Phosphorylation of the tumor suppressor CYLD by the breast cancer oncogene IKKε promotes cell transformation

doi:10.1016/j.molcel.2009.04.031

Mol Cell. Author manuscript; available in PMC 2010 May 14.

Published in final edited form as:

Mol Cell. 2009 May 14; 34(4): 461–472.

doi: 10.1016/j.molcel.2009.04.031

PMCID: PMC2746958

NIHMSID: NIHMS121328

Phosphorylation of the tumor suppressor CYLD by the breast cancer oncogene IKKε promotes cell transformation

Jessica E. Hutti,^1,² Rhine R. Shen,^3,⁴ Derek W. Abbott,⁵ Alicia Y. Zhou,^3,⁴ Kam M. Sprott,⁶ John M. Asara,^2,⁷ William C. Hahn,^3,^4,⁸ and Lewis C. Cantley^1,^2,^4,^8,⁹

¹Department of Systems Biology, Harvard Medical School, Boston, MA 02115

²Division of Signal Transduction, Beth Israel Deaconess Medical Center, Boston, MA 02115

³Department of Medical Oncology, Dana Farber Cancer Institute, Boston, MA 02115

⁴Broad Institute of Harvard and MIT, Cambridge, MA 02142

⁵Department of Pathology, Case Western Reserve University, Cleveland, OH 44106

⁶Cell Signaling Technology, Inc., Danvers, MA 01923

⁷Department of Pathology, Harvard Medical School, Boston, MA 02115

⁸These senior authors contributed equally to this work

⁹ To whom correspondence should be addressed: BIDMC Center for Life Sciences, 330 Brookline Ave., Room 412, Boston, MA 02115, Phone: 617-735-2601, Fax: 617-735-2646, Email: lcantley/at/hms.harvard.edu

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Summary

The non-canonical IKK family member IKKε is essential for regulating anti-viral signaling pathways and is a recently-discovered breast cancer oncoprotein. Although several IKKε targets have been described, direct IKKε substrates necessary for regulating cell transformation have not been identified. Here, we performed a screen for putative IKKε substrates using an unbiased proteomic and bioinformatic approach. Using a positional scanning peptide library assay we determined the optimal phosphorylation motif for IKKε and used bioinformatic approaches to predict IKKε substrates. Of these potential substrates, serine 418 of the tumor suppressor CYLD was identified as a likely site of IKKε phosphorylation. We confirmed that CYLD is directly phosphorylated by IKKε, and that IKKε phosphorylates serine 418 in vivo. Phosphorylation of CYLD at serine 418 decreases its deubiquitinase activity and is necessary for IKKε-driven transformation. Together, these observations define IKKε and CYLD as an oncogene-tumor suppressor network that participates in tumorigenesis.

Introduction

Abundant evidence supports the view that chronic inflammation contributes to cancer initiation (Basseres and Baldwin, 2006; Karin and Greten, 2005; Perkins, 2007). Subversion of many key regulatory steps involved in immune responses occurs in both hematopoietic and epithelial cancers. For example, a large number of inflammatory mediators act as oncogenes, (IKKβ, IKKε), tumor suppressors (CYLD), or pro-survival genes (p65, BCL-3) in specific cell types (Basseres and Baldwin, 2006; Karin and Greten, 2005; Perkins, 2007).

One pathway that plays a key role in inflammation and cancer is the NF-κB pathway. Canonical, NF-κB-driven, inflammation is initiated following cellular recognition of pathogens or proinflammatory cytokines. These proinflammatory stimuli activate divergent signaling pathways, all of which ultimately converge to activate the IκB kinase (IKK) complex (Hayden and Ghosh, 2004). The core IKK complex consists of the serine/threonine kinases IKKα and IKKβ, as well as a regulatory subunit, NEMO. Upon activation by a diverse set of stimuli, this kinase complex phosphorylates the NF-κB inhibitory protein IκBα, which facilitates ubiquitination of IκBα and subsequent proteasomal degradation (Hayden and Ghosh, 2004). Degradation of IκBα permits nuclear translocation of activated NF-κB and the transcriptional activation of pro-inflammatory target genes. However, in addition to this canonical mechanism of NF-κB activation, several alternative signaling pathways also converge to activate NF-κB (Hacker and Karin, 2006; Hiscott, 2007; Kawai and Akira, 2007). Understanding these non-canonical regulatory mechanisms will provide insights into the interplay between inflammation and cancer.

IKKε (IκB kinase epsilon) is one such non-canonical IKK family member that plays a critical role in the regulation of interferon signaling pathways. Both specialized membrane-bound Toll-like receptors and intracellular receptors which recognize viral nucleic acids such as dsRNA activate this serine/threonine kinase (Hacker and Karin, 2006; Hiscott, 2007; Pichlmair and Reis e Sousa, 2007; Thompson and Locarnini, 2007). Activated IKKε (and the related kinase TBK1) then phosphorylates interferon response factors 3 and 7 (IRF3 and IRF7), allowing their nuclear translocation and the transcriptional upregulation of genes involved in the type I interferon response (Fitzgerald et al., 2003; Hemmi et al., 2004; McWhirter et al., 2004; Sharma et al., 2003). IKKε also phosphorylates STAT1 following activation by IFNβ (Tenoever et al., 2007). The transcription factor NF-κB is also activated by IKKε, although the mechanism by which IKKε regulates the canonical NF-κB pathway is not well-understood (Peters et al., 2000; Shimada et al., 1999).

We and others have identified IKKε as a breast cancer oncogene amplified and overexpressed in over 30% of breast carcinomas and breast cancer cell lines (Adli and Baldwin, 2006; Boehm et al., 2007; Eddy et al., 2005). Forced expression of IKKε substitutes for AKT and cooperates with MEK signaling to transform immortal human cells and overexpression of IKKε alone is sufficient to drive transformation of NIH-3T3 cells. In addition, suppression of IKKε expression in breast cancer cell lines that harbor increased copy number leads to cell death (Boehm et al., 2007). While tumorigenicity induced by either IKKε or AKT requires NF-κB activation (Boehm et al., 2007), the specific substrates of IKKε that are involved in cell transformation remain undefined.

Familial cylindromatosis is an autosomal dominant disease characterized by the formation of benign skin tumors, primarily on the head and neck (van Balkom and Hennekam, 1994). This disease results from inheritance of a gene encoding a truncation mutant of the tumor suppressor CYLD, followed by loss of heterozygosity (Bignell et al., 2000). Decreased CYLD expression has also been identified in both hepatocellular and colon cancer cell lines and tumors (Hellerbrand et al., 2007). Furthermore, deletion of the CYLD locus has recently been shown to be associated with NF-κB activation in multiple myeloma cell lines and patient tissues (Annunziata et al., 2007; Keats et al., 2007). CYLD is a deubiquitinating enzyme (DUB) that removes Lys63-linked ubiquitin chains and acts as a negative regulator of NF-κB signaling (Brummelkamp et al., 2003; Kovalenko et al., 2003; Trompouki et al., 2003). CYLD deubiquitinates several NF-κB regulators, including TRAF2, TRAF6, and NEMO (Brummelkamp et al., 2003; Kovalenko et al., 2003; Trompouki et al., 2003) as well as BCL3, a member of the NF-κB family of transcription factors (Massoumi et al., 2006). Following ubiquitination, BCL-3 translocates from the cytoplasm to the nucleus where it heterodimerizes with the NF-κB subunits p50 or p52 and activates the transcription of cyclin D, a key regulator of the G1 restriction point (Westerheide et al., 2001). Similar to what is observed in kindreds afflicted with familial cylindromatosis, CYLD^-/- mice develop benign skin tumors following exposure to UV light or TPA (Massoumi et al., 2006). Other studies have shown that CYLD^-/-mice also develop multi-organ hyperinflammation and have increased susceptibility to inflammation-induced tumors (Reiley et al., 2007; Zhang et al., 2006). Little is known, however, about how CYLD activity is regulated.

Using a recently developed peptide library approach we determined the optimal motif for phosphorylation by IKKε and then incorporated this information into a bioinformatic search for likely IKKε protein substrates (Hutti et al., 2004; Turk et al., 2006). Many of the predicted substrates are known components of inflammatory and/or oncogenic signaling pathways, including the tumor suppressor CYLD. We show here that CYLD is a substrate of IKKε and that phosphorylation of CYLD by IKKε contributes to cell transformation.

Results

Determination of IKKε substrate specificity

IKKε has recently been identified as a breast cancer oncogene. However, the mechanism by which IKKε contributes to cell transformation is not known. Since IKKε is a serine/threonine kinase, we sought to determine whether the kinase activity of IKKε is necessary for its oncogenic activity. Wild-type IKKε or kinase-dead IKKε K38A was introduced into NIH-3T3 cells (Figure 1A). We found that cells expressing WT IKKε, but not IKKε K38A, exhibited robust anchorage-independent colony growth (Figure 1B). These results confirm that the kinase activity of IKKε is necessary for its activity as an oncogene.

Figure 1

Identification of the optimal IKKε phosphorylation motif

While several IKKε substrates involved in interferon signaling pathways have been identified, direct substrates required for the oncogenic functions of IKKε have not been reported. To address this question, we utilized proteomic and bioinformatic approaches to perform an unbiased screen for likely IKKε substrates. Specifically, we used a positional scanning peptide library assay recently developed in this laboratory to identify the optimal phosphorylation motif for IKKε (Hutti et al., 2004; Turk et al., 2006). This assay employs 198 biotinylated peptide libraries. Each library has a 1:1 mixture of serine and threonine fixed at the central position, and has one additional position fixed to one of the 20 naturally-occurring amino acids. All other positions contain a degenerate mixture of amino acids (excluding serine, threonine, and cysteine). Phosphothreonine and Phosphotyrosine were included at the fixed positions in order to facilitate the identification of kinases which have a requirement for priming phosphorylation events. Using recombinant GST-IKKε purified from HEK-293T cells, we performed kinase assays simultaneously on all 198 peptide libraries in solution using γ-³²P-ATP. Biotinylated peptides were captured with a streptavidin-coated membrane and the relative preference for each amino acid at each position was determined by the relative level of radiolabel incorporation into the corresponding peptides.

We found that IKKε exhibits strong sequence selectivity at multiple positions surrounding the phosphorylation site. IKKε strongly prefers substrates with a hydrophobic residue at the +1 position relative to the phosphorylation site (Figures 1C and 1F). This kinase also exhibits strong sequence selectivity for aromatic residues at the -2 position, and for bulky hydrophobic residues at the +3 position (Figures 1C and 1F). Kinase-dead IKKε K38A does not exhibit selectivity at these positions, confirming that peptides were not being phosphorylated by a contaminating kinase (Figure 1D).

To validate this motif, we generated a consensus peptide substrate, IKKε-Tide, and measured the phosphorylation of peptides bearing individual alanine substitutions. By comparing these results to those obtained using the consensus sequence we confirmed that substitution of amino acids at the +1 or -2 positions resulted in a decrease in the efficiency of peptide phosphorylation (Figure 1E). This motif has similarities to the sequence surrounding the autophosphorylation site in the activation loop of IKKε (DEKFVS₁₇₂VYGTE) due to the aromatic residue at -2 and aliphatic at +1, although this site is predicted to be suboptimal due to the lack of an aromatic residue at +3 (Peters et al., 2000).

IKKε has been shown to phosphorylate one of the essential serines on IκBα in vitro, but in vivo phosphorylation of IκBα by this kinase has not been described (Peters et al., 2000). Therefore, the role of IKKε in IκBα phosphorylation and degradation remains unclear. To examine this question, we performed kinase assays using a peptide substrate corresponding to the sequence surrounding Ser32 and Ser36 of IκBα. This peptide contains two potential phosphorylation sites, but neither site is within a sequence context that matches the optimal motif for IKKε. We found that this peptide was a poor in vitro substrate for IKKε when compared with the optimal peptide determined from the peptide library screen (Figure 1E). In contrast, when recombinant GST-IKKβ was used to phosphorylate the same set of peptides, the IκBα peptide was phosphorylated by IKKβ much more efficiently than IKKε-Tide (Figure S1). These observations suggest that IκBα is unlikely to be an important physiological substrate of IKKε. We recently demonstrated that, like IKKε, IKKβ prefers aromatic residues at the -2 position and hydrophobic residues at the +1 position (Hutti et al., 2007). However, the phosphorylation motifs for these kinases differ at the -4, -5, and +3 positions. Taken together, these observations demonstrate that while the substrate specificities of IKKβ and the related kinase IKKε have overlapping characteristics, the optimal substrate peptides for these kinases differ in substantial ways and therefore can be predicted to have different (though possibly overlapping) in vivo substrates.

Prediction of IKKε substrates

Spot intensities from the peptide library screen were then quantified (Table S1) and converted into a matrix which could be used with the bioinformatic search engine Scansite. Scansite (http://scansite.mit.edu) allows proteome-wide searches for sites which best match the data provided by the input matrix (Obenauer et al., 2003; Yaffe et al., 2001). Table 1 shows top-scoring candidate IKKε substrates obtained following the Scansite analysis, all of which scored in the top 0.05% of sites in the SwissProt database. Interestingly, a large number of predicted IKKε substrates are known to be involved in inflammatory and/or oncogenic signaling pathways. Of these potential substrates, the deubiquitinating enzyme CYLD was of particular interest, as it has been shown to have roles as both an inflammatory mediator and tumor suppressor, functions that could be downstream of IKKε (Bignell et al., 2000). Our bioinformatic analysis predicted that CYLD is likely to be phosphorylated by IKKε at Ser418.

Table 1

Candidate IKKε substrates identified by Scansite

CYLD is phosphorylated by IKKε at Ser418

To further facilitate the identification of novel IKKε substrates we raised antibodies against a collection of phosphopeptides biased towards the optimal IKKε phosphorylation motif We verified that these antibodies recognize known IKKε substrates in a kinase-dependent manner (data not shown). These antibodies were then used to determine whether CYLD is phosphorylated at a site matching the IKKε phosphorylation motif HEK-293T cells were cotransfected with Myc-epitope tagged CYLD (Myc-CYLD) and either GST-IKKε WT or kinase-dead IKKε K38A. CYLD was immunoprecipitated via its Myc tag and immunoblotted with the anti-IKKε phospho-substrate antibody. Figure 2A shows that the phospho-substrate antibody blotted CYLD which had been transfected with WT IKKε, but not IKKε K38A. CYLD treated with calf-intestinal phosphatase (CIP) following cotransfection with IKKε was no longer recognized by the phospho-substrate antibody, confirming that the IKKε phospho-substrate antibody specifically recognizes phosphorylated CYLD (Figure 2B).

Figure 2

CYLD co-immunoprecipitates with and is phosphorylated by IKKε

In order to determine whether IKKε can directly phosphorylate CYLD, an in vitro kinase assay was performed. Wild-type GST-IKKε or GST-IKKε K38A was purified from HEK-293T cells. Myc-CYLD was separately transfected into HEK-293T cells and immunoprecipitated. When the CYLD immunoprecipitate was incubated in an in vitro kinase assay with WT IKKε, strong phosphorylation of CYLD was observed (Figure 2C). This phosphorylation was not observed in the presence of IKKε K38A.

To determine whether IKKε and CYLD physically interact, Myc-CYLD was cotransfected into HEK-293T cells expressing GST- IKKε WT or K38A. CYLD was immunoprecipitated via its Myc tag and these immune complexes were blotted with an anti-GST antibody to identify IKKε. In CYLD immune complexes we identified both WT and kinase-dead IKKε (Figure 2D). Moreover, when we performed the reciprocal experiment we found that Myc-CYLD was also observed in the IKKε precipitates (Figure 2E).

While CYLD Ser418 was predicted by Scansite to be the optimal site for IKKε phosphorylation (ENRFHS₄₁₈LPFSL), two additional serines within the CYLD sequence were potential, though less optimal, IKKε phosphorylation sites (DSRFAS₅₄₇LQPVS and KKIFPS₇₇₂LELNI). Therefore, we used mass spectrometry to determine which residue(s) of CYLD is phosphorylated in vivo. Myc-CYLD and GST-IKKε were cotransfected into HEK-293T cells. CYLD was immunoprecipitated, subjected to SDS-PAGE, and Coomassie stained (Figure S2). The band corresponding to Myc-CYLD was excised, digested with trypsin or chymotrypsin, and subjected to microcapillary LC/MS/MS. A phosphopeptide consistent with phosphorylation at Ser418 was identified. This analysis confirmed that CYLD is phosphorylated at Ser418, the same site predicted by our bioinformatic analysis (Figure 3A). Ser418 is evolutionarily conserved in all sequenced mammals, as well as Gallus gallus and Xenopus tropicalis (Figure 3B). In addition, the -2F, +1L, and +3F relative to Ser418 (which correspond to the IKKε phosphorylation motif) are also conserved, providing further evidence for the evolutionary importance of this phosphorylation site.

Figure 3

CYLD is phosphorylated by IKKε at Ser418

To further verify that Ser418 is the critical site phosphorylated by IKKε, we created Myc-epitope tagged mutants of CYLD at Ser418 (S418A), Ser547 (S547A), and Ser772 (S772A). We introduced wild-type CYLD or each of these mutants into HEK-293T cells alone or with GST-IKKε and isolated CYLD immune complexes. Mutation of either Ser547 or Ser772 did not affect recognition of CYLD by the IKKε phospho-substrate antibody (Figure 3C). However, Myc-CYLD S418A was no longer recognized by the IKKε phospho-substrate antibody when cotransfected with IKKε (Figure 3C). In addition, when we introduced Myc-CYLD or Myc-CYLD S418A with IKKε into HEK-293T cells and performed an immunoprecipitation using the IKKε phospho-substrate antibody, we found that wild-type Myc-CYLD, but not Myc-CYLD S418A, was efficiently immunoprecipitated by the IKKε phospho-substrate antibody (Figure 3D). Finally, endogenous CYLD was immunoprecipitated from IKKε-transformed NIH-3T3 cells utilized in Figure 1. Immunoblotting with the IKKε phospho-substrate antibody revealed that CYLD is indeed phosphorylated in cells transformed by IKKε, but not in cells expressing IKKε K38A (Figure 3E). Together, these data confirm that CYLD Ser418 is phosphorylated in vivo in the presence of IKKε.

It has been reported that Ser418 of CYLD is a substrate of the canonical IKK family members, IKKβ and IKKα (Reiley et al., 2005). Therefore, in order to determine which IKK family member(s) can most efficiently phosphorylate CYLD, we cotransfected Myc-CYLD with individual IKK family members fused to GST. CYLD was immunoprecipitated using an anti-Myc antibody and blotted using the anti-IKKε phospho-substrate antibody. Figure S4 shows that CYLD was efficiently phosphorylated in cells transfected with IKKε. In contrast, CYLD is phosphorylated inefficiently in cells transfected with IKKα and IKKβ, even when greater than 5× more kinase is present (Figure S3). While it is therefore possible for any of these IKK family members to phosphorylate Ser418 of CYLD, CYLD is a much better substrate for IKKε than for IKKα or IKKβ. Our analysis of IKKε and IKKβ peptide substrate specificities also demonstrated that these kinases should be expected to have largely different, though possibly overlapping, substrate pools (Figures 1E and S1).

IKKε-mediated phosphorylation of CYLD at S418 facilitates transformation

Since CYLD is a known tumor suppressor and IKKε is a newly-discovered oncoprotein (Bignell et al., 2000; Boehm et al., 2007; Massoumi et al., 2006), we hypothesized that phosphorylation of CYLD by IKKε might play a role in the regulation of IKKε-mediated cell transformation. In Figure 1 we show that forced expression of IKKε induces a tumorigenic phenotype in NIH-3T3 cells. To determine whether suppression of CYLD also induced cell transformation in this experimental model, we suppressed murine CYLD using two distinct short hairpin RNAs (shRNA), each of which strongly suppresses endogenous levels of CYLD (Figure 4A). Suppression of CYLD induced substantial anchorage-independent growth of NIH-3T3 cells when compared to control NIH-3T3 cells (Figures 4B and 4C).

Figure 4

Suppression of CYLD protein expression is sufficient to induce transformation

To determine whether CYLD phosphorylation is required for IKKε-mediated transformation, we generated IKKε-transformed NIH-3T3 cells that stably express wild-type CYLD, CYLD S418A, or CYLD S772A (Figure 5A). We found that Flag-epitope tagged IKKε-expressing cells exhibited robust anchorage-independent growth that was 4-fold above that observed in control NIH-3T3 cells or cells expressing WT CYLD or CYLD S772A (Figure 5B). In contrast, in tumorigenic cells expressing Flag-epitope-tagged IKKε, co-expression of CYLD S418A suppressed anchorage-independent growth (Figure 5B). A similar result was observed in vivo when we evaluated the contribution of CYLD phosphorylation to IKKε-induced tumorigenicity. Introduction of IKKε-transformed NIH-3T3 cells into immunodeficient animals yielded tumor formation at a high penetrance (Figure 5C, data not shown). While expression of WT CYLD or CYLD S772A failed to significantly alter tumorigenicity, expression of CYLD S418A led to statistically-significantly smaller tumors (p<0.05) (Figure 5C). Taken together, these findings demonstrate that phosphorylation of CYLD by IKKε at serine 418 is necessary for IKKε to fully induce transformation.

Figure 5

CYLD phosphorylation at serine 418 is important for IKKε-mediated transformation

Phosphorylation of CYLD at Ser418 decreases CYLD activity

CYLD is a deubiquitinating enzyme which removes Lys63-linked ubiquitin chains from a large number of inflammatory mediators including TRAF2, TRAF6, and NEMO, as well as the pro-proliferation transcription factor BCL-3 (Brummelkamp et al., 2003; Kovalenko et al., 2003; Massoumi et al., 2006; Trompouki et al., 2003). Removal of ubiquitin chains by CYLD inactivates these substrates, inhibiting inflammatory signaling and cell cycle progression. Cell transformation induced by loss of CYLD is hypothesized to occur following the accumulation of a variety of ubiquitinated species, which leads to both increased cell proliferation and the increased transcription of NF-κB-regulated anti-apoptotic factors. Therefore, we sought to determine the effect of CYLD phosphorylation on ubiquitination of known CYLD substrates.

TRAF2 is a critical NF-κB mediator ubiquitinated via Lys63-linked ubiquitin chains following stimulation with TNFα, and CYLD removes these ubiquitin chains to prevent uncontrolled TNFα-induced inflammation. To determine the effect of CYLD phosphorylation on TRAF2 ubiquitination, HEK-293T cells were transfected with Myc-epitope tagged TRAF2 in combination with GFP-WT CYLD or CYLD S418A and GST-IKKε. In order to examine only changes in Lys63-linked ubiquitin chains, cells were also transfected with an HA-tagged ubiquitin variant which has all lysines mutated to arginine except for Lys63 (HA-Ub-K63). Cells were stimulated with TNFα for 10 minutes prior to lysis and CYLD-mediated deubiquitination of TRAF2 was assessed by immunoprecipitation. As expected, CYLD efficiently removed Lys63-linked ubiquitin chains from TRAF2 (Figure 6A). Interestingly, CYLD-mediated deubiquitination of TRAF2 was blocked in the presence of IKKε. In constrast, cotransfection of CYLD S418A with IKKε did not block deubiquitination of TRAF2 (Figure 6A). These data demonstrate that phosphorylated CYLD exhibits less deubiquitinating activity than unphosphorylated CYLD. Thus, IKKε regulates the deubiquitination of TRAF2 by phosphorylating CYLD.

Figure 6

Phosphorylation of CYLD at Ser418 decreases CYLD activity

The IKK regulatory subunit NEMO undergoes Lys63-specific ubiquitination at Lys285 when coexpressed with RIP2, resulting in increased NF-κB activity (Abbott et al., 2004; Abbott et al., 2007). CYLD efficiently removes these RIP2-induced ubiquitin chains from NEMO (Abbott et al., 2004). To determine whether phosphorylation of CYLD affects RIP2-induced NEMO ubiquitination, HEK-293T cells were cotransfected with Myc-NEMO, OMNI-RIP2, HA-Ubiquitin, GFP-CYLD, and GST-IKKε. As expected, cotransfection of RIP2 with NEMO increased NEMO ubiquitination, and this ubiquitination was suppressed by CYLD (Figure 6B). However, cotransfection of IKKε with CYLD resulted in the stabilization of NEMO ubiquitination, suggesting that phosphorylation of CYLD decreases its deubiquitinase activity. As in Figure 6A, cotransfection of CYLD S418A with IKKε failed to stabilize NEMO ubiquitination (Figure 6B). Together, these observations demonstrate that IKKε-mediated phosphorylation of CYLD at Ser418 inhibits CYLD deubiquitinase activity.

We next sought to determine what effect this phosphorylation exerts on NF-κB activity. We have shown previously that the gene encoding IKKε, IKBKE, is amplified and overexpressed in MCF-7 cells (Boehm et al., 2007). These cells were transiently transfected with Myc-TRAF2 alone or in combination with Myc-CYLD, Myc-CYLD S418A, or Myc-CYLD S772A and we performed a NF-κB-luciferase reporter assay. In cells expressing TRAF2 alone, we found substantial (14.9 fold) activation of the NF-κB reporter (Figure 6C, data not shown). Cotransfection with a sub-saturating amount of WT CYLD or CYLD S772A resulted in an approximately 50% decrease in TRAF2-induced NF-κB activation. However, co-transfection with CYLD S418A resulted in a statistically significantly larger decrease in NF-κB reporter activity (p<0.05) (Figure 6C). These results demonstrate that phosphorylation of CYLD at Ser418 suppresses CYLD activity, resulting in increased NF-κB transcriptional activation.

As further validation, we assessed whether CYLD phosphorylation modulates NF-κB activity in the context of transformation. NF-κB luciferase reporter activity was measured in IKKε-transformed NIH-3T3 cells overexpressing WT or mutant CYLD (S418A and S772A). IKKε-transformed cells exhibited strong NF-κB activation in comparison to control cells, and introduction of WT CYLD and S772A failed to induce a substantial change in NF-κB activity. As observed in MCF-7 cells, however, CYLD S418A more strongly inhibited IKKε-induced NF-κB activation. These findings indicate that IKKε-mediated phosphorylation of CYLD at serine 418 plays a role in the observed activation of NF-κB. As the decrease in NF-κB activation observed in the presence of CYLD S418A was not as robust as the decrease in transformation that was observed in Figure 5, it is likely that NF-κB is not the only pathway affected by phosphorylation of CYLD. However, these findings suggest that the regulation of CYLD phosphorylation by IKKε contributes to the NF-κB activity necessary for IKKε-mediated cell transformation.

To further confirm that cell transformation mediated by CYLD suppression is NF-κB dependent, we utilized the NIH-3T3 cells expressing shRNAs targeting murine CYLD which were described in Figure 4. When a non-phosphorylatable IκBα mutant (IκBα S32,36A), which acts as an NF-κB “superrepressor,” was stably expressed in these cells, we found a dramatic reduction in anchorage-independent colony growth (Figure 6E). Thus, suppression of CYLD induces NF-κB-dependent cell transformation similar to that induced by overexpression of IKKε.

Discussion

IKKε plays a central role in regulating innate immunity and also acts as an oncogene in a significant fraction of breast cancers. To gain a deeper understanding of the role(s) of IKKε in these biological processes, we used an unbiased proteomic and bioinformatic approach, which allowed us to identify the optimal substrate phosphorylation motif for IKKε. Based on these findings, a proteome-wide search for sites that correspond to the IKKε phosphorylation motif then led to the identification of a large number of putative IKKε substrates.

Of the candidate IKKε substrates identified, many have known roles in inflammatory and/or oncogenic signaling pathways (Table 1). In particular, serine 418 of the deubiquitinase and tumor suppressor CYLD emerged as a very likely site of IKKε phosphorylation. Here we showed direct in vitro phosphorylation of CYLD by IKKε, and have used mass spectrometry and an IKKε phospho-substrate antibody to show that Ser418 of CYLD is, in fact, the in vivo site phosphorylated by IKKε. While it has been reported that IKKα and IKKβ can phosphorylate Ser418 of CYLD (Reiley et al., 2005), we demonstrated that CYLD is phosphorylated much more efficiently by IKKε than by either of the canonical IKKs.

Upon finding that an oncoprotein is capable of strongly phosphorylating a known tumor suppressor, we predicted that CYLD phosphorylation at Ser418 might play an important role in cell transformation. Indeed, we found that introduction of CYLD S418A suppressed anchorage-independent growth in IKKε-transformed NIH-3T3 cells and hindered IKKε-driven tumor growth. The data presented here therefore define one potential mechanism by which IKKε induces tumorigenesis. Further examination showed that phosphorylation of CYLD at S418 decreases its deubiquitinase activity and increases NF-κB activation. Activation of NF-κB therefore appears to be one important mechanism by which CYLD phosphorylation regulates cell transformation. As CYLD also has functions independent of NF-κB it is likely that other signaling pathways may also be involved. Moreover, we recognize that IKKε may also modulate other substrates in addition to CYLD during cell transformation. Thus, although further work will be necessary to evaluate the full mechanisms by which IKKε and CYLD contribute to transformation, these observations connect an oncogene and a tumor suppressor that both regulate NF-κB to cell transformation.

The role of chronic inflammation in the development of inflammation-related cancers is well-known, but the mechanisms by which inflammatory mediators cause cell transformation are poorly understood (Basseres and Baldwin, 2006; Karin and Greten, 2005; Perkins, 2007). For kinases involved in these signaling pathways, such as the anti-viral mediator and oncoprotein IKKε, our inability to identify the complete repertoire of intracellular substrates has hindered progress towards understanding how these enzymes exert their oncogenic effects. The techniques described here, involving the identification of the phosphorylation motif for a kinase, followed by development of a phospho-substrate antibody and bioinformatic prediction of substrates, can provide an efficient and unbiased method for the identification and validation of physiologically relevant kinase substrates. We have validated this method through the identification of CYLD as a novel IKKε substrate and have shown that CYLD phosphorylation is necessary for full IKKε-mediated anchorage-independent growth. As expression of CYLD S418A does not completely suppress IKKε-driven cell transformation, CYLD is unlikely to be the only IKKε substrate important for cell transformation. However, the strength of the approach described here, as opposed to more targeted methods of substrate identification, is that it has simultaneously identified a large number of other potentially important IKKε substrates. Available in vitro assays should allow other putative substrates to be efficiently tested for their ability to modulate IKKε-driven cell transformation, leading to both an increased understanding of how IKKε controls oncogenesis, and, perhaps, new insights into therapeutic targets for IKKε-driven cancers.

Experimental Procedures

Antibodies, Plasmids, and Reagents

Anti-Myc (9E10), anti-CYLD, anti-RIP2, and anti-GFP were obtained from Santa Cruz Technology. Anti-Myc (rabbit) and anti-GST were from Cell Signaling Technology. Anti-Flag (M2) and anti-HA were from Covance. Anti-IKKε and anti-actin were from Sigma. Lentiviral shRNA constructs targeting murine CYLD and GFP were obtained from the RNAi Platform of the Broad Institute of Harvard and MIT. IKKε-Tide, IKKε-Y5A, and IKKε-L8A were created and HPLC purified under contract by the Tufts University Core Facility. The IκBα (IKK substrate) peptide was from Upstate. Myc-CYLD and Myc-TRAF2 were created by PCR cloning into the BamHI site of the 3XMyc (pEBB) vector. GST-IKKε was created by PCR cloning into the BamHI and NotI sites of the pEBG vector. Myc-CYLD S418A, Myc-CYLD S547A, Myc-CYLD S772A, and GST-IKKε K38A were created using a modification of the QuickChange Site-directed mutagenesis protocol (Stratagene). pBabe-CYLD WT and mutants were created by subcloning into the BamHI site of the pBabe vector. Flag-IKKε, Myc NEMO, OMNI-RIP2, HA-ubiquitin, HA-Ub K63 only, and IκBα S32,36A were described previously (Abbott et al., 2004; Boehm et al., 2007).

Cell Culture, Transfection, Immunoprecipitations, and Western Blotting

HEK-293T and MCF-7 cells were obtained from ATCC and grown in DMEM containing 10% FBS. NIH-3T3 cells were obtained from ATCC and grown in DMEM containing 10% bovine calf serum. Transfection was performed by polyethylenimine. For preparation of recombinant GST-IKKε and GST-IKKε K38A, and for preparation of all immunoprecipitates used in kinase assays and Western blots, cells were lysed in 50 mM Tris (pH 7.5), 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 1 mM EGTA, 1 mM β-glycerophosphate, 1 mM PMSF, 1 mM sodium orthovanadate, 1 μg/mL leupeptin, 1 μg/mL pepstatin, and 10 nM Calyculin A. For preparation of Myc-CYLD for LC/MS/MS, immunoprecipitates were washed in the above lysis buffer containing 0.25% deoxycholic acid and 1M NaCl.

In vitro kinase assays

Kinase buffer contained 50 mM Tris (pH 7.5), 12 mM MgCl₂, 1 mM β-glycerophosphate, 100 μM ATP, and 10 μCi γ-³²P-ATP/reaction. Reactions were incubated at 30°C for 1h. The positional scanning peptide library assay was performed as described previously (Hutti et al., 2004; Turk et al., 2006).

Identification of in vivo Phosphorylation Sites by Mass Spectrometry

Coomassie stained gel bands containing CYLD were excised and subjected to in-gel digestion and reversed-phase microcapillary LC/MS/MS using a LTQ 2D linear ion trap (Thermo Scientific) in data-dependent acquisition and positive ion mode at a flow rate of 275nL/min using a 10 cm C₁₈ microcapillary column. The column was equilibrated and peptides were loaded using 0.1% acetic acid/0.9% acetonitrile/99% water then eluted with a gradient from 5% buffer B (acetonitrile) to 38% B, followed by 95% B for washing. MS/MS spectra were searched against the reversed SwissProt protein database using Sequest (Proteomics Browser Software, Thermo Scientific) with differential modifications for Ser, Thr and Tyr phosphorylation (+79.97) and Met oxidation (+15.99). Phosphorylation sites were identified if top ranking sequences matched to CYLD with the following Sequest scoring thresholds: 2+ ions, Xcorr ≥ 2.0, Sf ≥ 0.4, P ≥ 0; 3+ ions, Xcorr ≥ 2.75, Sf ≥ 0.5, P ≥ 0 against the forward database. Passing MS/MS spectra were manually inspected to be sure that b- and y- fragment ions aligned with the assigned protein database sequence. The exact sites of phosphorylation were aided using GraphMod software (PBS). For phosphorylation sites in regions exhibiting low amino acid coverage, targeted ion MS/MS (TIMM) was performed by setting the ion trap to perform MS/MS on expected m/z values for predicted peptides. Ser418 (FHpSLPFSLTK) was not observed from a data dependent acquisition, but was detected via database searching from a TIMM experiment targeting the doubly charged ion at m/z 628.81.

NF-κB reporter assays

MCF-7 cells

MCF-7 cells were transfected with an NF-κB-luciferase reporter (Clontech) and Renilla luciferase (to standardize transfection efficiency). Assays were performed according to the manufacturer's instructions (Promega).

NIH-3T3 cells

NF-κB activity was measured using the Dual-Glo Luciferase assay (Promega). IKKε-transformed NIH-3T3 cells stably expressing WT or mutant CYLD were transiently transfected with pTRH1-NF-κB-Luciferase reporter and pRL-SV40-Renilla. Additional ectopic WT and mutant CYLD was also introduced in these experiments using the expression constructs MYC-CYLD, Myc-CYLD S418A, or Myc-CYLD S772A.

In vitro cell transformation assays

Growth of NIH-3T3 cells in soft agar was determined by plating 5 × 10⁴ cells in triplicate in 0.4% Noble agar and DMEM containing 10% bovine calf serum. Colonies greater than 100 μm in diameter were counted microscopically 21 days after plating.

Tumorigenicity assay

2 × 10⁶ cells were subcutaneously injected into immunodeficient mice (Balb/c Nude, Charles River Laboratories) anesthetized with isofluorane. Two mice were used per group, and three injection sites were made per mouse. Tumors were measured at 21 days post-injection.

IKKε phospho-substrate antibody

Rabbits were immunized with a phosphopeptide library with fixed residues corresponding to the IKKε phosphorylation motif and antibodies were purified as has been described previously (Doppler et al., 2005).

Supplementary Material

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Acknowledgments

We thank members of the Cantley and Hahn labs for technical assistance and critical comments. This work was supported by NIH R01 GM-56203 (L.C.C.), NIH R01 CA130988 (W.C.H.), DoD Breast Cancer Research Program grant W81XWH-07-01-0408 (W.C.H.), a grant from Susan G. Komen for the Cure (R.R.S.), and a Burroughs-Wellcome Career Award for Biomedical Scientists (D.W.A).

Footnotes

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