (upper panel). Input of the proteins was assessed by immunoblotting with the indicated antibodies (lower 3 panels). (d) Anti-IKK precipitates from the indicated cells were immunoblotted with anti-phospho-IKK-Ser 181 and anti-IKK antibodies. (e) Anti-IKK precipitates from the indicated cells were incubated with GST-IB(1–54) and -32P-ATP. The reaction products were analysed by SDS–PAGE and autoradiography (upper panels). The precipitates were also immunoblotted with an anti-IKK antibody (lower panels). (f) GST or GST–IKK bound to glutathione beads was incubated with IKK in the absence and presence of MUC1-CD or MUC1-CD(mSRM). The precipitated complexes were suspended in kinase buffer containing ATP and incubated for 30 min at 30 °C. The reaction products were immunoblotted with an anti-phospho-IKK-Ser 181 antibody. Input of the GST–IKK, IKK and MUC1-CD proteins is shown in Fig. 3c. Full scans of the gels in a, b, c and f are shown in Supplementary Fig. S6-3.

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To determine whether the association between MUC1-CD and the IKK–IKK complex is sufficient to activate the NF-B p65 pathway, ZR-75-1-MUC1 siRNA cells were transfected to express Flag-tagged MUC1-CD or MUC1-CD (mSRM) (Supplementary Information, Fig. S3a). When compared with ZR-75-1-MUC1 siRNA cells transfected with the empty vector, expression of MUC1-CD was associated with an increase in the formation of IKK–IKK complexes (Supplementary Information, Fig. S3b), increased phosphorylation of IKK on Ser 181 and of IB (Supplementary Information, Figs. S3c and S3d) and increased targeting of NF-B p65 to the nucleus (Supplementary Fig. S3e). Expression of MUC1-CD (mSRM) was less effective than MUC1-CD in inducing the formation of IKK–IKK complexes (Supplementary Information, Fig. S3b). MUC1-CD-induced phosphorylation of IKK on Ser 181, phosphorylation of IB and targeting of NF-B p65 to the nucleus were each attenuated by mutation of the SRM (Supplementary Information, Figs. S3c–e). These findings indicate that MUC1-CD is sufficient to activate the classical IKK–NF-B p65 pathway.

Binding of MUC1-C to the IKK–IKK complex may represent a physiological response in non-transformed cells that is constitutively activated by the overexpression of MUC1 in carcinoma cells. To address this hypothesis, studies were performed on the non-transformed mammary epithelial cell line, MCF-10A^{9, 10}, in which MUC1 is expressed at levels lower than those found in MCF-7 and ZR-75-1 cells (Supplementary Information, Fig. S4a) and where binding of MUC1-C to IKK or IKK is minimal (Supplementary Information, Fig. S4b). IKK and IKK are responsible for signalling to NF-B in the response to tumour necrosis factor alpha (TNF) and other pro-inflammatory cytokines. Significantly, stimulation of the MCF-10A cells with TNF was associated with increased binding of MUC1-C to IKK and IKK (Supplementary Information, Fig. S4b). In contrast, TNF had no apparent effect on binding of MUC1-C to IKK or IKK in MCF-7 cells (data not shown). Treatment of MCF-10A cells with TNF was also associated with phosphorylation and degradation of IB (Fig. 4a), and targeting of NF-B p65 to the nucleus (Fig. 4a). Transient silencing of MUC1 attenuated phosphorylation of IKK on Ser 181, phosphorylation of IB and nuclear targeting of NF-B p65 in the response to TNF stimulation (Fig. 4b). Previous work has demonstrated that the effects of TNF on mammary epithelial cells are of physiological importance for mammary gland morphogenesis during puberty and pregnancy^{11, 12, 13, 14, 15}. To determine whether these effects of TNF are regulated by MUC1, primary mouse mammary epithelial cells (MMECs) were transfected with control or mouse-specific Muc1 siRNAs (Fig. 4c). Silencing of Muc1 in MMECs blocked TNF-induced phosphorylation of IKK and IB (Fig. 4c). Muc1 was also necessary for TNF-induced targeting of NF-B p65 to the nucleus (Fig. 4c).

Figure 4: MUC1-C contributes to NF-B activation in the response of MCF-10A cells to TNF.

(a) MCF-10A cells were left untreated or stimulated with 20 ng ml^-1 TNF for the indicated times. Cytosolic and nuclear fractions were immunoblotted with the indicated antibodies. (b) MCF-10A cells were transfected with control siRNA or MUC1 siRNA pools for 72 h. Whole cell lysates, cytosolic fractions and nuclear fractions were immunoblotted with the indicated antibodies. (c) Primary mouse mammary epithelial cells were transfected with control or Muc1 siRNA pools for 72 h. Total RNA was isolated for determination of Muc1 and -actin mRNA levels by RT-PCR (upper panel). Whole cell lysates and cytoplasmic and nuclear fractions were immunoblotted with the indicated antibodies. (d, e) MCF-10A cells were transfected with control siRNA, TRADD siRNA, TRAF2 siRNA or RIP1 siRNA pools for 72 h. The transfected cells were left untreated or stimulated with TNF for 30 min. Lysates were directly immunoblotted with the indicated antibodies. Lysates were also precipitated with an anti-TNF-R1 antibody (d) or an anti-IKK antibody (e) and the precipitates were immunoblotted with the indicated antibodies. (f) MCF-10A cells were transfected with control siRNA or RIP1 siRNA pools for 72 h and then stimulated with TNF. Whole-cell- or nuclear-lysates were immunoblotted with the indicated antibodies. Full scans of the gels in a, b, d and f are shown in Supplementary Fig. S6-4.

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Binding of TNF to TNF-R1 is associated with the recruitment of TNF-R1-associated death domain protein (TRADD) and TNF receptor-associated factor 2 (TRAF2) to the receptor complex². In turn, TRAF2 recruits IKKs to the complex. Stimulation of MCF-10A cells with TNF induced the association of MUC1-C with TNF-R1, TRADD and TRAF2 (Supplementary Information, Fig. S4c), indicating that MUC1-C is recruited to the TNF-R1 complex. Silencing of TRADD or TRAF2 demonstrated that both of these proteins are required for TNF-induced recruitment of MUC1-C (Fig. 4d). The death-domain kinase receptor-interacting protein 1 (RIP1) is also recruited to the TNF-R1 complex, where it functions as a scaffold for IKK activation². Notably, silencing of RIP1 had little, if any, effect on the recruitment of MUC1-C (Fig. 4d). Silencing of TRADD or TRAF2, but not RIP1, also blocked the TNF-induced association of MUC1-C with IKK (Fig. 4e). In TNF-treated fibroblasts, HeLa cells and Jurkat cells^{16, 17, 18, 19, 20}, which are null for MUC1 expression^{6, 21, 22}, RIP1 mediates IKK activation by binding directly to IKK and is essential for induction of NF-B signalling. In contrast, we found that silencing of RIP1 in MCF-10A cells had little effect on TNF-induced activation of NF-B p65 (Fig. 4f). These results indicate that in MCF-10A cells, TNF recruits MUC1-C to the TNF-R1 complex by a TRADD- and TRAF2-dependent mechanism and that MUC1-C functions independently of RIP1 in the activation of IKK.

To determine whether MUC1-dependent phosphorylation of IKK on Ser 181 is mediated by transforming growth factor--activated kinase 1 (TAK1), which phosphorylates IKK on Ser 181 and activates the NF-B pathway in response to TNF stimulation^{23, 24}, TAK1 was silenced in MCF-10A cells (Fig. 5a). These results demonstrate that TNF stimulation is associated with phosphorylation of IKK on Ser 181 by a TAK1-mediated mechanism (Fig. 5a). TNF-induced phosphorylation of IB and targeting of NF-B p65 to the nucleus were also dependent on TAK1 (Fig. 5a). The TAK1-binding proteins TAB1, TAB2 and TAB3 function as adaptor molecules in linking TRAFs to TAK1 activation^{23, 25, 26}. Silencing of TAB1 in MCF-10A cells had no effect on TNF-induced phosphorylation of IKK on Ser 181, phosphorylation and degradation of IB, or targeting of NF-B p65 to the nucleus (Supplementary Information, Fig. S5). However, silencing of TAB2 (Fig. 5b), but not its homologue TAB3 (ref. 26 and data not shown), was associated with attenuation of TNF-induced phosphorylation of IKK on Ser 181 and activation of the NF-B p65 pathway. In this regard, TAB2 but not TAB1, is critical for TNF signalling^{23, 27, 28}. Together with our other findings, these results support a model in which MUC1-C is necessary for TNF-induced recruitment of TAK1 to the TNF-R1 complex. Consistent with such a model, silencing of MUC1 blocked recruitment of TAK1 to TNF-R1 in TNF-stimulated MCF-10A cells (Fig. 5c, upper). Notably, studies in Jurkat cells have shown that RIP1 is necessary for recruitment of TAK1 to the TNF-R1 complex²⁹. Our studies showed that silencing of RIP1 in MCF-10A cells had little, if any effect, on recruitment of TAK1 to TNF-R1 (Fig. 5c, lower). This indicates that MUC1, but not RIP1, is essential for TNF-induced TAK1 phosphorylation of IKK. Silencing of MUC1 in MCF-10A cells was also associated with TNF-induced apoptosis (Fig. 5d), a response blocked by activation of the NF-B pathway³⁰. These findings indicate that MUC1 is involved in activation of TAK1–IKK–NF-B p65 signalling by TNF and thereby attenuation of the apoptotic response.

Figure 5: MUC1 is necessary for TNF-induced recruitment of TAK1 to the TNF-R1 complex.

(a–c) MCF-10A cells were transfected with control siRNA or TAK1 (a), TAB2 (b), MUC1 (c, upper) or RIP1 (c, lower) siRNA pools for 72 h and then stimulated with TNF. Whole cell lysates, cytosolic fractions and nuclear fractions were immunoblotted with the indicated antibodies (a,b). Whole cell lysates were precipitated with anti-TNF-R1. The precipitates and lysates not subjected to immunoprecipitation were blotted with the indicated antibodies (c). (d) MCF-10A cells were transfected with control siRNA or MUC1 siRNA pools for 72 h. The transfected cells were left untreated or treated with TNF for 24 h and then monitored for DNA content. The results are expressed as percentage apoptotic cells (mean s.d., n = 3) with sub-G1 DNA. (e) Proposed model for the effects of MUC1 on activation of the IKK–IKK complex and the NF-B p65 pathway. Full scans of the gels in a and b are shown in Supplementary Fig. S6-5

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The present studies support a model in which MUC1-C binds directly to IKK and IKK to activate the IKK complex (Fig. 5e). As shown for heat shock proteins, which associate with multiple targets³¹, the evidence to date indicates that MUC1-C maintains effectors of growth and survival pathways (-catenin, p53) and nuclear hormone receptors (ER) in stabilized and active configurations^{4, 5, 6, 7}. We found that expression of MUC1 in carcinoma cells constitutively induces multiple processes, including the formation of IKK–IKK complexes, phosphorylation of IKK on Ser 181, IKK-mediated phosphorylation of IB and targeting of NF-B p65 to the nucleus (Fig. 5e). A variety of human tumours aberrantly overexpress MUC1 and exhibit constitutive activation of the NF-B pathway^{21, 32, 33, 34}. The present findings indicate that MUC1 may contribute, at least in part, to activation of NF-B in these tumours to promote cell survival. Studies performed in non-malignant MCF-10A mammary epithelial cells demonstrate that MUC1-C associates with the TNF-R1 complex in the response to TNF stimulation and functions in the recruitment of TAK1, TAK1-mediated phosphorylation of IKK, assembly of the IKK–IKK complex and trans-autophosphorylation of IKK. Our results also indicate that TAB2 is necessary for MUC1-C function, whereas RIP1 is not required for TNF-induced recruitment of MUC1-C to the TNF-R1 complex, binding of MUC1-C to IKK or activation of NF-B. Moreover, MUC1, and not RIP1, was shown to be necessary for recruitment of TAK1 to the TNF-R1 complex. These findings indicate that MUC1-C functions independently of RIP1 in TNF-induced IKK activation. The findings further indicate that there may be more than one IKK complex, depending on cell type, and that the binding partner (for example RIP1 or MUC1-C) may vary, depending on availability and affinity for the IKKs. In summary, our findings indicate that overexpression of MUC1 as found in human tumours is important for sustained IKK–NF-B signalling and that MUC1-mediated activation of this pathway may be exploited by malignant cells for survival under adverse conditions.

Acknowledgements

This work was supported by Grant CA97098, CA42802 and CA100707 awarded by the National Cancer Institute (Bethesda, MD). The authors thank Michael Karin (University of California San Diego, CA) for the GST–IKK plasmid, Richard Gaynor (Lilly Research Laboratories, Indianapolis, IN) for the GST–IKK plasmid, and Al Baldwin (University of North Carolina, Chapel Hill, NC) for the wild-type and mutant pNF-B-Luc reporters. Kamal Chauhan is acknowledged for technical support.

Received 17 August 2007; Accepted 10 October 2007; Published online 25 November 2007.

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1. Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115, USA.
2. Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA.

Correspondence to: Donald Kufe¹ e-mail: donald_kufe@dfci.harvard.edu

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