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TNF-Induced Recruitment and Activation of the IKK Complex Require Cdc37 and Hsp90
Molecular Cell, Vol. 9, 401–410, February, 2002, Copyright 2002 by Cell Press
TNF-Induced Recruitment and Activation of the IKK Complex Require Cdc37 and Hsp90 Guoqing Chen, Ping Cao, and David V. Goeddel1 Tularik, Incorporated Two Corporate Drive South San Francisco, California 94080
Summary The IKK complex, containing two catalytic subunits IKK␣ and IKK and a regulatory subunit NEMO, plays central roles in signal-dependent activation of NF-B. We identify Cdc37 and Hsp90 as two additional components of the IKK complex. IKK␣/IKK/NEMO and Cdc37/Hsp90 form an ⵑ900 kDa heterocomplex, which is assembled via direct interactions of Cdc37 with Hsp90 and with the kinase domain of IKK␣/IKK. Geldanamycin (GA), an antitumor agent that disrupts the formation of this heterocomplex, prevents TNF-induced activation of IKK and NF-B. GA treatment reduces the size of the IKK complex and abolishes TNF-dependent recruitment of the IKK complex to TNF receptor 1 (TNF-R1). Therefore, heterocomplex formation with Cdc37/Hsp90 is a prerequisite for TNF-induced activation and trafficking of IKK from the cytoplasm to the membrane. Introduction NF-B plays prominent roles in inducible expression of genes involved in diverse biological processes, including development, immune, and inflammatory responses, cell growth and death, stress responses, and oncogenesis (for recent reviews, see Ghosh, 1999; Maniatis, 1999; Karin and Ben-Neriah, 2000; Karin and Delhase, 2000). In resting cells, the majority of NF-B is sequestered in the cytoplasm in complexes with a class of inhibitory proteins termed IB. Upon exposure of cells to a variety of extracellular stimuli, including tumor necrosis factor (TNF) and interleukin-1 (IL-1), IB proteins become phosphorylated, resulting in ubiquitination and degradation of IB. This allows NF-B to translocate into the nucleus, where it activates transcription of target genes. The protein kinase responsible for stimulus-dependent phosphorylation of IB has been identified using different approaches (DiDonato et al., 1997; Mercurio et al., 1997; Regnier et al., 1997; Woronicz et al., 1997; Zandi et al., 1997). Biochemical purification defines the IB kinase (IKK) activity as an ⵑ700–900 kDa protein complex, consisting of at least two catalytic subunits, IKK␣ and IKK, and a regulatory subunit, NEMO (NF-B essential modulator, also known as IKK␥/IKKAP1/FIP3) (Chen et al., 1996; Rothwarf et al., 1998; Yamaoka et al., 1998; Zandi et al., 1998; Li et al., 1999; Mercurio et al., 1999). IKK␣ and IKK are two highly homologous kinases, both containing a conserved N-terminal kinase domain and a C-terminal region with a leucine zipper (LZ) and a helixloop-helix (HLH) motif. While the LZ motif is responsible 1
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for dimerization of IKK␣ and IKK, both the LZ and HLH motifs are important for modulating the kinase activity of IKK (Woronicz et al., 1997; Zandi et al., 1997, 1998; Delhase et al., 1999). NEMO contains multiple putative coiled-coil motifs and a potential zinc finger motif at the C terminus. NEMO is an essential component of the IKK complex because NEMO-deficient cells are unable to activate IKK or NF-B in response to a wide set of stimuli, including TNF and IL-1. The large 700–900 kDa IKK complex also does not form in cells lacking NEMO (Yamaoka et al., 1998). Accumulating evidence suggests that the IKK complex represents a converging point for transducing diverse upstream NF-B-activating stimuli (Karin, 1999a, 1999b). However, the detailed mechanism by which IKK is activated in response to various stimuli, in particular TNF and IL-1, remains unclear. A simple model for IKK activation may involve an upstream activating kinase, or so-called IKK kinase (IKK-K) (Mercurio and Manning, 1999; Israel, 2000). However, the identity of such an IKK-K, if it exists, remains elusive. Like the signal transducing molecules TRADD (Hsu et al., 1996b), RIP (Hsu et al., 1996a), and TRAF2 (Shu et al., 1996), the IKK complex is also recruited to TNF receptor 1 (TNF-R1) upon TNF induction (Devin et al., 2000; Zhang et al., 2000). Signal-dependent recruitment of IKK to TNF-R1 requires TRAF2, while RIP primarily mediates IKK activation. Interestingly, enforced oligomerization of RIP, NEMO, IKK␣, or IKK has been shown to be sufficient for inducing IKK and NF-B activation (Poyet et al., 2000). We have identified Cdc37 and Hsp90, which together form a kinase-specific chaperone, as additional components of the ⵑ900 kDa IKK complex. Geldanamycin (GA), an Hsp90 binding agent, reduces the size of the IKK complex by disrupting the association of IKK/NEMO with Cdc37/Hsp90. GA treatment also abolishes TNFdependent recruitment of the IKK complex to TNF-R1 and blocks TNF-induced activation of IKK. These results indicate that Cdc37 and Hsp90 play a physiological role in TNF-dependent translocation and activation of the IB kinases. Results Characterization of HeLa Flag-IKK Stable Cells To facilitate purification of the IKK complex, we generated various lines of HeLa cells that stably express either wild type (WT) or kinase-dead (KA mutant) forms of Flagtagged IKK␣ and IKK. We monitored both TNF- and IL-1-induced NF-B activation in these stable cells by transient transfection with a NF-B-dependent luciferase reporter (Figure 1A). In comparison with parental HeLa cells, HeLa cells stably expressing wild type IKK␣ (␣WT) demonstrated almost normal NF-B activation, while IKKWT stable cells (WT) displayed relatively higher basal and induced NF-B activities. In contrast, stable expression of kinase-inactive IKK␣ (␣KA) or IKK (KA) significantly reduced basal and cytokine-induced NF-B activities. In spite of these differences, each of
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Figure 1. Characterization of HeLa Cells Stably Expressing Flag-IKK (A) NF-B activation in Flag-IKK stable cells. Parental HeLa cells (HeLa) or cells that stably express Flag-tagged wild-type IKK␣ (␣WT), IKK (WT), or kinase-inactive IKK␣ (␣KA), IKK (KA) were cotransfected with an NF-B-dependent luciferase reporter and a control Renilla luciferase reporter. Reporter activities were measured 4 hr after cells were stimulated with TNF or IL-1. Relative luciferase activities were normalized to the control Renilla luciferase activities. Each bar is the average plus the standard deviation of three independent duplicate experiments. (B) IKK activation in Flag-IKK stable cells. Extracts from above HeLa and stable cell lines that had been treated with (⫹) or without (⫺) TNF were immunoprecipitated (IP) with anti-Flag antibodies. Each immunoprecipitate was analyzed by anti-Flag immunoblotting (IB) and in vitro kinase assay (KA) using GST-IB␣ (1–54) as the IKK substrate. Equal amounts of total cell extracts (TCE) from these cells were also immunoblotted with anti-Flag, anti-IKK␣/anti-IKK (IKK␣/), and anti-NEMO (served as a loading control) antibodies (lower three panels) to determine expression levels.
these HeLa cell lines exhibited roughly similar ratios (induced to basal) of NF-B induction by TNF or IL-1. These results suggest that in the cell lines examined here, stable expression of various Flag-IKKs exerts only minor effects on the endogenous NF-B signaling pathway. This behavior may be explained by the relatively low levels of overexpression of various Flag-IKK proteins, particularly in the ␣KA and KA cell lines, in comparison with the endogenous levels of IKK␣ and IKK (Figure 1B, lower three panels). In addition, immunoprecipitation and in vitro kinase assay revealed that the kinase activity of the Flag-IKK-containing complexes was TNF-inducible in all four cell lines (Figure 1B, upper 2 panels). The observed IKK activation by TNF in ␣KA and KA cells is likely due to the presence of endogenous IKK␣ and/or IKK in the Flag-IKK-containing complexes. Purification of the IKK Complex from Flag-IKK Stable Cells We next attempted to purify the IKK complex from the Flag-IKK stable cell lines. A representative purification of the IKK complex from the ␣KA cell line is illustrated in Figure 2. For each purification, we harvested 40 liters of cells grown in suspension after cells were treated with TNF or left untreated. Cells were then gently lysed under isotonic conditions using 0.1% nonionic detergent NP-40 to open the plasma membrane and release the cytosol. Cleared lysates were loaded onto an antiFlag affinity column. After extensive washing, bound proteins were eluted using Flag peptides. Aliquots of the eluates derived from TNF stimulated (⫹) or unstimulated (⫺) cells were separated by SDS-PAGE and visualized by Coomassie blue staining (Figure 2A). Similar polypeptide species were observed for all purifications.
To further purify the IKK complex, we fractionated the Flag eluates by gel filtration with a Superose 6 column (Figure 2B). Each fraction was then analyzed by immunoblotting with anti-IKK␣/anti-IKK (mix) and anti-NEMO antibodies and in vitro kinase assays to monitor TNFdependent IKK activation. The peak levels of NEMO and TNF-induced IKK activity coeluted in fraction 10 (marked by arrowheads), corresponding to an exclusion size of ⵑ900 kDa. This result is consistent with previous findings that IB kinase activity exists as a 700–900 kDa protein complex (Chen et al., 1996; DiDonato et al., 1997; Mercurio et al., 1997). To determine if known proteins implicated in NF-B signaling were present in our purified IKK complex, we immunoblotted these peak fractions with a panel of antibodies against TRADD (Hsu et al., 1995), RIP (Hsu et al., 1996a), TRAF2 (Hsu et al., 1996b), TRAF6 (Cao et al., 1996), I-TRAF (Rothe et al., 1996), MEKK1 (Mercurio et al., 1997), NIK (Malinin et al., 1997), AKT (Ozes et al., 1999), ACT1/CIKS (Leonardi et al., 2000; Li et al., 2000), MKP1 (Mercurio et al., 1997), and IB␣ (Mercurio et al., 1997). We failed to detect any of these molecules in the purified IKK complexes (data not shown). Purified IKK Complexes Contain Cdc37 and Hsp90 We combined fractions containing peak IKK activities (fractions 8 ⫹ 9 and 10 ⫹ 11) and resolved them by preparative SDS-PAGE (Figure 2C). Coomassie blue staining revealed almost identical polypeptides present in the TNF-stimulated and -unstimulated IKK complexes, except that a faint ⵑ60 kDa polypeptide (labeled p60) was detected only in the unstimulated complex. We excised all visible bands and digested them with trypsin. The tryptic peptide mixture from each in-gel digest was analyzed by mass spectrometry. The resulting peptide mass fingerprints were compared with
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Figure 2. Purification of the IKK Complex from Flag-IKK Stable Cells (A) A Coomassie blue-stained SDS-PAGE gel containing samples affinity purified from TNF-unstimulated (⫺) or -stimulated (⫹) ␣KA stable cells. Samples represent Flag peptide eluates from the anti-Flag affinity columns and were used as the starting material (SM) for further purification shown in (B) and (C). (B) Gel filtration chromatography to isolate the IKK complex. The Flag peptide eluates from (A) were further fractionated on a Superose 6 gel filtration column. Each fraction was analyzed by immunoblotting (IB) with anti-IKK␣/anti-IKK (IKK␣/) and anti-NEMO antibodies and in vitro kinase assays (KA) to monitor the IKK enzymatic activities. An anti-Flag immunoblotting was also performed, revealing a pattern very similar to that of anti-IKK (data not shown). Elution positions of protein standards run in a parallel experiment are indicated above corresponding fractions. Arrowheads mark the peaks of purified IKK complexes. (C) Identification of protein components of the purified IKK complexes. The peak fractions (8 ⫹ 9 and 10 ⫹ 11) from (B) were pooled, concentrated, and resolved by preparative SDS-PAGE. Coomassie blue-stained bands were excised, and their identities (as indicated on the right) were determined by peptide mass fingerprint mapping.
the theoretical tryptic peptide masses derived from the NCBInr database. This allowed us to unambiguously identify IKK␣ and NEMO in the purified complex (Figure 2C, right). IKK was not found in the purified complex by peptide mass fingerprint mapping, even though a small amount of IKK was indeed detected in this purified IKK complex by immunoblotting (data not shown). The presence of IKK explains why TNF-induced IKK activation was observed for the IKK complex purified from the ␣KA cell line (Figure 2B). These results also suggest that the majority of the IKK complex isolated from the ␣KA cell line is composed of overexpressed Flag-IKK␣ and endogenous NEMO. In parallel to these findings, complexes containing predominantly FlagIKK and NEMO were purified from the Flag-IKK stable cell lines (data not shown). In addition to IKK␣/IKK and NEMO, we consistently observed the presence of Hsp90, Cdc37, and -tubulin in the IKK complexes purified from different Flag-IKK stable cells (Figure 2 and data not shown). Cdc37 and Hsp90 have been previously shown to function as a kinase-specific chaperone (Csermely et al., 1998; Pratt et al., 1999). Coomassie blue staining revealed similar amounts of Cdc37, Hsp90, IKK, and NEMO present in the purified IKK complexes (Figure 2C and data not shown), suggesting that Cdc37/Hsp90 and IKK␣/IKK/ NEMO form a heterocomplex at a stoichiometric ratio of 1:1. Conversely, both -tubulin and p60 appear to represent substoichiometic components of the IKK complex. The in-gel digest of p60 generated a peptide mass fingerprint that failed to match any known protein in the database. Purification of the Endogenous IKK Complex We have verified the copurification of Cdc37 and Hsp90 with the IKK complexes isolated from the Flag-IKK sta-
ble cells by immunoblotting (data not shown). To exclude the possibility that this copurification is due to an artifact of Flag-IKK overexpression in these cells, we purified the endogenous IKK complex from nontransfected HeLa cells by anti-IKK␣ affinity chromatography. Gel filtration analysis of the purified endogenous IKK complex demonstrated that Cdc37/Hsp90 precisely coeluted with IKK␣, IKK, and NEMO at a peak corresponding to an exclusion size of 900 kDa (Figure 3A, arrowhead). Interestingly, Hsp90, Cdc37, and IKK␣/ were also present in some smaller molecular weight fractions, perhaps reflecting the IKK complexes that lacked NEMO and were associated with a different set of proteins, or that were disrupted during the fractionation process. Aliquots of fractions 9, 10, and 11 derived from the gel filtration column were resolved by SDS-PAGE and visualized by silver staining (Figure 3B). Similar amounts of Hsp90, IKK␣, IKK, Cdc37, and NEMO were detected in the peak fraction of the purified endogenous IKK complex (arrowhead). Thus, all our purification results provide evidence to strongly support the existence of a stoichiometric heterocomplex of IKK␣/IKK/NEMO and Cdc37/Hsp90 in the cell. Cdc37/Hsp90 Associates with the Kinase Domains of IKK The presence of Cdc37/Hsp90 in the IKK complex was further confirmed by an endogenous coimmunoprecipitation experiment (Figure 4A). Both Cdc37 and Hsp90 were coprecipitated from HeLa cell extracts with antiNEMO and anti-IKK antibodies, but not with a control antibody. In addition, the specific interaction of Cdc37/ Hsp90 with IKK/NEMO is independent of TNF stimulation. To further assess the stability of this interaction, we subjected equal amounts of anti-NEMO immunoprecipitates to extensive washes with the lysis buffer alone
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Figure 3. Purification of the Endogenous IKK Complex (A) Gel filtration analysis of the endogenous IKK complex purified from HeLa cells by antiIKK␣ affinity chromatography. Gel filtration was performed as in Figure 2B, and each fraction was analyzed by immunoblotting with anti-Hsp90, anti-IKK␣/anti-IKK (IKK␣/), anti-Cdc37, and anti-NEMO antibodies. The arrowhead denotes the peak of the purified endogenous IKK complex that contains IKK, NEMO, Cdc37, and Hsp90. (B) A silver stained SDS-PAGE gel containing aliquots of fractions 9, 10, and 11 from the gel filtration fractionation in (A). The identity of each polypeptide band in fraction 10 was determined by mass spectrometry (as indicated on the right). Asterisks denote protein bands that either nonspecifically bind the anti-IKK␣ affinity matrix or are absent in the peak of the IKK complex.
or the buffer containing 1 M NaCl, 1% Triton X-100, or 2 M urea (Figure 4B). Immunoblotting analyses showed that the association of IKK/NEMO with Cdc37/Hsp90 was vulnerable to high salt and denaturing agent washes, while it tolerated most detergent washes. This finding explains the earlier failure to identify Cdc37 and Hsp90 as components of the IKK complex (DiDonato et al., 1997; Mercurio et al., 1997), as these purifications
usually incorporated an initial step of stringent wash with high salt or denaturing agent. To map the regions of the IKKs that interact with Cdc37/ Hsp90, we transiently expressed various deletions of Flagtagged IKK␣ and IKK and immunoprecipitated cell extracts with anti-Flag antibodies. Endogenous Cdc37 and Hsp90 were detected in the immunoprecipitates containing the full-length Flag-IKK␣ and Flag-IKK (Figure Figure 4. Cdc37 Targets Hsp90 to the Kinase Domains of IKK
(A) Coimmunoprecipitation of endogenous IKK/NEMO with Cdc37/Hsp90. Extracts from TNF-unstimulated (⫺) or -stimulated (⫹) HeLa cells were immunoprecipitated (IP) with either anti-NEMO (NEMO), anti-IKK (IKK), or a control IgG1 (CTRL) monoclonal antibody. Each immunoprecipitate was immunoblotted (IB) with rabbit polyclonal antibodies against Hsp90, IKK␣/IKK (IKK␣/), Cdc37, and NEMO. (B) Stability of the interaction between IKK/ NEMO and Cdc37/Hsp90. Equal amounts of those anti-NEMO precipitates in (A) were washed extensively with the buffer alone (lane 1) or the buffer containing either 1.0 M NaCl (lane 2), 1% Triton X-100 (lane 3), or 2.0 M urea (lane 4) and subsequently analyzed by immunoblotting as shown in (A). Asterisks in both (A) and (B) mark those nonspecific bands. (C) Cdc37/Hsp90 interacts with the kinase domains of IKK. HeLa cells were transfected with vector alone or vectors expressing various deletions of Flag-tagged IKK␣ or IKK. Cell extracts were immunoprecipitated with anti-Flag antibodies, and coprecipitating Cdc37 and Hsp90 proteins were determined by immunoblotting with anti-Hsp90 and antiCdc37 antibodies. Expression levels of IKK␣ and IKK variants were monitored by immunoblotting with anti-Flag (biotinylated) antibody. (D) A direct interaction between Cdc37 and the kinase domains of IKK. Equal amounts of purified HA-Cdc37 (INPUT) were incubated with the glutathione beads containing GST alone, GST-IKK␣/IKK-kinase domain (KD), or GST-NEMO fusion proteins. After extensive washing, amounts of HA-Cdc37 retained on beads were determined by anti-HA immunoblotting. The bottom is a Coomassie blue (CBB)-stained SDS-polyacrylamide gel containing purified HA-Cdc37 and various GST fusion proteins.
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Figure 5. TNF-Induced Activation of NF-B and IKK Requires Cdc37/Hsp90 (A) Effects of GA treatment on the heterocomplex formation between IKK/NEMO and Cdc37/Hsp90. Extracts of HeLa cells that had been treated with DMSO or GA were immunoprecipitated (IP) with a control IgG1 (CTRL) or anti-NEMO (NEMO) monoclonal antibody. The presence of Hsp90, Cdc37, IKK␣/IKK, and NEMO in the immunocomplexes was determined by immunoblotting (IB) as described in Figure 4A, except that a goat anti-Cdc37 polyclonal antibody was used. (B) Effects of GA treatment on NF-B- and AP1-dependent transcription. HeLa cells were cotransfected with a NF-B- (left) or AP1-driven (right) luciferase reporter along with a control Renilla luciferase reporter. 24 hr later, cells were treated with DMSO or GA prior to TNF and IL-1 induction. Relative luciferase activities were measured as described in Figure 1A. (C) Effects of GA treatment on IKK activation. Following the pretreatment with DMSO or GA, HeLa cells were stimulated with (⫹) or without (⫺) TNF. The IKK complexes were isolated by anti-NEMO immunoprecipitation and further analyzed by in vitro kinase assays (KA) using GST-IB␣ (1–54) as the IKK substrate (bottom). The IKK protein level in each precipitate was also determined by immunoblotting. (D) Effects of GA treatment on JNK activation. Both immunopreciptation and in vitro kinase assay were performed essentially as described in (C), except that an anti-JNK1 monoclonal antibody and the GST-Jun (1–79) substrate were used for each assay.
4C). Furthermore, IKK deletions containing only the kinase domain were also capable of binding Cdc37 and Hsp90, while those without the kinase domain failed to do so. A direct interaction between the IKKs and Cdc37 was further ascertained by a GST pull-down assay (Figure 4D). Purified HA-tagged Cdc37 was retained on beads containing GST-IKK␣ or GST-IKK-kinase domain, but not on beads with GST alone (the faint band likely represents the shadow of overloaded input) or GST-NEMO. Given the high input, the binding between Cdc37 and IKK appears very weak. We found that Cdc37 interacts more strongly with IKK in the presence of Hsp90 and that purified Hsp90 by itself associates poorly with GST-IKK fusions (data not shown). This suggests a cooperative binding of Cdc37/Hsp90 to IKK. Thus, Hsp90 is targeted to the IKK complex via a direct interaction between Cdc37 and the catalytic region of IKK␣/. TNF-Induced Activation of IKK and NF-B Requires Cdc37/Hsp90 To examine the possible physiological role in TNF signaling of the association of IKK/NEMO with Cdc37/Hsp90, we employed an antitumor agent, geldanamycin (GA). GA was previously shown to interact and interfere with the normal functions of Hsp90 (Whitesell et al., 1994). Using anti-NEMO immunoprecipitation, we isolated the IKK complexes from HeLa cells that had been pretreated with either DMSO or GA (Figure 5A). Subsequent immu-
noblotting analysis showed that the treatment with GA almost completely disrupted the interaction between IKK/NEMO and Hsp90/Cdc37, but DMSO exhibited no detectable effect. Thus, GA provides a powerful tool to explore in vivo functions of the association of IKK/NEMO with Cdc37/Hsp90. We next studied the impact of GA treatment on NF-B activation using transient transfection with a NF-B-driven luciferase reporter. GA treatment not only potently blocked both TNF- and IL-1-induced NF-B activation, but also moderately inhibited (2- to 3-fold) the unstimulated NFB activity (Figure 5B, left). To determine the specificity of GA inhibition on NF-B activation, we performed a similar experiment with an AP1-dependent luciferase reporter (Figure 5B, right). In this case, GA treatment only partially inhibited TNF- and IL-1-induced AP1-mediated reporter activities. These effects were much less severe than those on NF-B activation. In addition, GA treatment had minimal effects on transcription of the internal control Renilla luciferase reporter (⬍2-fold). These results indicate that GA selectively impedes the NF-B activation pathway. We also performed in vitro immunocomplex kinase assays to measure the effects of GA treatment on TNFinduced IKK activation (Figure 5C). HeLa cells were treated with GA or DMSO prior to TNF induction, and in vitro kinase assays were performed on the immunoprecipitated IKK complexes using exogenous GST-IB␣ fusion proteins as substrates. TNF stimulation resulted
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Figure 6. Roles for Hsp90/Cdc37 in Complex Assembly and Receptor Recruitment of IKK (A and B) Gel filtration analysis of the IKK complexes. Equal amounts of total extracts from HeLa cells that had been treated with DMSO (A) or GA (B) were fractionated on a Superose 6 column as described in Figure 2B. The IKK complex sizes were monitored by immunoblotting (IB) analysis of each fraction with antibodies against IKK␣ and IKK (IKK␣/) and NEMO. (C) TNF-induced recruitment of IKK to TNF-R1 requires Hsp90/Cdc37. HeLa cells were treated with (⫹) or without (⫺) TNF after cells had been preincubated with DMSO or GA. Equal amounts of cell extracts were immunoprecipitated (IP) with anti-TNF-R1 (985) monoclonal antibodies. TNF-R1 and associated IKK, Hsp90, RIP, TRAF2, and TRADD were detected by immunoblotting with the indicated polyclonal antibodies (right panels). Total cell extracts (TCE) were also prepared and immunoblotted in the same way to monitor the effects of GA treatment on cellular levels of these proteins (left panels). Asterisks indicate the protein bands that are nonspecifically recognized by respective antibodies. Brackets mark the higher molecular weight species of RIP and TRAF2 that become visible only after TNF stimulation.
in a dramatic increase of IKK enzymatic activities. However, the pretreatment with GA almost completely abolished this robust activation of IKK in response to TNF. As a control, we also investigated if GA treatment might affect c-Jun N-terminal kinase 1 (JNK1) activation by TNF (Figure 5D). Similar to the DMSO control, GA exhibited almost no effect on TNF-induced JNK1 activation, even though the unstimulated JNK1 activity was slightly reduced by GA treatment. In addition, GA had been previously shown to block TNF-induced degradation of IB, indicating that GA acts on this arm of NF-B response (Lewis et al., 2000). These results together demonstrate that the specific formation of a heterocomplex between IKK/NEMO and Cdc37/Hsp90 is essential for TNF-induced activation of IKK and NF-B. Role for Cdc37/Hsp90 in Assembly of the IKK Complex To investigate if GA-mediated disruption of the interaction between IKK/NEMO and Cdc37/Hsp90 might affect the formation and composition of the IKK complex, we fractionated equal amounts of extracts from HeLa cells that had been treated with DMSO or GA by gel filtration. Immunoblotting revealed that the peak of the IKK complex in GA-treated cells (Figure 6B, fraction 12) eluted with an exclusion size smaller than in DMSO-treated cells (Figure 6A, fraction 11). Similarly, GA treatment causes a reduction in size of the purified IKK complex that lacks Cdc37/Hsp90 (data not shown). However, no apparent effect was observed on the IKK activity when GA was directly added to the in vitro kinase assays with the purified IKK complexes (data not shown). This suggests that GA blocks the process of IKK activation by TNF rather than inhibits the IKK enzymatic activity per se. GA treatment also resulted in the presence of
significantly increased amounts of NEMO in the lower molecular weight fractions in comparison with those from DMSO-treated cells (compare fractions 15–17 in Figures 6A and 6B). The dramatic effects of GA on the size of the endogenous IKK complex and the redistribution of IKK␣/ and NEMO demonstrate a critical role for Cdc37/Hsp90 in the assembly of functional IKK complexes under physiological conditions. TNF-Induced Recruitment of IKK to TNF-R1 Requires Cdc37/Hsp90 We also examined if Cdc37/Hsp90 plays a role in shuttling the IKK complex from the cytoplasm to the plasma membrane. HeLa cells were treated with DMSO or GA and then briefly stimulated with or without TNF. TNFR1-associated signaling complexes were isolated from these cells by immunoprecipitation using a monoclonal antibody against the extracellular domain of TNF-R1. Immunoblotting demonstrated TNF-dependent recruitment of the signaling molecules TRADD, RIP, and TRAF2 to TNF-R1 in DMSO-treated cells (Figure 6C, lanes 5 and 6). Additionally, IKK and Hsp90 were also recruited to TNF-R1 after TNF stimulation, confirming that the IKK/NEMO-Cdc37/Hsp90 heterocomplex is recruited to TNF-R1 in a ligand-dependent manner. In GA-treated cells, recruitment of TRAF2 and TRADD to TNF-R1 appeared unchanged (Figure 6C, lanes 7 and 8). However, GA treatment almost fully abrogated TNF-dependent recruitment of IKK, Hsp90, and RIP molecules to TNF-R1. To determine the effects of GA treatment on expression levels of these proteins, we prepared total cell extracts and immunoblotted them with corresponding antibodies (Figure 6C, left panels). GA treatment had little or no effect on the cellular levels of IKK, TRAF2,
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plex in response to TNF. These results reveal a physiological role for the Cdc37/Hsp90 chaperone in transducing NF-B activating signals.
Figure 7. Kinetics of GA-Mediated Inhibition of IKK Recruitment to TNF-R1 and Complex Formation with Cdc37/Hsp90 Equal numbers of HeLa cells were treated with GA for the indicated times (0–10 hr) and then stimulated with TNF for 10 min. Aliquots of extracts prepared from these cells were immunoprecipitated (IP) with either anti-TNF-R1 (A) or anti-NEMO (B) monoclonal antibodies. Anti-TNF-R1 immunoprecipitates were further analyzed by immunoblotting (IB) with anti-IKK and anti-TRADD polyclonal antibodies (A), while anti-NEMO immunoprecipitates were immunoblotted with anti-IKK and anti-Cdc37 polyclonal antibodies (B).
TRADD, and TNF-R1 proteins, while it modestly upregulated Hsp90 expression. In contrast, RIP expression levels were greatly diminished in GA-treated cells, suggesting a role for Cdc37/Hsp90 in stabilizing RIP. This is in an agreement with a previous report that RIP is also an Hsp90-associated kinase and GA treatment selectively causes RIP degradation (Lewis et al., 2000). Thus, the defective recruitment of RIP to TNF-R1 in GAtreated cells could be due simply to the rapid turnover of RIP when it is dissociated from Hsp90. However, RIP is not required for IKK recruitment to TNF-R1 (Devin et al., 2000), and GA treatment has no obvious effect on the levels of IKK and NEMO proteins. In addition, time course experiments indicate that the kinetics of GAinduced inhibition of IKK recruitment to TNF-R1 correlate with those of GA-mediated disruption of the IKKCdc37/Hsp90 complex (Figure 7). GA treatment for 5 hr significantly reduced the amount of IKK recruited to TNF-R1 and the amount of Cdc37 associated with IKK, whereas 10 hr treatment almost completely abolished both interactions. Taken together, these results support an important role for Cdc37/Hsp90 in trafficking the IKK complex from the cytoplasm to the membrane. It is also possible that Cdc37/Hsp90 may be required for the interaction of the IKK complex with components of the upstream TNF-R1 signaling complex. Discussion We report the purification and identification of Cdc37 and Hsp90 as additional components of the IKK complex. Several lines of evidence argue that the formation of a heterocomplex with Cdc37/Hsp90 is essential for assembly, translocation, and activation of the IKK com-
Functions of Cdc37/Hsp90 in IKK Activation Hsp90, one of most abundant cytosolic proteins in eukaryotic cells, plays an important role in the maturation, activation, and trafficking of proteins involved in signal transduction, cell cycle control, development, and transcriptional regulation (Csermely et al., 1998; Pratt et al., 1999). Unlike the more general Hsp70 and Hsp60 chaperones, Hsp90 appears to be a dedicated chaperone for only a limited number of client proteins, including steroid hormone receptors and protein kinases. Hsp90 functions in concert with a set of cochaperones that link Hsp90 to distinct classes of client proteins. For example, the mammalian homolog of the yeast cell cycle control protein Cdc37 acts as a kinase-specific targeting subunit for Hsp90 (Dai et al., 1996; Stepanova et al., 1996). A growing list of kinases, including Src family tyrosine kinases and the serine/threonine kinases Raf and MEK, are known to exist as heterocomplexes with Cdc37/ Hsp90. Cdc37/Hsp90 contributes to the stabilization, activation, and/or translocation of these kinases. IKK␣ and IKK can now be added to the list of kinases whose functions and activities are dependent on Cdc37/ Hsp90. We find that the Hsp90 chaperone has no apparent effect on the stability of IKK. In contrast, Hsp90 affects the integrity of RIP, another important kinase in the TNF/NF-B signaling pathway. This is in an agreement with a previous study showing that GA selectively induces degradation of RIP and blocks TNF-induced NF-B activation (Lewis et al., 2000). IKK␣, IKK, and NEMO are recruited to TNF-R1 following TNF stimulation (Devin et al., 2000; Zhang et al., 2000). Our current study extends these findings by revealing that Cdc37/Hsp90 is part of the IKK complex and is also recruited to TNFR1 in a stimulus-dependent manner. Given that RIP is dispensable for IKK recruitment to TNF-R1, our results reveal a critical role for Cdc37/Hsp90 in shuttling IKK to the membrane. GA treatment causes the dissociation of Cdc37/ Hsp90 from the IKK complex, a reduction in size of the IKK complex, and the inhibition of TNF-induced activation of IKK and NF-B. These findings point out a physiological role for Cdc37/Hsp90 in the TNF/NF-B signaling pathway. The potent blockade of NF-B signaling by GA is likely due to multiple effects on the pathway. First, GA disrupts the interaction of IKK with Cdc37/Hsp90 and prevents the recruitment of IKK to the membrane. Second, GA treatment causes degradation of RIP, resulting in the assembly of a TNF-R1 signaling complex lacking RIP and defective in IKK activation. It is possible that Cdc37/Hsp90 may also provide a link to components of the upstream TNF-R1 complex, allowing the recruitment of IKK to the receptor. For example, both RIP and IKK associate with Cdc37/Hsp90, which may serve as a bridging factor to bring them into proximity. Interestingly, a protein highly homologous to Hsp90 has been proposed to directly bind the intracellular region of TNF-R1 (Song et al., 1995).
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Stoichiometry of the IKK-Cdc37/Hsp90 Complex Our gel filtration analysis has revealed that the endogenous IKK complex exists as an ⵑ900 kDa heterocomplex containing Cdc37/Hsp90. The size is consistent with that reported previously for the native IKK complex isolated from HeLa cells (DiDonato et al., 1997; Mercurio et al., 1997). While the native IKK complex contains a heterodimer of IKK␣ and IKK (Mercurio et al., 1997; Rothwarf et al., 1998; Woronicz et al., 1997), purified recombinant IKK␣ or IKK exists as a homodimer with a native molecular weight (MW) of 230–250 kDa (Zandi et al., 1998). Hsp90 is also a dimeric protein with a native MW of ⵑ250 kDa (Pratt et al., 1999). Cdc37 has a size of 130–150 kDa under native conditions, suggesting that it is a dimer or trimer (Kimura et al., 1997). NEMO is also known to form a dimer or trimer (Rothwarf et al., 1998). Our purifications suggest that there are roughly equal amounts of IKK, NEMO, Cdc37, and Hsp90 in the purified IKK complex. Based on these data, we predict that the complete IKK complex should contain a heterodimer of IKK␣ and IKK, a homodimer of Hsp90, and two to three molecules of NEMO and Cdc37. Such a complex would have an estimated MW of ⵑ800 kDa, matching the size of the native IKK complex and providing further evidence for that Cdc37 and Hsp90 are bona fide subunits of the IKK complex. Mechanisms of IKK Activation Although this study and others have shown a correlation between the recruitment of IKK to TNF-R1 and the activation of IKK, the exact mechanism of IKK activation is still an enigma. It is plausible that the recruitment of the IKK complex to TNF-R1 may represent an initial step. Once recruited and activated, IKK may be released from the receptor, reentering the cytoplasm and in trans activating residual inactive IKK molecules. It is possible that, like many signaling molecules, IKK may traverse the cytoplasm simply by diffusion and become trapped at the sites of action by protein-protein interactions (e.g., those involved in the formation of TNF-R1 signaling complexes at the internal surface of the membrane). It is also possible that the IKK complex may move through the cytoplasm via a transport system involving Cdc37/ Hsp90, even though nothing is known about how this movement is initiated or directed to the cell membrane. RIP plays an essential role in TNF-mediated IKK activation (Devin et al., 2000; Hsu et al., 1996a; Kelliher et al., 1998; Ting et al., 1996; Zhang et al., 2000). Following the recruitment of IKK to TNF-R1, therefore, a RIPdependent process or factor may directly trigger IKK activation. One possibility is that an upstream kinase (e.g., IKK-K) is brought to the receptor complex and activated in a RIP-dependent manner. It has been shown that enforced oligomerization of RIP, NEMO, IKK␣, or IKK is sufficient for inducing IKK activation (Poyet et al., 2000). These results suggest that such an intermediate factor or IKK-K may not be necessary for IKK activation. RIP itself may cause some conformational changes of the IKK complex, resulting in IKK activation. However, it will be difficult to test this hypothesis under physiological conditions. To fully understand the mechanism of TNF-mediated IKK activation, it will be crucial to determine which activ-
ity in the TNF-R1 signaling complex is directly responsible for activating IKK. The diversity of NF-B stimuli forecasts the existence of different upstream activators involved in signal-dependent IKK activation. It is thus reasonable to postulate that each of these NF-B signaling pathways may utilize a pathway-specific factor to directly trigger IKK activation. To date, however, most of these putative direct activators of IKK have not been identified. Determining if the IKK complex contains additional components, in particular substoichiometric and/ or transiently associated subunits, will improve our understanding of the mechanism of IKK activation as well as the specificity and diversity of NF-B signaling. Experimental Procedures Reagents Recombinant human TNF and IL-1 were obtained from Genentech (South San Francisco, CA) and BioSource International (Camarillo, CA), respectively. Geldanamycin (GA), anti-Flag M2 affinity gel, Flag peptide, anti-Flag M2, and biotinylated anti-Flag M2 monoclonal antibodies were purchased from Sigma. Rabbit anti-IKK␣, anti-IKK, anti-NEMO, anti-Hsp90, anti-TRAF2, anti-HA, anti-TRADD, antiTNF-R1, and anti-RIP polyclonal antibodies as well as goat antiCdc37 polyclonal antibody were products from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit polyclonal anti-Cdc37 antibody was purchased from StressGen (Victoria, BC, Canada). Anti-NEMO (3F6) monoclonal antibody was provided by Z. Cao (Tularik), and antiJNK1 monoclonal antibody was purchased from Pharmingen (San Diego, CA). Anti-TNF-R1 (985) monoclonal antibody has been described previously (Hsu et al., 1996b). Anti-HA matrix (3F10) was purchased from Roche Molecular Biochemicals (Indianapolis, IN), and HA peptides were synthesized by L. Huang (Tularik). Purified GSTc-Jun (1–79) proteins were products from Santa Cruz Biotechnology, and GST-IB␣ (1–54) proteins were provided by W. Liu (Tularik). Plasmids Expression vectors for Flag-tagged wild-type IKK␣ and IKK, and their kinase inactive (KA) mutants, have been described previously (Ling et al., 1998). pRK7-N-Flag-IKK␣ and pRK5-C-Flag-IKK constructs for expressing various Flag-tagged IKK deletions, as well as pGEX4T1-NEMO, pGEX4T1-IKK␣ (1–304), and pGEX4T1-IKK (1–316) for producing GST-NEMO and -IKK fusion proteins, were generated by PCR. The full-length Cdc37 cDNA was isolated from human HeLa Marathon-Ready cDNA library by PCR (Clontech, Palo Alto, CA) and further cloned into pVL1392 vector (Pharmingen) for generating recombinant baculovirus expressing HA-tagged Cdc37. NF-B-dependent (pNF-B5x-Luc) and AP1-dependent (pAP17x-Luc) firefly luciferase reporters were products from Stratagene. The Renilla luciferase reporter utilized to normalize transfection efficiency in reporter assays has been described (Chen et al., 1999). Transfection and Reporter Assays HeLa cell lines stably expressing Flag-IKKs were generated using standard methods. Transient transfection was performed as described previously (Regnier et al., 1997), except using the Effectene transfection reagent (Qiagen, Valencia, CA). Dual-luciferase assays were conducted with the kit from Promega. Luciferase activities were normalized to the control Renilla luciferase activities as described (Chen et al., 1999). For reporter assays in the GA-treated cells, HeLa cells were transfected with indicated reporters. 24 hr posttransfection, cells were incubated with 0.5 M of GA in serumfree medium for 15 hr before TNF or IL-1 stimulation. GA concentration was maintained at 0.5 M throughout stimulation. Protein Purification To purify the IKK complex, 40 liters of Flag-IKK HeLa cells were grown in suspension and harvested after cells were treated with or without TNF (100 ng/ml) for 10 minutes. Cell pellets were gently lysed in 10 volumes of the isotonic protein lysis buffer IP150 containing 50 mM HEPES (pH 7.6), 150 mM NaCl, 1 mM EDTA, 0.1% NP-40, 10%
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glycerol, 1 mM DTT, 0.5 mM PMSF, 20 mM -glycerophosphate, 20 mM p-nitrophenyl phosphate, 1 mM Na3VO4, and 1⫻ complete protease inhibitors (Roche Molecular Biochemicals). Cleared lysates were precipitated with anti-Flag M2 affinity gel at a volume ratio of 200:1. After extensive washing, beads were loaded onto a column, and bound proteins were eluted with 4-bead volumes of the IP150 buffer containing 0.3 mg/ml of Flag peptide. Flag eluates were fractionated with a Superose 6 HR 30/10 column connected to a FPLC system (Amersham Pharmacia, Piscataway, NJ). The column was precalibrated with standards blue dextran (2,000 kDa), thyroglobulin (670 kDa), and -amylase (200 kDa). Similarly, the endogenous IKK complex was isolated from untransfected HeLa cells using an antiIKK␣ affinity matrix (Santa Cruz Biotechnology). GST pull-down assays were performed as described previously (Chen et al., 1999). Mass Spectrometry Analysis Coomassie blue-stained protein bands of interest were excised from the gel, washed, and dehydrated. Dried gel slices were reduced with DTT, and free sulfhydryls were alkylated by iodoacetamide prior to in-gel digest with trypsin. Aliquots of tryptic peptides derived from each in-gel digest were spotted on the matrix-assisted laser desorption/ionization (MALDI) sample plate. Mass spectra were acquired using a Voyager-DE STR Biospectrometry Workstation (PerSeptive Biosystems, Framingham, MA), operated in reflectron mode with delayed extraction. Proteins were identified by comparing observed peptide mass fingerprints with those theoretically derived from NCBInr database using the Protein Prospector AutoMS-Fit algorithm. Immunoprecipitation and Kinase Assays For immunoprecipitation of overexpressed proteins, cells were transfected with 0.5 g of each indicated expression vector. 24 hr later, cells were lysed in the buffer IP150. Cleared cell extracts were incubated with 2 g of anti-Flag antibody and 20 l of protein G agarose beads (Upstate Biotechnology, Lake Placid, NY). For precipitating endogenous IKK complexes, 2 g of anti-NEMO (3F6) or isotype-matched monoclonal antibodies were used. For TNF-R1 immunoprecipitation, HeLa cells were induced with TNF (100 ng/ ml) for 5 min and lysed in the IP150 buffer containing 1% Triton X-100. Total cell extracts were prepared with the boiling SDS-PAGE sample buffer. For kinase assays, each immunopreciptate was incubated with or without 1 g of exogenous substrate GST-IB␣ (1–54) or GST-c-Jun (1–79) in 20 l of kinase buffer containing 20 mM HEPES (pH 7.6), 20 mM MgCl2, 20 mM -glycerophosphate, 1 mM DTT, 10 M ATP, and 5 Ci [␥-32P]ATP. Samples were separated by SDSPAGE, and 32P-labeled proteins were visualized by autoradiography. Acknowledgments We thank Zhaodan Cao, Xiong Gao, Lei Ling, Wei Liu, and Linda Huang for providing various reagents and Miki Rich for DNA sequencing. We are also grateful to Shyun Li for assistance with generating the HeLa Flag-IKK stable cell lines and to Xiaohong Liu for assistance with gel filtration chromatography. We thank Zhulun Wang and Nigel Walker for their help in the preparation of figures. Received June 1, 2001; revised November 12, 2001. References Cao, Z., Xiong, J., Takeuchi, M., Kurama, T., and Goeddel, D.V. (1996). TRAF6 is a signal transducer for interleukin-1. Nature 383, 443–446. Chen, Z.J., Parent, L., and Maniatis, T. (1996). Site-specific phosphorylation of IB␣ by a novel ubiquitination-dependent protein kinase activity. Cell 84, 853–862. Chen, G., Fernandez, J., Mische, S., and Courey, A.J. (1999). A functional interaction between the histone deacetylase Rpd3 and the corepressor Groucho in Drosophila development. Genes Dev. 13, 2218–2230. Csermely, P., Schnaider, T., Soti, C., Prohaszka, Z., and Nardai, G. (1998). The 90-kDa molecular chaperone family: structure, function,
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TNF-Induced Recruitment and Activation of the IKK Complex Require Cdc37 and Hsp90 Guoqing Chen, Ping Cao, and David V. Goeddel1 Tularik, Incorporated Two Corporate Drive South San Francisco, California 94080
Summary The IKK complex, containing two catalytic subunits IKK␣ and IKK and a regulatory subunit NEMO, plays central roles in signal-dependent activation of NF-B. We identify Cdc37 and Hsp90 as two additional components of the IKK complex. IKK␣/IKK/NEMO and Cdc37/Hsp90 form an ⵑ900 kDa heterocomplex, which is assembled via direct interactions of Cdc37 with Hsp90 and with the kinase domain of IKK␣/IKK. Geldanamycin (GA), an antitumor agent that disrupts the formation of this heterocomplex, prevents TNF-induced activation of IKK and NF-B. GA treatment reduces the size of the IKK complex and abolishes TNF-dependent recruitment of the IKK complex to TNF receptor 1 (TNF-R1). Therefore, heterocomplex formation with Cdc37/Hsp90 is a prerequisite for TNF-induced activation and trafficking of IKK from the cytoplasm to the membrane. Introduction NF-B plays prominent roles in inducible expression of genes involved in diverse biological processes, including development, immune, and inflammatory responses, cell growth and death, stress responses, and oncogenesis (for recent reviews, see Ghosh, 1999; Maniatis, 1999; Karin and Ben-Neriah, 2000; Karin and Delhase, 2000). In resting cells, the majority of NF-B is sequestered in the cytoplasm in complexes with a class of inhibitory proteins termed IB. Upon exposure of cells to a variety of extracellular stimuli, including tumor necrosis factor (TNF) and interleukin-1 (IL-1), IB proteins become phosphorylated, resulting in ubiquitination and degradation of IB. This allows NF-B to translocate into the nucleus, where it activates transcription of target genes. The protein kinase responsible for stimulus-dependent phosphorylation of IB has been identified using different approaches (DiDonato et al., 1997; Mercurio et al., 1997; Regnier et al., 1997; Woronicz et al., 1997; Zandi et al., 1997). Biochemical purification defines the IB kinase (IKK) activity as an ⵑ700–900 kDa protein complex, consisting of at least two catalytic subunits, IKK␣ and IKK, and a regulatory subunit, NEMO (NF-B essential modulator, also known as IKK␥/IKKAP1/FIP3) (Chen et al., 1996; Rothwarf et al., 1998; Yamaoka et al., 1998; Zandi et al., 1998; Li et al., 1999; Mercurio et al., 1999). IKK␣ and IKK are two highly homologous kinases, both containing a conserved N-terminal kinase domain and a C-terminal region with a leucine zipper (LZ) and a helixloop-helix (HLH) motif. While the LZ motif is responsible 1
Correspondence: [email protected]
for dimerization of IKK␣ and IKK, both the LZ and HLH motifs are important for modulating the kinase activity of IKK (Woronicz et al., 1997; Zandi et al., 1997, 1998; Delhase et al., 1999). NEMO contains multiple putative coiled-coil motifs and a potential zinc finger motif at the C terminus. NEMO is an essential component of the IKK complex because NEMO-deficient cells are unable to activate IKK or NF-B in response to a wide set of stimuli, including TNF and IL-1. The large 700–900 kDa IKK complex also does not form in cells lacking NEMO (Yamaoka et al., 1998). Accumulating evidence suggests that the IKK complex represents a converging point for transducing diverse upstream NF-B-activating stimuli (Karin, 1999a, 1999b). However, the detailed mechanism by which IKK is activated in response to various stimuli, in particular TNF and IL-1, remains unclear. A simple model for IKK activation may involve an upstream activating kinase, or so-called IKK kinase (IKK-K) (Mercurio and Manning, 1999; Israel, 2000). However, the identity of such an IKK-K, if it exists, remains elusive. Like the signal transducing molecules TRADD (Hsu et al., 1996b), RIP (Hsu et al., 1996a), and TRAF2 (Shu et al., 1996), the IKK complex is also recruited to TNF receptor 1 (TNF-R1) upon TNF induction (Devin et al., 2000; Zhang et al., 2000). Signal-dependent recruitment of IKK to TNF-R1 requires TRAF2, while RIP primarily mediates IKK activation. Interestingly, enforced oligomerization of RIP, NEMO, IKK␣, or IKK has been shown to be sufficient for inducing IKK and NF-B activation (Poyet et al., 2000). We have identified Cdc37 and Hsp90, which together form a kinase-specific chaperone, as additional components of the ⵑ900 kDa IKK complex. Geldanamycin (GA), an Hsp90 binding agent, reduces the size of the IKK complex by disrupting the association of IKK/NEMO with Cdc37/Hsp90. GA treatment also abolishes TNFdependent recruitment of the IKK complex to TNF-R1 and blocks TNF-induced activation of IKK. These results indicate that Cdc37 and Hsp90 play a physiological role in TNF-dependent translocation and activation of the IB kinases. Results Characterization of HeLa Flag-IKK Stable Cells To facilitate purification of the IKK complex, we generated various lines of HeLa cells that stably express either wild type (WT) or kinase-dead (KA mutant) forms of Flagtagged IKK␣ and IKK. We monitored both TNF- and IL-1-induced NF-B activation in these stable cells by transient transfection with a NF-B-dependent luciferase reporter (Figure 1A). In comparison with parental HeLa cells, HeLa cells stably expressing wild type IKK␣ (␣WT) demonstrated almost normal NF-B activation, while IKKWT stable cells (WT) displayed relatively higher basal and induced NF-B activities. In contrast, stable expression of kinase-inactive IKK␣ (␣KA) or IKK (KA) significantly reduced basal and cytokine-induced NF-B activities. In spite of these differences, each of
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Figure 1. Characterization of HeLa Cells Stably Expressing Flag-IKK (A) NF-B activation in Flag-IKK stable cells. Parental HeLa cells (HeLa) or cells that stably express Flag-tagged wild-type IKK␣ (␣WT), IKK (WT), or kinase-inactive IKK␣ (␣KA), IKK (KA) were cotransfected with an NF-B-dependent luciferase reporter and a control Renilla luciferase reporter. Reporter activities were measured 4 hr after cells were stimulated with TNF or IL-1. Relative luciferase activities were normalized to the control Renilla luciferase activities. Each bar is the average plus the standard deviation of three independent duplicate experiments. (B) IKK activation in Flag-IKK stable cells. Extracts from above HeLa and stable cell lines that had been treated with (⫹) or without (⫺) TNF were immunoprecipitated (IP) with anti-Flag antibodies. Each immunoprecipitate was analyzed by anti-Flag immunoblotting (IB) and in vitro kinase assay (KA) using GST-IB␣ (1–54) as the IKK substrate. Equal amounts of total cell extracts (TCE) from these cells were also immunoblotted with anti-Flag, anti-IKK␣/anti-IKK (IKK␣/), and anti-NEMO (served as a loading control) antibodies (lower three panels) to determine expression levels.
these HeLa cell lines exhibited roughly similar ratios (induced to basal) of NF-B induction by TNF or IL-1. These results suggest that in the cell lines examined here, stable expression of various Flag-IKKs exerts only minor effects on the endogenous NF-B signaling pathway. This behavior may be explained by the relatively low levels of overexpression of various Flag-IKK proteins, particularly in the ␣KA and KA cell lines, in comparison with the endogenous levels of IKK␣ and IKK (Figure 1B, lower three panels). In addition, immunoprecipitation and in vitro kinase assay revealed that the kinase activity of the Flag-IKK-containing complexes was TNF-inducible in all four cell lines (Figure 1B, upper 2 panels). The observed IKK activation by TNF in ␣KA and KA cells is likely due to the presence of endogenous IKK␣ and/or IKK in the Flag-IKK-containing complexes. Purification of the IKK Complex from Flag-IKK Stable Cells We next attempted to purify the IKK complex from the Flag-IKK stable cell lines. A representative purification of the IKK complex from the ␣KA cell line is illustrated in Figure 2. For each purification, we harvested 40 liters of cells grown in suspension after cells were treated with TNF or left untreated. Cells were then gently lysed under isotonic conditions using 0.1% nonionic detergent NP-40 to open the plasma membrane and release the cytosol. Cleared lysates were loaded onto an antiFlag affinity column. After extensive washing, bound proteins were eluted using Flag peptides. Aliquots of the eluates derived from TNF stimulated (⫹) or unstimulated (⫺) cells were separated by SDS-PAGE and visualized by Coomassie blue staining (Figure 2A). Similar polypeptide species were observed for all purifications.
To further purify the IKK complex, we fractionated the Flag eluates by gel filtration with a Superose 6 column (Figure 2B). Each fraction was then analyzed by immunoblotting with anti-IKK␣/anti-IKK (mix) and anti-NEMO antibodies and in vitro kinase assays to monitor TNFdependent IKK activation. The peak levels of NEMO and TNF-induced IKK activity coeluted in fraction 10 (marked by arrowheads), corresponding to an exclusion size of ⵑ900 kDa. This result is consistent with previous findings that IB kinase activity exists as a 700–900 kDa protein complex (Chen et al., 1996; DiDonato et al., 1997; Mercurio et al., 1997). To determine if known proteins implicated in NF-B signaling were present in our purified IKK complex, we immunoblotted these peak fractions with a panel of antibodies against TRADD (Hsu et al., 1995), RIP (Hsu et al., 1996a), TRAF2 (Hsu et al., 1996b), TRAF6 (Cao et al., 1996), I-TRAF (Rothe et al., 1996), MEKK1 (Mercurio et al., 1997), NIK (Malinin et al., 1997), AKT (Ozes et al., 1999), ACT1/CIKS (Leonardi et al., 2000; Li et al., 2000), MKP1 (Mercurio et al., 1997), and IB␣ (Mercurio et al., 1997). We failed to detect any of these molecules in the purified IKK complexes (data not shown). Purified IKK Complexes Contain Cdc37 and Hsp90 We combined fractions containing peak IKK activities (fractions 8 ⫹ 9 and 10 ⫹ 11) and resolved them by preparative SDS-PAGE (Figure 2C). Coomassie blue staining revealed almost identical polypeptides present in the TNF-stimulated and -unstimulated IKK complexes, except that a faint ⵑ60 kDa polypeptide (labeled p60) was detected only in the unstimulated complex. We excised all visible bands and digested them with trypsin. The tryptic peptide mixture from each in-gel digest was analyzed by mass spectrometry. The resulting peptide mass fingerprints were compared with
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Figure 2. Purification of the IKK Complex from Flag-IKK Stable Cells (A) A Coomassie blue-stained SDS-PAGE gel containing samples affinity purified from TNF-unstimulated (⫺) or -stimulated (⫹) ␣KA stable cells. Samples represent Flag peptide eluates from the anti-Flag affinity columns and were used as the starting material (SM) for further purification shown in (B) and (C). (B) Gel filtration chromatography to isolate the IKK complex. The Flag peptide eluates from (A) were further fractionated on a Superose 6 gel filtration column. Each fraction was analyzed by immunoblotting (IB) with anti-IKK␣/anti-IKK (IKK␣/) and anti-NEMO antibodies and in vitro kinase assays (KA) to monitor the IKK enzymatic activities. An anti-Flag immunoblotting was also performed, revealing a pattern very similar to that of anti-IKK (data not shown). Elution positions of protein standards run in a parallel experiment are indicated above corresponding fractions. Arrowheads mark the peaks of purified IKK complexes. (C) Identification of protein components of the purified IKK complexes. The peak fractions (8 ⫹ 9 and 10 ⫹ 11) from (B) were pooled, concentrated, and resolved by preparative SDS-PAGE. Coomassie blue-stained bands were excised, and their identities (as indicated on the right) were determined by peptide mass fingerprint mapping.
the theoretical tryptic peptide masses derived from the NCBInr database. This allowed us to unambiguously identify IKK␣ and NEMO in the purified complex (Figure 2C, right). IKK was not found in the purified complex by peptide mass fingerprint mapping, even though a small amount of IKK was indeed detected in this purified IKK complex by immunoblotting (data not shown). The presence of IKK explains why TNF-induced IKK activation was observed for the IKK complex purified from the ␣KA cell line (Figure 2B). These results also suggest that the majority of the IKK complex isolated from the ␣KA cell line is composed of overexpressed Flag-IKK␣ and endogenous NEMO. In parallel to these findings, complexes containing predominantly FlagIKK and NEMO were purified from the Flag-IKK stable cell lines (data not shown). In addition to IKK␣/IKK and NEMO, we consistently observed the presence of Hsp90, Cdc37, and -tubulin in the IKK complexes purified from different Flag-IKK stable cells (Figure 2 and data not shown). Cdc37 and Hsp90 have been previously shown to function as a kinase-specific chaperone (Csermely et al., 1998; Pratt et al., 1999). Coomassie blue staining revealed similar amounts of Cdc37, Hsp90, IKK, and NEMO present in the purified IKK complexes (Figure 2C and data not shown), suggesting that Cdc37/Hsp90 and IKK␣/IKK/ NEMO form a heterocomplex at a stoichiometric ratio of 1:1. Conversely, both -tubulin and p60 appear to represent substoichiometic components of the IKK complex. The in-gel digest of p60 generated a peptide mass fingerprint that failed to match any known protein in the database. Purification of the Endogenous IKK Complex We have verified the copurification of Cdc37 and Hsp90 with the IKK complexes isolated from the Flag-IKK sta-
ble cells by immunoblotting (data not shown). To exclude the possibility that this copurification is due to an artifact of Flag-IKK overexpression in these cells, we purified the endogenous IKK complex from nontransfected HeLa cells by anti-IKK␣ affinity chromatography. Gel filtration analysis of the purified endogenous IKK complex demonstrated that Cdc37/Hsp90 precisely coeluted with IKK␣, IKK, and NEMO at a peak corresponding to an exclusion size of 900 kDa (Figure 3A, arrowhead). Interestingly, Hsp90, Cdc37, and IKK␣/ were also present in some smaller molecular weight fractions, perhaps reflecting the IKK complexes that lacked NEMO and were associated with a different set of proteins, or that were disrupted during the fractionation process. Aliquots of fractions 9, 10, and 11 derived from the gel filtration column were resolved by SDS-PAGE and visualized by silver staining (Figure 3B). Similar amounts of Hsp90, IKK␣, IKK, Cdc37, and NEMO were detected in the peak fraction of the purified endogenous IKK complex (arrowhead). Thus, all our purification results provide evidence to strongly support the existence of a stoichiometric heterocomplex of IKK␣/IKK/NEMO and Cdc37/Hsp90 in the cell. Cdc37/Hsp90 Associates with the Kinase Domains of IKK The presence of Cdc37/Hsp90 in the IKK complex was further confirmed by an endogenous coimmunoprecipitation experiment (Figure 4A). Both Cdc37 and Hsp90 were coprecipitated from HeLa cell extracts with antiNEMO and anti-IKK antibodies, but not with a control antibody. In addition, the specific interaction of Cdc37/ Hsp90 with IKK/NEMO is independent of TNF stimulation. To further assess the stability of this interaction, we subjected equal amounts of anti-NEMO immunoprecipitates to extensive washes with the lysis buffer alone
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Figure 3. Purification of the Endogenous IKK Complex (A) Gel filtration analysis of the endogenous IKK complex purified from HeLa cells by antiIKK␣ affinity chromatography. Gel filtration was performed as in Figure 2B, and each fraction was analyzed by immunoblotting with anti-Hsp90, anti-IKK␣/anti-IKK (IKK␣/), anti-Cdc37, and anti-NEMO antibodies. The arrowhead denotes the peak of the purified endogenous IKK complex that contains IKK, NEMO, Cdc37, and Hsp90. (B) A silver stained SDS-PAGE gel containing aliquots of fractions 9, 10, and 11 from the gel filtration fractionation in (A). The identity of each polypeptide band in fraction 10 was determined by mass spectrometry (as indicated on the right). Asterisks denote protein bands that either nonspecifically bind the anti-IKK␣ affinity matrix or are absent in the peak of the IKK complex.
or the buffer containing 1 M NaCl, 1% Triton X-100, or 2 M urea (Figure 4B). Immunoblotting analyses showed that the association of IKK/NEMO with Cdc37/Hsp90 was vulnerable to high salt and denaturing agent washes, while it tolerated most detergent washes. This finding explains the earlier failure to identify Cdc37 and Hsp90 as components of the IKK complex (DiDonato et al., 1997; Mercurio et al., 1997), as these purifications
usually incorporated an initial step of stringent wash with high salt or denaturing agent. To map the regions of the IKKs that interact with Cdc37/ Hsp90, we transiently expressed various deletions of Flagtagged IKK␣ and IKK and immunoprecipitated cell extracts with anti-Flag antibodies. Endogenous Cdc37 and Hsp90 were detected in the immunoprecipitates containing the full-length Flag-IKK␣ and Flag-IKK (Figure Figure 4. Cdc37 Targets Hsp90 to the Kinase Domains of IKK
(A) Coimmunoprecipitation of endogenous IKK/NEMO with Cdc37/Hsp90. Extracts from TNF-unstimulated (⫺) or -stimulated (⫹) HeLa cells were immunoprecipitated (IP) with either anti-NEMO (NEMO), anti-IKK (IKK), or a control IgG1 (CTRL) monoclonal antibody. Each immunoprecipitate was immunoblotted (IB) with rabbit polyclonal antibodies against Hsp90, IKK␣/IKK (IKK␣/), Cdc37, and NEMO. (B) Stability of the interaction between IKK/ NEMO and Cdc37/Hsp90. Equal amounts of those anti-NEMO precipitates in (A) were washed extensively with the buffer alone (lane 1) or the buffer containing either 1.0 M NaCl (lane 2), 1% Triton X-100 (lane 3), or 2.0 M urea (lane 4) and subsequently analyzed by immunoblotting as shown in (A). Asterisks in both (A) and (B) mark those nonspecific bands. (C) Cdc37/Hsp90 interacts with the kinase domains of IKK. HeLa cells were transfected with vector alone or vectors expressing various deletions of Flag-tagged IKK␣ or IKK. Cell extracts were immunoprecipitated with anti-Flag antibodies, and coprecipitating Cdc37 and Hsp90 proteins were determined by immunoblotting with anti-Hsp90 and antiCdc37 antibodies. Expression levels of IKK␣ and IKK variants were monitored by immunoblotting with anti-Flag (biotinylated) antibody. (D) A direct interaction between Cdc37 and the kinase domains of IKK. Equal amounts of purified HA-Cdc37 (INPUT) were incubated with the glutathione beads containing GST alone, GST-IKK␣/IKK-kinase domain (KD), or GST-NEMO fusion proteins. After extensive washing, amounts of HA-Cdc37 retained on beads were determined by anti-HA immunoblotting. The bottom is a Coomassie blue (CBB)-stained SDS-polyacrylamide gel containing purified HA-Cdc37 and various GST fusion proteins.
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Figure 5. TNF-Induced Activation of NF-B and IKK Requires Cdc37/Hsp90 (A) Effects of GA treatment on the heterocomplex formation between IKK/NEMO and Cdc37/Hsp90. Extracts of HeLa cells that had been treated with DMSO or GA were immunoprecipitated (IP) with a control IgG1 (CTRL) or anti-NEMO (NEMO) monoclonal antibody. The presence of Hsp90, Cdc37, IKK␣/IKK, and NEMO in the immunocomplexes was determined by immunoblotting (IB) as described in Figure 4A, except that a goat anti-Cdc37 polyclonal antibody was used. (B) Effects of GA treatment on NF-B- and AP1-dependent transcription. HeLa cells were cotransfected with a NF-B- (left) or AP1-driven (right) luciferase reporter along with a control Renilla luciferase reporter. 24 hr later, cells were treated with DMSO or GA prior to TNF and IL-1 induction. Relative luciferase activities were measured as described in Figure 1A. (C) Effects of GA treatment on IKK activation. Following the pretreatment with DMSO or GA, HeLa cells were stimulated with (⫹) or without (⫺) TNF. The IKK complexes were isolated by anti-NEMO immunoprecipitation and further analyzed by in vitro kinase assays (KA) using GST-IB␣ (1–54) as the IKK substrate (bottom). The IKK protein level in each precipitate was also determined by immunoblotting. (D) Effects of GA treatment on JNK activation. Both immunopreciptation and in vitro kinase assay were performed essentially as described in (C), except that an anti-JNK1 monoclonal antibody and the GST-Jun (1–79) substrate were used for each assay.
4C). Furthermore, IKK deletions containing only the kinase domain were also capable of binding Cdc37 and Hsp90, while those without the kinase domain failed to do so. A direct interaction between the IKKs and Cdc37 was further ascertained by a GST pull-down assay (Figure 4D). Purified HA-tagged Cdc37 was retained on beads containing GST-IKK␣ or GST-IKK-kinase domain, but not on beads with GST alone (the faint band likely represents the shadow of overloaded input) or GST-NEMO. Given the high input, the binding between Cdc37 and IKK appears very weak. We found that Cdc37 interacts more strongly with IKK in the presence of Hsp90 and that purified Hsp90 by itself associates poorly with GST-IKK fusions (data not shown). This suggests a cooperative binding of Cdc37/Hsp90 to IKK. Thus, Hsp90 is targeted to the IKK complex via a direct interaction between Cdc37 and the catalytic region of IKK␣/. TNF-Induced Activation of IKK and NF-B Requires Cdc37/Hsp90 To examine the possible physiological role in TNF signaling of the association of IKK/NEMO with Cdc37/Hsp90, we employed an antitumor agent, geldanamycin (GA). GA was previously shown to interact and interfere with the normal functions of Hsp90 (Whitesell et al., 1994). Using anti-NEMO immunoprecipitation, we isolated the IKK complexes from HeLa cells that had been pretreated with either DMSO or GA (Figure 5A). Subsequent immu-
noblotting analysis showed that the treatment with GA almost completely disrupted the interaction between IKK/NEMO and Hsp90/Cdc37, but DMSO exhibited no detectable effect. Thus, GA provides a powerful tool to explore in vivo functions of the association of IKK/NEMO with Cdc37/Hsp90. We next studied the impact of GA treatment on NF-B activation using transient transfection with a NF-B-driven luciferase reporter. GA treatment not only potently blocked both TNF- and IL-1-induced NF-B activation, but also moderately inhibited (2- to 3-fold) the unstimulated NFB activity (Figure 5B, left). To determine the specificity of GA inhibition on NF-B activation, we performed a similar experiment with an AP1-dependent luciferase reporter (Figure 5B, right). In this case, GA treatment only partially inhibited TNF- and IL-1-induced AP1-mediated reporter activities. These effects were much less severe than those on NF-B activation. In addition, GA treatment had minimal effects on transcription of the internal control Renilla luciferase reporter (⬍2-fold). These results indicate that GA selectively impedes the NF-B activation pathway. We also performed in vitro immunocomplex kinase assays to measure the effects of GA treatment on TNFinduced IKK activation (Figure 5C). HeLa cells were treated with GA or DMSO prior to TNF induction, and in vitro kinase assays were performed on the immunoprecipitated IKK complexes using exogenous GST-IB␣ fusion proteins as substrates. TNF stimulation resulted
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Figure 6. Roles for Hsp90/Cdc37 in Complex Assembly and Receptor Recruitment of IKK (A and B) Gel filtration analysis of the IKK complexes. Equal amounts of total extracts from HeLa cells that had been treated with DMSO (A) or GA (B) were fractionated on a Superose 6 column as described in Figure 2B. The IKK complex sizes were monitored by immunoblotting (IB) analysis of each fraction with antibodies against IKK␣ and IKK (IKK␣/) and NEMO. (C) TNF-induced recruitment of IKK to TNF-R1 requires Hsp90/Cdc37. HeLa cells were treated with (⫹) or without (⫺) TNF after cells had been preincubated with DMSO or GA. Equal amounts of cell extracts were immunoprecipitated (IP) with anti-TNF-R1 (985) monoclonal antibodies. TNF-R1 and associated IKK, Hsp90, RIP, TRAF2, and TRADD were detected by immunoblotting with the indicated polyclonal antibodies (right panels). Total cell extracts (TCE) were also prepared and immunoblotted in the same way to monitor the effects of GA treatment on cellular levels of these proteins (left panels). Asterisks indicate the protein bands that are nonspecifically recognized by respective antibodies. Brackets mark the higher molecular weight species of RIP and TRAF2 that become visible only after TNF stimulation.
in a dramatic increase of IKK enzymatic activities. However, the pretreatment with GA almost completely abolished this robust activation of IKK in response to TNF. As a control, we also investigated if GA treatment might affect c-Jun N-terminal kinase 1 (JNK1) activation by TNF (Figure 5D). Similar to the DMSO control, GA exhibited almost no effect on TNF-induced JNK1 activation, even though the unstimulated JNK1 activity was slightly reduced by GA treatment. In addition, GA had been previously shown to block TNF-induced degradation of IB, indicating that GA acts on this arm of NF-B response (Lewis et al., 2000). These results together demonstrate that the specific formation of a heterocomplex between IKK/NEMO and Cdc37/Hsp90 is essential for TNF-induced activation of IKK and NF-B. Role for Cdc37/Hsp90 in Assembly of the IKK Complex To investigate if GA-mediated disruption of the interaction between IKK/NEMO and Cdc37/Hsp90 might affect the formation and composition of the IKK complex, we fractionated equal amounts of extracts from HeLa cells that had been treated with DMSO or GA by gel filtration. Immunoblotting revealed that the peak of the IKK complex in GA-treated cells (Figure 6B, fraction 12) eluted with an exclusion size smaller than in DMSO-treated cells (Figure 6A, fraction 11). Similarly, GA treatment causes a reduction in size of the purified IKK complex that lacks Cdc37/Hsp90 (data not shown). However, no apparent effect was observed on the IKK activity when GA was directly added to the in vitro kinase assays with the purified IKK complexes (data not shown). This suggests that GA blocks the process of IKK activation by TNF rather than inhibits the IKK enzymatic activity per se. GA treatment also resulted in the presence of
significantly increased amounts of NEMO in the lower molecular weight fractions in comparison with those from DMSO-treated cells (compare fractions 15–17 in Figures 6A and 6B). The dramatic effects of GA on the size of the endogenous IKK complex and the redistribution of IKK␣/ and NEMO demonstrate a critical role for Cdc37/Hsp90 in the assembly of functional IKK complexes under physiological conditions. TNF-Induced Recruitment of IKK to TNF-R1 Requires Cdc37/Hsp90 We also examined if Cdc37/Hsp90 plays a role in shuttling the IKK complex from the cytoplasm to the plasma membrane. HeLa cells were treated with DMSO or GA and then briefly stimulated with or without TNF. TNFR1-associated signaling complexes were isolated from these cells by immunoprecipitation using a monoclonal antibody against the extracellular domain of TNF-R1. Immunoblotting demonstrated TNF-dependent recruitment of the signaling molecules TRADD, RIP, and TRAF2 to TNF-R1 in DMSO-treated cells (Figure 6C, lanes 5 and 6). Additionally, IKK and Hsp90 were also recruited to TNF-R1 after TNF stimulation, confirming that the IKK/NEMO-Cdc37/Hsp90 heterocomplex is recruited to TNF-R1 in a ligand-dependent manner. In GA-treated cells, recruitment of TRAF2 and TRADD to TNF-R1 appeared unchanged (Figure 6C, lanes 7 and 8). However, GA treatment almost fully abrogated TNF-dependent recruitment of IKK, Hsp90, and RIP molecules to TNF-R1. To determine the effects of GA treatment on expression levels of these proteins, we prepared total cell extracts and immunoblotted them with corresponding antibodies (Figure 6C, left panels). GA treatment had little or no effect on the cellular levels of IKK, TRAF2,
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plex in response to TNF. These results reveal a physiological role for the Cdc37/Hsp90 chaperone in transducing NF-B activating signals.
Figure 7. Kinetics of GA-Mediated Inhibition of IKK Recruitment to TNF-R1 and Complex Formation with Cdc37/Hsp90 Equal numbers of HeLa cells were treated with GA for the indicated times (0–10 hr) and then stimulated with TNF for 10 min. Aliquots of extracts prepared from these cells were immunoprecipitated (IP) with either anti-TNF-R1 (A) or anti-NEMO (B) monoclonal antibodies. Anti-TNF-R1 immunoprecipitates were further analyzed by immunoblotting (IB) with anti-IKK and anti-TRADD polyclonal antibodies (A), while anti-NEMO immunoprecipitates were immunoblotted with anti-IKK and anti-Cdc37 polyclonal antibodies (B).
TRADD, and TNF-R1 proteins, while it modestly upregulated Hsp90 expression. In contrast, RIP expression levels were greatly diminished in GA-treated cells, suggesting a role for Cdc37/Hsp90 in stabilizing RIP. This is in an agreement with a previous report that RIP is also an Hsp90-associated kinase and GA treatment selectively causes RIP degradation (Lewis et al., 2000). Thus, the defective recruitment of RIP to TNF-R1 in GAtreated cells could be due simply to the rapid turnover of RIP when it is dissociated from Hsp90. However, RIP is not required for IKK recruitment to TNF-R1 (Devin et al., 2000), and GA treatment has no obvious effect on the levels of IKK and NEMO proteins. In addition, time course experiments indicate that the kinetics of GAinduced inhibition of IKK recruitment to TNF-R1 correlate with those of GA-mediated disruption of the IKKCdc37/Hsp90 complex (Figure 7). GA treatment for 5 hr significantly reduced the amount of IKK recruited to TNF-R1 and the amount of Cdc37 associated with IKK, whereas 10 hr treatment almost completely abolished both interactions. Taken together, these results support an important role for Cdc37/Hsp90 in trafficking the IKK complex from the cytoplasm to the membrane. It is also possible that Cdc37/Hsp90 may be required for the interaction of the IKK complex with components of the upstream TNF-R1 signaling complex. Discussion We report the purification and identification of Cdc37 and Hsp90 as additional components of the IKK complex. Several lines of evidence argue that the formation of a heterocomplex with Cdc37/Hsp90 is essential for assembly, translocation, and activation of the IKK com-
Functions of Cdc37/Hsp90 in IKK Activation Hsp90, one of most abundant cytosolic proteins in eukaryotic cells, plays an important role in the maturation, activation, and trafficking of proteins involved in signal transduction, cell cycle control, development, and transcriptional regulation (Csermely et al., 1998; Pratt et al., 1999). Unlike the more general Hsp70 and Hsp60 chaperones, Hsp90 appears to be a dedicated chaperone for only a limited number of client proteins, including steroid hormone receptors and protein kinases. Hsp90 functions in concert with a set of cochaperones that link Hsp90 to distinct classes of client proteins. For example, the mammalian homolog of the yeast cell cycle control protein Cdc37 acts as a kinase-specific targeting subunit for Hsp90 (Dai et al., 1996; Stepanova et al., 1996). A growing list of kinases, including Src family tyrosine kinases and the serine/threonine kinases Raf and MEK, are known to exist as heterocomplexes with Cdc37/ Hsp90. Cdc37/Hsp90 contributes to the stabilization, activation, and/or translocation of these kinases. IKK␣ and IKK can now be added to the list of kinases whose functions and activities are dependent on Cdc37/ Hsp90. We find that the Hsp90 chaperone has no apparent effect on the stability of IKK. In contrast, Hsp90 affects the integrity of RIP, another important kinase in the TNF/NF-B signaling pathway. This is in an agreement with a previous study showing that GA selectively induces degradation of RIP and blocks TNF-induced NF-B activation (Lewis et al., 2000). IKK␣, IKK, and NEMO are recruited to TNF-R1 following TNF stimulation (Devin et al., 2000; Zhang et al., 2000). Our current study extends these findings by revealing that Cdc37/Hsp90 is part of the IKK complex and is also recruited to TNFR1 in a stimulus-dependent manner. Given that RIP is dispensable for IKK recruitment to TNF-R1, our results reveal a critical role for Cdc37/Hsp90 in shuttling IKK to the membrane. GA treatment causes the dissociation of Cdc37/ Hsp90 from the IKK complex, a reduction in size of the IKK complex, and the inhibition of TNF-induced activation of IKK and NF-B. These findings point out a physiological role for Cdc37/Hsp90 in the TNF/NF-B signaling pathway. The potent blockade of NF-B signaling by GA is likely due to multiple effects on the pathway. First, GA disrupts the interaction of IKK with Cdc37/Hsp90 and prevents the recruitment of IKK to the membrane. Second, GA treatment causes degradation of RIP, resulting in the assembly of a TNF-R1 signaling complex lacking RIP and defective in IKK activation. It is possible that Cdc37/Hsp90 may also provide a link to components of the upstream TNF-R1 complex, allowing the recruitment of IKK to the receptor. For example, both RIP and IKK associate with Cdc37/Hsp90, which may serve as a bridging factor to bring them into proximity. Interestingly, a protein highly homologous to Hsp90 has been proposed to directly bind the intracellular region of TNF-R1 (Song et al., 1995).
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Stoichiometry of the IKK-Cdc37/Hsp90 Complex Our gel filtration analysis has revealed that the endogenous IKK complex exists as an ⵑ900 kDa heterocomplex containing Cdc37/Hsp90. The size is consistent with that reported previously for the native IKK complex isolated from HeLa cells (DiDonato et al., 1997; Mercurio et al., 1997). While the native IKK complex contains a heterodimer of IKK␣ and IKK (Mercurio et al., 1997; Rothwarf et al., 1998; Woronicz et al., 1997), purified recombinant IKK␣ or IKK exists as a homodimer with a native molecular weight (MW) of 230–250 kDa (Zandi et al., 1998). Hsp90 is also a dimeric protein with a native MW of ⵑ250 kDa (Pratt et al., 1999). Cdc37 has a size of 130–150 kDa under native conditions, suggesting that it is a dimer or trimer (Kimura et al., 1997). NEMO is also known to form a dimer or trimer (Rothwarf et al., 1998). Our purifications suggest that there are roughly equal amounts of IKK, NEMO, Cdc37, and Hsp90 in the purified IKK complex. Based on these data, we predict that the complete IKK complex should contain a heterodimer of IKK␣ and IKK, a homodimer of Hsp90, and two to three molecules of NEMO and Cdc37. Such a complex would have an estimated MW of ⵑ800 kDa, matching the size of the native IKK complex and providing further evidence for that Cdc37 and Hsp90 are bona fide subunits of the IKK complex. Mechanisms of IKK Activation Although this study and others have shown a correlation between the recruitment of IKK to TNF-R1 and the activation of IKK, the exact mechanism of IKK activation is still an enigma. It is plausible that the recruitment of the IKK complex to TNF-R1 may represent an initial step. Once recruited and activated, IKK may be released from the receptor, reentering the cytoplasm and in trans activating residual inactive IKK molecules. It is possible that, like many signaling molecules, IKK may traverse the cytoplasm simply by diffusion and become trapped at the sites of action by protein-protein interactions (e.g., those involved in the formation of TNF-R1 signaling complexes at the internal surface of the membrane). It is also possible that the IKK complex may move through the cytoplasm via a transport system involving Cdc37/ Hsp90, even though nothing is known about how this movement is initiated or directed to the cell membrane. RIP plays an essential role in TNF-mediated IKK activation (Devin et al., 2000; Hsu et al., 1996a; Kelliher et al., 1998; Ting et al., 1996; Zhang et al., 2000). Following the recruitment of IKK to TNF-R1, therefore, a RIPdependent process or factor may directly trigger IKK activation. One possibility is that an upstream kinase (e.g., IKK-K) is brought to the receptor complex and activated in a RIP-dependent manner. It has been shown that enforced oligomerization of RIP, NEMO, IKK␣, or IKK is sufficient for inducing IKK activation (Poyet et al., 2000). These results suggest that such an intermediate factor or IKK-K may not be necessary for IKK activation. RIP itself may cause some conformational changes of the IKK complex, resulting in IKK activation. However, it will be difficult to test this hypothesis under physiological conditions. To fully understand the mechanism of TNF-mediated IKK activation, it will be crucial to determine which activ-
ity in the TNF-R1 signaling complex is directly responsible for activating IKK. The diversity of NF-B stimuli forecasts the existence of different upstream activators involved in signal-dependent IKK activation. It is thus reasonable to postulate that each of these NF-B signaling pathways may utilize a pathway-specific factor to directly trigger IKK activation. To date, however, most of these putative direct activators of IKK have not been identified. Determining if the IKK complex contains additional components, in particular substoichiometric and/ or transiently associated subunits, will improve our understanding of the mechanism of IKK activation as well as the specificity and diversity of NF-B signaling. Experimental Procedures Reagents Recombinant human TNF and IL-1 were obtained from Genentech (South San Francisco, CA) and BioSource International (Camarillo, CA), respectively. Geldanamycin (GA), anti-Flag M2 affinity gel, Flag peptide, anti-Flag M2, and biotinylated anti-Flag M2 monoclonal antibodies were purchased from Sigma. Rabbit anti-IKK␣, anti-IKK, anti-NEMO, anti-Hsp90, anti-TRAF2, anti-HA, anti-TRADD, antiTNF-R1, and anti-RIP polyclonal antibodies as well as goat antiCdc37 polyclonal antibody were products from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit polyclonal anti-Cdc37 antibody was purchased from StressGen (Victoria, BC, Canada). Anti-NEMO (3F6) monoclonal antibody was provided by Z. Cao (Tularik), and antiJNK1 monoclonal antibody was purchased from Pharmingen (San Diego, CA). Anti-TNF-R1 (985) monoclonal antibody has been described previously (Hsu et al., 1996b). Anti-HA matrix (3F10) was purchased from Roche Molecular Biochemicals (Indianapolis, IN), and HA peptides were synthesized by L. Huang (Tularik). Purified GSTc-Jun (1–79) proteins were products from Santa Cruz Biotechnology, and GST-IB␣ (1–54) proteins were provided by W. Liu (Tularik). Plasmids Expression vectors for Flag-tagged wild-type IKK␣ and IKK, and their kinase inactive (KA) mutants, have been described previously (Ling et al., 1998). pRK7-N-Flag-IKK␣ and pRK5-C-Flag-IKK constructs for expressing various Flag-tagged IKK deletions, as well as pGEX4T1-NEMO, pGEX4T1-IKK␣ (1–304), and pGEX4T1-IKK (1–316) for producing GST-NEMO and -IKK fusion proteins, were generated by PCR. The full-length Cdc37 cDNA was isolated from human HeLa Marathon-Ready cDNA library by PCR (Clontech, Palo Alto, CA) and further cloned into pVL1392 vector (Pharmingen) for generating recombinant baculovirus expressing HA-tagged Cdc37. NF-B-dependent (pNF-B5x-Luc) and AP1-dependent (pAP17x-Luc) firefly luciferase reporters were products from Stratagene. The Renilla luciferase reporter utilized to normalize transfection efficiency in reporter assays has been described (Chen et al., 1999). Transfection and Reporter Assays HeLa cell lines stably expressing Flag-IKKs were generated using standard methods. Transient transfection was performed as described previously (Regnier et al., 1997), except using the Effectene transfection reagent (Qiagen, Valencia, CA). Dual-luciferase assays were conducted with the kit from Promega. Luciferase activities were normalized to the control Renilla luciferase activities as described (Chen et al., 1999). For reporter assays in the GA-treated cells, HeLa cells were transfected with indicated reporters. 24 hr posttransfection, cells were incubated with 0.5 M of GA in serumfree medium for 15 hr before TNF or IL-1 stimulation. GA concentration was maintained at 0.5 M throughout stimulation. Protein Purification To purify the IKK complex, 40 liters of Flag-IKK HeLa cells were grown in suspension and harvested after cells were treated with or without TNF (100 ng/ml) for 10 minutes. Cell pellets were gently lysed in 10 volumes of the isotonic protein lysis buffer IP150 containing 50 mM HEPES (pH 7.6), 150 mM NaCl, 1 mM EDTA, 0.1% NP-40, 10%
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glycerol, 1 mM DTT, 0.5 mM PMSF, 20 mM -glycerophosphate, 20 mM p-nitrophenyl phosphate, 1 mM Na3VO4, and 1⫻ complete protease inhibitors (Roche Molecular Biochemicals). Cleared lysates were precipitated with anti-Flag M2 affinity gel at a volume ratio of 200:1. After extensive washing, beads were loaded onto a column, and bound proteins were eluted with 4-bead volumes of the IP150 buffer containing 0.3 mg/ml of Flag peptide. Flag eluates were fractionated with a Superose 6 HR 30/10 column connected to a FPLC system (Amersham Pharmacia, Piscataway, NJ). The column was precalibrated with standards blue dextran (2,000 kDa), thyroglobulin (670 kDa), and -amylase (200 kDa). Similarly, the endogenous IKK complex was isolated from untransfected HeLa cells using an antiIKK␣ affinity matrix (Santa Cruz Biotechnology). GST pull-down assays were performed as described previously (Chen et al., 1999). Mass Spectrometry Analysis Coomassie blue-stained protein bands of interest were excised from the gel, washed, and dehydrated. Dried gel slices were reduced with DTT, and free sulfhydryls were alkylated by iodoacetamide prior to in-gel digest with trypsin. Aliquots of tryptic peptides derived from each in-gel digest were spotted on the matrix-assisted laser desorption/ionization (MALDI) sample plate. Mass spectra were acquired using a Voyager-DE STR Biospectrometry Workstation (PerSeptive Biosystems, Framingham, MA), operated in reflectron mode with delayed extraction. Proteins were identified by comparing observed peptide mass fingerprints with those theoretically derived from NCBInr database using the Protein Prospector AutoMS-Fit algorithm. Immunoprecipitation and Kinase Assays For immunoprecipitation of overexpressed proteins, cells were transfected with 0.5 g of each indicated expression vector. 24 hr later, cells were lysed in the buffer IP150. Cleared cell extracts were incubated with 2 g of anti-Flag antibody and 20 l of protein G agarose beads (Upstate Biotechnology, Lake Placid, NY). For precipitating endogenous IKK complexes, 2 g of anti-NEMO (3F6) or isotype-matched monoclonal antibodies were used. For TNF-R1 immunoprecipitation, HeLa cells were induced with TNF (100 ng/ ml) for 5 min and lysed in the IP150 buffer containing 1% Triton X-100. Total cell extracts were prepared with the boiling SDS-PAGE sample buffer. For kinase assays, each immunopreciptate was incubated with or without 1 g of exogenous substrate GST-IB␣ (1–54) or GST-c-Jun (1–79) in 20 l of kinase buffer containing 20 mM HEPES (pH 7.6), 20 mM MgCl2, 20 mM -glycerophosphate, 1 mM DTT, 10 M ATP, and 5 Ci [␥-32P]ATP. Samples were separated by SDSPAGE, and 32P-labeled proteins were visualized by autoradiography. Acknowledgments We thank Zhaodan Cao, Xiong Gao, Lei Ling, Wei Liu, and Linda Huang for providing various reagents and Miki Rich for DNA sequencing. We are also grateful to Shyun Li for assistance with generating the HeLa Flag-IKK stable cell lines and to Xiaohong Liu for assistance with gel filtration chromatography. We thank Zhulun Wang and Nigel Walker for their help in the preparation of figures. Received June 1, 2001; revised November 12, 2001. References Cao, Z., Xiong, J., Takeuchi, M., Kurama, T., and Goeddel, D.V. (1996). TRAF6 is a signal transducer for interleukin-1. Nature 383, 443–446. Chen, Z.J., Parent, L., and Maniatis, T. (1996). Site-specific phosphorylation of IB␣ by a novel ubiquitination-dependent protein kinase activity. Cell 84, 853–862. Chen, G., Fernandez, J., Mische, S., and Courey, A.J. (1999). A functional interaction between the histone deacetylase Rpd3 and the corepressor Groucho in Drosophila development. Genes Dev. 13, 2218–2230. Csermely, P., Schnaider, T., Soti, C., Prohaszka, Z., and Nardai, G. (1998). The 90-kDa molecular chaperone family: structure, function,
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