The K48-K63 Branched Ubiquitin Chain Regulates NF-kB Signaling

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1 Article The K48-K63 Branched Ubiquitin Chain Regulates NF-kB Signaling Graphical Abstract Authors Fumiaki Ohtake, Yasushi Saeki, Satoshi Ishido, Jun Kanno, Keiji Tanaka Correspondence (F.O.), (K.T.) In Brief Functional significance of branched polyubiquitin chains is poorly understood. Ohtake et al. identified polyubiquitin chains branched at lysine 48 (K48) and K63. The K48-K63 branched chain acts as a unique coding signal that specifically affects recognition by downstream reader proteins to enhance NF-kB signaling, representing the crosstalk of the ubiquitin code. Highlights d Ubiquitin chains branched at lysine 48 (K48) and K63 are abundant in mammalian cells d d d The E3 ubiquitin ligase HUWE1 cooperates with TRAF6 to assemble branched chains The K48-K63 branched chains formed in response to IL1b amplify NF-kB signaling The K48-K63 branch protects K63 linkages from CYLDmediated deubiquitylation Ohtake et al., 2016, Molecular Cell 64, October 20, 2016 ª 2016 Elsevier Inc.

2 Molecular Cell Article The K48-K63 Branched Ubiquitin Chain Regulates NF-kB Signaling Fumiaki Ohtake, 1,2,4, * Yasushi Saeki, 1 Satoshi Ishido, 3 Jun Kanno, 2 and Keiji Tanaka 1, * 1 Laboratory of Protein Metabolism, Tokyo Metropolitan Institute of Medical Science, Tokyo , Japan 2 Division of Cellular and Molecular Toxicology, Biological Safety Research Center, National Institute of Health Sciences, Tokyo , Japan 3 Department of Microbiology, Hyogo College of Medicine, Nishinomiya, Hyogo , Japan 4 Lead Contact *Correspondence: otake-fm@igakuken.or.jp (F.O.), tanaka-kj@igakuken.or.jp (K.T.) SUMMARY Polyubiquitin chains of different topologies regulate diverse cellular processes. K48- and K63-linked chains, the two most abundant chain types, regulate proteolytic and signaling pathways, respectively. Although recent studies reported important roles for heterogeneous chains, the functions of branched ubiquitin chains remain unclear. Here, we show that the ubiquitin chain branched at K48 and K63 regulates nuclear factor kb (NF-kB) signaling. A massspectrometry-based quantification strategy revealed that K48-K63 branched ubiquitin linkages are abundant in cells. In response to interleukin-1b, the E3 ubiquitin ligase HUWE1 generates K48 branches on K63 chains formed by TRAF6, yielding K48-K63 branched chains. The K48-K63 branched linkage permits recognition by TAB2 but protects K63 linkages from CYLD-mediated deubiquitylation, thereby amplifying NF-kB signals. These results reveal a previously unappreciated cooperation between K48 and K63 linkages that generates a unique coding signal: ubiquitin chain branching differentially controls readout of the ubiquitin code by specific reader and eraser proteins to activate NF-kB signaling. INTRODUCTION Ubiquitylation is a versatile post-translational modification with diverse cellular roles. Ubiquitin is conjugated to substrate proteins via a cascade of E1, E2, and E3 enzymes and removed by deubiquitylation enzymes (DUBs) (Hershko and Ciechanover, 1998). The functional diversity of this modification is based on the distinct topologies of various types of ubiquitylation. Substrates are modified either by monoubiquitin or polyubiquitin. Ubiquitin can be linked through any of seven lysine residues or the first methionine, yielding eight potential types of homogeneous polyubiquitin chains (Husnjak and Dikic, 2012). Among them, K48- and K63-linked polyubiquitin chains (hereinafter, the K48 chain and K63 chain, respectively) are the two most abundant polyubiquitin chain types. K48 chains tag substrates for proteasomal degradation, whereas K63 chains act as proteasome-independent signals for endocytosis, DNA damage responses, and immune responses. Other chains also regulate specific processes: linear (M1) chains regulate nuclear factor k B (NF-kB) activation, whereas K11 and K29 chains promote proteasomal degradation. Collectively, these diverse functions of ubiquitylation are referred to as the ubiquitin code (Komander and Rape, 2012). The ubiquitin code is interpreted via specific recognition of ubiquitin chain linkages by reader proteins containing various types of ubiquitin-binding domains (UBDs) (Husnjak and Dikic, 2012; Komander and Rape, 2012). The NZF domain of TAB2 specifically recognizes K63-linked diubiquitin through interaction with the Ile44-containing hydrophobic patches of both the proximal and distal ubiquitin moieties (Kulathu et al., 2009; Sato et al., 2009). Similarly, the UBAN domain of NEMO (also known as IKKg) binds with high affinity to M1- linked diubiquitin through interactions with the Ile44 and Phe4 patches. Certain DUBs also recognize specific chain linkages for disassembly of chains and signal termination. In addition, E2s/E3s noncovalently associate with ubiquitin for assembly of specific chains. NF-kB is a central regulator of a wide range of biological pathways, including immune and inflammatory responses, cell survival, and senescence (Wertz and Dixit, 2010; Zinngrebe et al., 2014). Activation of NF-kB involves at least three types of ubiquitin chains. In response to the proinflammatory cytokine interleukin-1b (IL1b) or activators of Toll-like receptors (TLRs), a complex of adaptor proteins containing MyD88, IRAK1, IRAK2, and IRAK4 is formed, and the RING-type E3 ubiquitin ligase TRAF6 assembles K63 chains in cooperation with UBC13-UEV1A, an E2 complex specific for the K63 linkage (Deng et al., 2000). In addition, M1 chains are assembled by LUBAC to substrates such as NEMO and IRAK1. Subsequently, the TAB2/3 subunit of the TAK1 complex and the NEMO subunit of the IKK complex specifically recognize K63 and M1 linkages, respectively, to form signaling complexes. This, in turn, induces phosphorylation and activation of IKK, resulting in the phosphorylation of inhibitor of NF-kB (IkB) for subsequent K48-linked polyubiquitylation and degradation, a critical event in releasing active NF-kB for gene regulation. NF-kB activation is tightly controlled within cells; negative regulators include K63/M1-specific DUB CYLD; the Molecular Cell 64, , October 20, 2016 ª 2016 Elsevier Inc. 251

3 Figure 1. Identification of K48-K63 Branched Ubiquitin Chains (A) Relative amounts of eight types of polyubiquitin linkages in whole-cell lysates from untreated U2OS_shUb cells. Data are derived from lane 1 of Figure 2D. (B) Schematic representation of heterogenous ubiquitin chains. Branched, mixed, and multiple chains are indistinguishable in mass-spectrometric analysis. (C) The ubiquitin R54A mutation enables identification and quantification of K48-K63 branched linkages, unbranched K48 linkages (not branched at K63), and unbranched K63 linkages (not branched at K48). (D) Point mutations in R54 do not affect abundance of K48 or K63 chains in cells. 293F cells were transfected with the indicated FLAG-Ub derivatives and immunoprecipitated using anti-flag antibody. IP, immunoprecipitation. (E) Identification of K48-K63 branched ubiquitin chains. 293F cells were transfected with FLAG-Ub(R54A), and anti-flag immunoprecipitants were subjected to mass-spectrometric analysis. The MS/MS spectrum corresponding to the signature peptide derived from K48-K63 branched linkages (aa 43 72, GlyGly modified at K48 and K63) was obtained. (legend continued on next page) 252 Molecular Cell 64, , October 20, 2016

4 M1-specific DUB OTULIN; and A20, which can inhibit NF-kB signaling in catalytic and non-catalytic manners (Wertz and Dixit, 2010). Ubiquitin chain topologies are classified into four types of architectures: homogeneous chains, multiple chains in which one substrate is separately modified by distinct chains, mixed chains in which a tandem chain contains two linkage types, and branched chains (Komander and Rape, 2012). In addition to the best studied homogeneous chains, recent studies characterized the roles of mixed and multiple chains. Mixed chains composed of two proteasome-related linkages (K29 and K48) or two NF-kB-related linkages (M1 and K63) are formed during the UFD pathway and NF-kB activation, respectively (Emmerich et al., 2013; Johnson et al., 1995), and multiple short chains on a substrate serve as a proteasomal degradation signal (Swatek and Komander, 2016). By contrast, the nature of branched ubiquitin chains is largely unknown. Ubiquitin chains of different linkages can be branched by conjugation at two or more acceptor sites within a single ubiquitin moiety (Peng et al., 2003). In vitro, branched chains containing multiple linkages can inhibit proteasomal degradation (Kim et al., 2007) and stabilize substrates (Ben-Saadon et al., 2006). In mammalian cells, the anaphase-promoting complex/cyclosome (APC/C) assembles branched chains containing the K11 linkage, which promotes proteasomal degradation through increasing the density of ubiquitin signals (Meyer and Rape, 2014). However, few studies, to date, have characterized such branched ubiquitin chains. A major difficulty in the characterization of branched ubiquitin chains is the lack of techniques for their accurate identification and quantification (Ben-Saadon et al., 2006; Swatek and Komander, 2016). Hence, knowledge of the cellular abundance and functional importance of branched ubiquitin chains remains scarce. Despite characterization of a specific E3 (APC/C), the global abundance of branched chains in cells is unknown. In addition, a previous study suggested that cells contain factors that actively prevent the formation of branched chains (Kim et al., 2007). Furthermore, it is unclear whether a branched linkage acts as the simple addition of two distinct constituent signals or as a unique coding signal that specifically affects recognition by downstream reader proteins. In this study, we developed a mass-spectrometry-based strategy for the identification and quantification of K48-K63 branched ubiquitin linkages. We found that the K48-K63 branched linkage is abundant in mammalian cells. Moreover, the E3 ubiquitin ligase HUWE1 generates K48 branches on K63 chains pre-formed by TRAF6 to assemble K48-K63 branched chains, which regulate NF-kB signaling. These findings establish the K48-K63 branched chain as a unique type of ubiquitin modification that is functionally distinct from mixed or multiple chains, further expanding our knowledge of the diversity of the ubiquitin code. RESULTS Identification of K48-K63 Branched Ubiquitin Chains To initiate our characterization of branched chains, we focused on putative ubiquitin chains branched at K48 and K63 for two reasons. First, K48 and K63 chains are the two most abundant (Figure 1A) and functionally significant linkages. Second, in contrast to other atypical chains implicated in proteasomal degradation, the individual modifications are functionally distinct (Komander and Rape, 2012). First, we developed a system for unambiguously identifying branched ubiquitin linkages. Heterogeneous chains can be branched chains, mixed chains, or multiple chains (Figure 1B). These alternatives are, in principle, hard to distinguish by conventional methods. Therefore, we decided to utilize mass-spectrometric analysis. In general, specific ubiquitin linkages can be identified as trypsin-digested peptides containing the GlyGly-modified lysine at a specific residue (Ordureau et al., 2015; Peng et al., 2003; Phu et al., 2011). However, for ubiquitin chains branched at K48 and K63, the ubiquitin moiety is cleaved at arginine-54 (Arg54), generating separate signatures of the K48 and K63 linkages (Figure S1A). Therefore, even if the ubiquitin chain is branched at K48 and K63, we cannot distinguish whether the observed signature peptides are derived from branched chains, mixed chains, or multiple chains. However, we noticed that ubiquitin, in which Arg54 was mutated to Ala, would generate a trypsinized peptide containing both GlyGly-modified K48 and K63 from branched chains (Figure 1C). Theoretically, the K48-K63 branched linkage, K63 linkage not branched at K48 (unbranched K63 linkage), and K48 linkage not branched at K63 (unbranched K48 linkage) (Figure 1C) can be distinguished using R54A mutant ubiquitin. A genetic analysis in yeast demonstrated that the R54A mutant of ubiquitin does not confer defects in cell growth, heat and cold sensitivity, or endocytosis (Sloper-Mould et al., 2001). Indeed, a comprehensive analysis revealed that no point mutation at Arg54 (i.e., replacement of Arg with any residue) causes a significant growth defect (Roscoe et al., 2013). Meyer and Rape (2014) inserted a TEV (Tobacco etch virus)-cleavage site between Glu53 and Arg54 to observe branched chains in vitro and in mammalian cells. These results indicated that Arg54 is a less important residue for ubiquitin functions. To confirm this, we tested whether Ub(R54A) is functionally intact. In an in vitro ubiquitylation assay with several E2 enzymes (chain-nonselective UBCH5, K11-specific UBE2S, K48-specific CDC34, and K63- specific UBC13-UEV1A), Ub(R54A) did not exhibit obvious defects in specific chain elongation (Figure S1B). In 293F cells, levels of K48 and K63 chains and the interaction with E1 remained constant in immunoprecipitates from cells expressing Ub(R54A) and several other Arg54 derivatives (Figure 1D). In sharp contrast, the R42A mutant, which is toxic in yeast (Roscoe et al., 2013), caused abnormal accumulation of ubiquitylated (F) Identification of endogenous K48-K63 branched ubiquitin chains. 293F cell lysates were immunoprecipitated with anti-ub (FK2), and the precipitants were digested with fewer amounts of trypsin. Using PRM, MS/MS fragment ions derived from the signature peptide mis-cleaved at Arg54 (aa 43 72, GlyGly modified at K48 and K63) were observed with the same retention time. See also Figures S1 and S7 and Table S1. Molecular Cell 64, , October 20,

5 Figure 2. Quantification of K48-K63 Branched Ubiquitin Linkage in Cells (A) Ubiquitin replacement strategy for wild-type and R54A ubiquitin. U2OS_shUb_ r Ub(WT) and U2OS_shUb_ r Ub(R54A) cells were incubated with doxycycline (Dox, 1 ng/ml) for 5 days, and whole-cell lysates were analyzed by western blotting. In the upper panel, r Ub(WT) and r Ub(R54A) are expressed at similar levels. In the lower panel, the replaced r Ub is expressed at a similar level to endogenous ubiquitin in intact cells. 254 Molecular Cell 64, , October 20, 2016 (legend continued on next page)

6 species (Figure 1D). These in vitro and in vivo analyses confirm that Ub(R54A) is basically functional. Therefore, we expressed FLAG-tagged Ub(R54A) in 293F cells and analyzed immunopurified Ub(R54A) by mass spectrometry (MS). We performed targeted acquisition of tandem mass spectrometry (MS/MS) spectra (Ohtake et al., 2015) and unambiguously identified MS/MS spectra for K48- and K63-ubiquitylated peptides (amino acids [aa] 43 72, GlyGly modifiedatk48andk63)(figure 1E; Table S1). We confirmed that the MS/MS fragment ions were identified at similar retention times (Figure S1C). These results suggest that ubiquitin is assembled into K48-K63 branched chains in cells. Unbranched K63 linkage and unbranched K48 linkage were also identified (Figure S1D). To detect endogenous K48-K63 branched chains, immunoprecipitated ubiquitin conjugates from intact 293F cells were partially digested with trypsin using 100-fold less enzyme than required for complete digestion. Using parallel reaction monitoring (PRM) (Ohtake et al., 2015), we observed MS/MS fragment ions for K48- and K63-ubiquitylated peptide (aa 43 72) miscleaved at Arg54, whose patterns were similar to those from R54A (Figure 1F). Quantification Strategy for K48-K63 Branched Ubiquitin Linkages in Cells Next, we established a quantification system for K48-K63 branched ubiquitin linkages based on PRM using standard absolute quantification (AQUA) peptides as internal spikes (Ohtake et al., 2015; Tsuchiya et al., 2013). To replace wild-type (WT) ubiquitin, we utilized a U2OS cell line, U2OS_shUb, in which the four endogenous ubiquitin genes (UBB, UBC, UBA52, and RPS27A) are knocked down with tetracycline-inducible short hairpin RNAs (shrnas), and exogenous ubiquitin genes (UBA52 and hemagglutinin [HA]-tagged RPS27A) containing a desired mutation are simultaneously induced to achieve replacement (Xu et al., 2009). Consistent with other reports using this strategy (Tarantino et al., 2014), we observed doxycycline (Dox)-dependent expression of exogenous ubiquitin (Figure 2A). Exogenous wild-type and R54A ubiquitin were expressed at similar levels and accumulated similarly in response to short (1-hr) proteasome inhibition. Moreover, expression levels of endogenous and replaced ubiquitin were unchanged (lower panel in Figure 2A). In the analysis of the K48-unbranched K63 linkage, a substantial percentage of N-terminal glutamine (Gln49) in the signature peptide (aa 49 72, with GlyGly at K63) was spontaneously converted to pyroglutamate (pyroglu), hampering accurate quantification (Figure 2B). To uniformly convert Gln into pyroglu, we treated samples with glutaminyl-peptide cyclotransferase (QPCT), which catalyzes the conversion of protein and peptide N-terminal glutamine to pyroglu (Xu et al., 2013), following trypsin digestion. Comparison of the peak areas of MS/MS fragment ions revealed that QPCT efficiently converted Gln49 to pyroglu within the K48-unbranched K63-linkage signature peptide (Figure 2B). For absolute quantification, we synthesized heavy-labeled AQUA peptides for the K48-K63 branched, unbranched K48, and unbranched K63 linkages harboring pyroglu at position Gln49 (Figure S2A). We confirmed co-elution of these AQUA peptides with the corresponding peptides derived from immunoprecipitated ubiquitin from U2OS cells harboring the R54A transgene (Figure 2C). MS/MS fragment ions used for quantification were selected based on their intensity and specificity (Figure S2A). The K48-K63 Branched Ubiquitin Chain Is an Abundant Modification in Cells U2OS_shUb cells harboring the ubiquitin (R54A) transgene denoted U2OS_shUb_ r Ub(R54A) cells were incubated in the presence or absence of Dox for 5 days and then treated or not treated with MG132 for 1 hr; whole-cell lysates were subjected to PRM quantification (Figure S2B). We confirmed Dox-dependent knockdown of endogenous K48 and K63 linkages derived from wild-type ubiquitin (Figure 2Di). Concomitantly, signature peptides derived from Ub(R54A) were present in a Dox-dependent manner (Figure 2Dii). Total ubiquitin (EST peptide) and other abundant modifications such as the K11 and K29 linkages were unchanged by the replacement (Figure 2Diii). Our quantification revealed that approximately 20% of the K63 linkages were actually K48-K63 branched linkages (lane 3 in Figure 2Dii). Although K63 chains are generally thought to be independent of proteasome targeting, there was a small but reproducible increase in the level of endogenous K63 linkages upon proteasome inhibition (lanes 1 and 2 in Figure 2Di). In Dox-treated cells, K48-K63 branched linkages accumulated upon proteasome inhibition, whereas unbranched K63 linkages were rather decreased (lanes 3 and 4 in Figure 2Dii). In cells treated with MG132, nearly 50% of all K63 linkages were the K48-K63 branched linkages. We confirmed that total K63 linkages, including endogenous, unbranched, and branched linkages, were present at levels similar to those in nonreplaced cells (i.e., cells expressing wild-type ubiquitin) (Figure 2Div). Although our quantification method cannot distinguish higher order chain architectures such as sequence of linkages (B) Strategy for quantifying K48-unbranched K63 linkages derived from Ub(R54A). (i) Scheme for enzymatic conversion of N-terminal Gln in a peptide into pyroglu. (ii) Immunoprecipitated Ub(R54A) was subjected to in-gel trypsin digestion. For the QPCT(+) sample, peptides were incubated with QPCT. Total peak areas of selected MS/MS fragment ions derived from Gln-containing or pyroglu-containing signature peptide from an unbranched K63 linkage (aa 55 72, GlyGly modified at K63) were compared. (C) Co-elution of light (sample-derived) and heavy (AQUA) peptides for absolute quantification. Ub was immunoprecipitated from Dox-treated U2OS_shUb_ r Ub(R54A) cells and subjected to a PRM analysis. (D) The K48-K63 branched ubiquitin linkage is abundant in cells. Dox-treated U2OS_shUb_ r Ub(R54A) cells were treated/untreated with 20 mm MG132 for 1 hr, and whole-cell lysates were subjected to a PRM analysis. Signature peptides specific to endogenous Ub (i) or replaced Ub(R54A) (ii), or common to both (iii), were quantified. (iv) Total K63 linkages containing branched, unbranched, and endogenous (un-replaced) species were present at comparable levels in Dox-treated and untreated cells. Error bars indicate means ± SEM (n = 3). See also Figure S2. Molecular Cell 64, , October 20,

7 Figure 3. IL1b-Dependent Assembly of the K48-K63 Branched Chain (A) IL1b-dependent assembly of polyubiquitin chains on endogenous TRAF6 complex. Dox-treated U2OS_shUb_ r Ub(R54A) cells were treated with IL1b for 20 min and immunoprecipitated using anti-traf6 antibody. IP, immunoprecipitation; IB, immunoblot; WCL whole-cell lysate. (legend continued on next page) 256 Molecular Cell 64, , October 20, 2016

8 and chain length, these results suggest that the K48-K63 branched ubiquitin linkage is an abundant modification in mammalian cells. IL1b-Dependent Assembly of the K48-K63 Branched Ubiquitin Chains Next, we investigated the physiological roles of the K48-K63 branched chain. Because a substantial percentage of total K63 linkages are K48-K63 branched linkages, we speculated that K63 chain-regulated signaling pathways are modulated by branching. In particular, we focused on the IL1b-stimulated NF-kB signaling cascade because K63 chains are indispensable for this pathway (Tarantino et al., 2014; Xu et al., 2009). Upon IL1b stimulation, TRAF6 assembles K63 chains in cooperation with UBC13-UEV1A (Deng et al., 2000). K63 chains are conjugated to TRAF6 itself or to adaptor proteins such as IRAK1, or they are unanchored (Wertz and Dixit, 2010). Therefore, we purified endogenous TRAF6 immunocomplexes from U2OS_shUb_ r Ub(R54A) cells and subjected it to PRM analysis (Figure 3A). We confirmed that IkB was degraded normally in these cells in response to IL1b (Figure 3A). PRM quantification revealed that TRAF6 was modified by K63 chains in an IL1b-dependent manner and, to a lesser extent, by M1 chains (Figure 3Bi). K48 chains were also present in both IL1b-treated and untreated cells. This is not surprising, because TRAF6 is turned over through the proteasome pathway at steady state. Strikingly, quantification of Ub(R54A)-derived peptides revealed that 30% of total K63 linkages were K48-K63 branched linkages, whereas 70% were unbranched K63 linkages (Figure 3Bii). In contrast to the unbranched K48 linkage, modification of TRAF6 with the K48-K63 branched linkage is stimulated by IL1b (Figure S3A). These data indicate that a fraction of TRAF6-modified K63 chains is branched. Because our quantification method is based on a bottom-up strategy, it yields limited information regarding higher order chain architectures on TRAF6. To overcome this issue, we next performed a complementary method, ubiquitin chain restriction assay using DUBs (UbiCRest) (Hospenthal et al., 2015). TRAF6-attached polyubiquitin chains were digested by K63- specific AMSH, K48-specific OTUB1, or universal DUB USP2. AMSH treatment massively decreased the level of both highand low-molecular-weight polyubiquitin chains on TRAF6 (corresponding to long and short polyubiquitin chains, respectively), indicating that K63 linkages are attached proximally to TRAF6 (Figure 3C). In sharp contrast, OTUB1 treatment diminished high-molecular-weight polyubiquitin chains and concomitantly caused the accumulation of short ubiquitin chains, indicating that the K48 linkage decorates pre-formed K63 linkages on TRAF6. Consistent with the data obtained in U2OS cells, FLAG-TRAF6 immunoprecipitated from 293F cells expressing HA-Ub(R54A) was modified with K48-K63 branched chains (Figure 3D). To obtain insight into the mechanism of branched chain assembly, we subjected a catalytically inactive TRAF6 mutant, C70A, to the same analysis. As established previously, the TRAF6(C70A) mutant was defective in polyubiquitin chain assembly in vitro (Figure 3E). Expression of TRAF6(C70A) in 293F cells resulted in lower levels of unbranched K63 linkages than TRAF6(WT), as expected. We also found that assembly of K48-K63 branched linkages on TRAF6 was dependent on the catalytic activity of TRAF6 (Figure 3D). This suggests that an unknown E3 ubiquitin ligase (or ligases) might assemble K48-K63 branched chains in cooperation with TRAF6. Although global levels of K48-K63 branched linkages are increased upon proteasome inhibition, TRAF6-attached K48- K63 branched linkages are not upregulated by MG132 treatment (Figure 3F). This indicates that the role of K48-K63 branched linkage may be different, depending on substrates and/or contexts. The E3 Ubiquitin Ligase HUWE1 Assembles K48-K63 Branched Chains in Cooperation with TRAF6 The results described earlier suggested that one or more unknown E3 ligases cooperate with TRAF6 to assemble branched chains. We reasoned that such enzymes could be recruited to active TRAF6 by interacting either with activated TRAF6 or TRAF6-conjugated polyubiquitin chains. To test this idea, we performed a proteomic identification of FLAG-TRAF6-interacting proteins. To screen for interacting proteins selective for active TRAF6, we subtracted spectrum counts for TRAF6(WT) from those of TRAF6(C70A) for each interactant (Figure 4A; Figure S4A; Table S2). In addition to TAK1, TAB2, and TAB3 (Figure S4B), all of which associate with TRAF6-conjugated K63 chains via TAB2 (Zinngrebe et al., 2014), we identified the E3 ligase HUWE1 as a TRAF6(WT)-enriched interacting protein (Figure 4A). The results of the proteomic screen were validated by immunoprecipitation: endogenous HUWE1 was co-immunoprecipitated with TRAF6(WT) but only weakly with TRAF6(C70A) (Figure 4B). Consistent with this, co-immunoprecipitation of HUWE1 with IRAK1 was significantly enhanced by (B) IL1b-dependent assembly of the K48-K63 branched chain on TRAF6. (i) Immunopurified TRAF6 in (A) was subjected to PRM analysis. The data were normalized against the amount of total ubiquitin chains in each experiment. Error bars indicate means ± SEM (n = 4). (ii) Percentage of branched and unbranched K63 linkages were calculated for each experiment. Error bars indicate means ± SEM (n = 4). (C) (i) Quality control of specificity of DUBs used in the assay. (ii) TRAF6 immunoprecipitated from U2OS cells treated with IL1b for 20 min were subjected to ubiquitin restriction assay with AMSH, OTUB1, and USP2. Cleavage of ubiquitin chains were analyzed by immunoblotting. (iii) The density of smears in (ii) were quantified. (D) Catalytic activity of TRAF6 is required for K48-K63 branch assembly. FLAG-TRAF6, either wild-type or bearing the C70A mutation, was co-expressed with HA- Ub(R54A) in 293F cells, and immunopurified TRAF6 was subjected to PRM quantification. Error bars indicate means ± SD (n = 3 for WT, and n = 2 for C70A). (E) Confirmation of catalytic deficiency of TRAF6(C70A). Recombinant TRAF6 (WT or C70A) (1 mg) was incubated with E1 (50 ng), UBC13 (50 ng), UEV1A (50 ng), and Ub(R54A) (2 mg) for 3.5 hr at 30 C in an in vitro ubiquitylation assay and immunoblotted using anti-ub antibody (Dako). (F) 293F cells were transfected with FLAG-TRAF6 and HA-Ub(R54A), treated with MG132 for 2 hr and subjected to immunoprecipitation using anti-flag antibody. Error bars indicate means ± SEM (n = 3). See also Figure S3. Molecular Cell 64, , October 20,

9 258 Molecular Cell 64, , October 20, 2016 (legend on next page)

10 co-expression of TRAF6 (Figure 4C). Knockdown of TAK1, TAB2/3, or TAB1 did not impair the HUWE1-TRAF6 association (Figure S4C). HUWE1 (also known as Mule, ARF-BP1, Lasu1, or HectH9) is a HECT-type ubiquitin ligase of 482 kda. HUWE1 is involved in multiple biological pathways, including cell growth, apoptosis, and the DNA damage response, and it ubiquitylates proteins such as p53, Myc, and Mcl-1 (Adhikary et al., 2005; Chen et al., 2005; Zhong et al., 2005). Total and conditional HUWE1-knockout mice exhibit defects in embryogenesis and B cell functions, respectively (Hao et al., 2012). However, the involvement of HUWE1 in NF-kB signaling has not been previously investigated. An in vitro ubiquitylation assay using a single-lysine ubiquitin series revealed that HUWE1 mainly assembles K48-linked chains and, to a lesser extent, other chains such as K6 and K11 (Figure 4D). We asked whether HUWE1 can assemble K48 branches on preformed K63 chains in vitro. First, recombinant Ub(R54A) was reacted with TRAF6 and UBC13-UEV1A, and then HUWE1 and UBCH7 were added to the reaction. Quantification revealed that addition of HUWE1 to TRAF6 resulted in efficient generation of K48-K63 branched chains (Figure 4E). When TRAF6-conjugated and unanchored K63 chains were separately reacted with HUWE1, both type of chains generate K48-K63 branched chains at a similar level (Figure S4D). To further explore the mechanistic basis of branched chain assembly, we asked whether HUWE1 directly associates with TRAF6 and/or TRAF6-modified ubiquitin chains. Recombinant TRAF6 (either wild-type or C70A mutant) was first subjected to self-ubiquitylation with UBC13-UEV1A, and either K63-ubiquitylated or unmodified TRAF6 was used as the bait in a subsequent pull-down assay with purified HUWE1 protein (Figure S4E). HUWE1 did not directly associate with unmodified TRAF6 but did interact with K63-ubiquitylated TRAF6 (Figure 4F, lanes 1 3). TRAF6(C70A) did not interact with HUWE1, even after reaction with UBC13-UEV1A (Figure 4F, lanes 4 5). HUWE1 possesses a ubiquitin-binding UBA-UIM domain. In vitro, the UBA-UIM domain did not interact with monoubiquitin, but did interact with di-, tri-, and tetra-k63 chains at levels comparable to those of two representative K63 readers, the TAB2-NZF domain and RAP80-tUIM domains (Figure 4G). Indeed, HUWE1 lacking the UBA-UIM domain [HUWE1(DUBA/UIM)] exhibited a significantly weaker interaction with K63 chain-modified TRAF6 (Figure 4F, lanes 6 8). Next, we asked whether the recognition of K63 chains conjugated to TRAF6 by HUWE1 is involved in branched-chain assembly. In a sequential reaction in which TRAF6 modified by UBC13- UEV1A was subsequently incubated with either HUWE1(WT) or HUWE1(DUBA/UIM), we found that HUWE1(DUBA/UIM) was significantly impaired in generating branched linkages to TRAF6- modified K63-chains (Figure 4H), although its catalytic activity itself is intact, as assessed by free ubiquitin chain formation (Figure S4F). We also performed an experiment in which Ub(R54A) was sequentially reacted with TRAF6-UBC13/UEV1A and/or HUWE1 UBCH7 to directly assess which chain type must be assembled first. We found that HUWE1 assembled branched linkages on K63 chains pre-formed by TRAF6 (Figure 4I, lane 3) but not in the reversed order (lane 4). Moreover, HUWE1 could not assemble branched chains on unmodified TRAF6 (lane 2). These results indicate that HUWE1 s recognition of preformed K63 chains is required for TRAF6/HUWE1-mediated branch formation. HUWE1 Positively Regulates NF-kB Signaling Next, we assessed the impact of HUWE1 on NF-kB signaling. To this end, we first asked whether HUWE1 regulates ubiquitin chains attached to TRAF6. Consistent with the in vitro results, IL1b-dependent modification of TRAF6 with K48-K63 branched linkages was markedly impaired by knockdown of HUWE1 (Figure 5A). In addition, knockdown of HUWE1 also decreased Figure 4. HUWE1 Assembles K48-K63 Branched Ubiquitin Chains in Cooperation with TRAF6 (A) Identification of HUWE1 as a TRAF6-interacting protein. FLAG-TRAF6 (WT or C70A)-associated proteins were immunoprecipitated from 293F cells and identified by LC-MS. The difference in spectrum counts (PSM) identified from TRAF6(WT) and the PSM identified from TRAF6(C70A) is plotted versus the PSM from TRAF6(WT). The red line indicates 2-fold enrichment of PSM in TRAF6(WT). A replicate experiment is shown in Figure S4A. (B) Endogenous HUWE1 associates with catalytically active TRAF6. FLAG-TRAF6-interacting proteins from 293F cells were analyzed using the indicated antibodies. IP, immunoprecipitation. (C) Endogenous HUWE1 interacts with IRAK1 via TRAF6. Cells were transfected with FLAG-IRAK1 and HA-TRAF6 as indicated and were immunoprecipitated with anti-flag antibody. (D) HUWE1 mainly assembles K48 chains in vitro. Recombinant HUWE1 HECT (0.3 mg) was incubated with E1 (50 ng), UbcH7 (50 ng), and a single-lysine Ub mutant (5 mg) for 30 min at 30 C in an in vitro ubiquitylation assay. The products were immunoblotted using anti-ub antibody (Dako). (E) HUWE1 assembles K48-K63 branched ubiquitin chains in cooperation with TRAF6 in vitro. Ub(R54A) (2 mg) was incubated with TRAF6 (0.4 mg), UBC13 (100 ng), UEV1A (100 ng), and E1 (50 ng) for 4 hr; mixed with HUWE1 HECT ( ng) and UbcH7 (100 ng); and incubated for an additional 30 min. The samples were analyzed by PRM. Error bars indicate means ± SD (n = 2). (F) HUWE1 interacts with K63-chain-modified TRAF6 through the UBA-UIM domain. Recombinant GST-TRAF6 (WT or C70A) bound on glutathione sepharose was incubated with/without E1, Ubc13-Uev1a, Ub, and ATP. After washing, purified HUWE1 (WT or DUBA/UIM) was added, and a GST pull-down assay was performed. (G) The HUWE1-UBA/UIM domain interacts with K63-linked Ub 2 4 but not with monoubiquitin. K63 chains were synthesized using Ub(WT) or R54A using Ubc13- Uev1a, and a GST pull-down assay was performed using the indicated recombinant ubiquitin-binding domains. IB, immunoblot. (H) Recombinant GST (glutathione S-transferase)-TRAF6 was incubated with/without E1, Ubc13-Uev1a, and Ub(R54A) in an in vitro ubiquitylation assay for 1 hr. After washing, purified HUWE1 (WT or DUBA/UIM) and UbcH7 were used for the second reaction for 1 hr. Branched linkage formation was quantified. Error bars indicate means ± SEM (n = 3). (I) Branched chain formation as in (H), but TRAF6-Ubc13/Uev1a or HUWE1-UbcH7 was sequentially added in the indicated orders. After the first and second reactions, GST-TRAF6 bound to beads was washed to remove enzymes. Error bars indicate means ± SEM (n = 3). See also Figure S4 and Table S2. Molecular Cell 64, , October 20,

11 Figure 5. HUWE1 Positively Regulates NF-kB Signaling (A) HUWE1 regulates the K48-K63 branched chain as well as the K63 and M1 chains. U2OS_shUb_ r Ub(R54A) cells transfected with sihuwe1 or a scrambled sirna were subjected to ubiquitin replacement by Dox. The cells were then treated with IL1b for 20 min and immunoprecipitated using anti-traf6 antibody. After 260 Molecular Cell 64, , October 20, 2016 (legend continued on next page)

12 the levels of unbranched K63 linkages and total K63 linkages modified on TRAF6 (Figure 5A), although HUWE1 itself exerted minimal K63-chain assembly activity in vitro (Figure 4D). HUWE1 knockdown also decreased the levels of M1 chains, which are reported to be attached on K63 chains (Emmerich et al., 2013). To confirm the involvement of HUWE1 in TRAF6- mediated K63 signaling, we next investigated NF-kB-mediated gene expression. IL1b-dependent induction of the NF-kB target genes TNF-a (also known as TNF) and IL8 (also known as CXCL8) was impaired by HUWE1 knockdown using two independent small interfering RNAs (sirnas) (Figure 5B). TRAF6 knockdown exerted more profound effects on target gene expression, and HUWE1 double knockdown did not further decrease gene induction (Figure 5B). Consistent with this, in an NF-kB-responsive luciferase assay, reporter induction following expression of TRAF6 or IRAK1 was decreased by the knockdown of HUWE1 (Figures 5C and S5A). TAB2- and TAB1-dependent NF-kB activity was not affected by HUWE1 knockdown, as in the case of the Ubc13 knockdown (Figure 5C), presumably because overexpression of these factors can bypass the decrease in the level of K63 chains (Zhang et al., 2011). We also confirmed that IL1b-induced phosphorylation of IkB, IKK, and p38 was impaired in HUWE1-knockdown cells (Figure 5D), indicating that HUWE1 participates in the IL1b signaling cascade upstream of IkB phosphorylation. To assess the assembly of the TAK1-TAB2/3 signaling complex on K63 chains, we analyzed the association of TAK1 and TAB2 with TRAF6. Association with TAK1 and TAB2, as well as K63 polyubiquitylation, of TRAF6 was decreased by the knockdown of HUWE1, with a concomitant decrease in the level of branched linkages (Figure 5E). We obtained similar results with immunoprecipitation of TAK1 (Figure S5B). Taken together, these results suggest that HUWE1 mediates assembly of K48- K63 branched ubiquitin linkages on TRAF6 and is necessary for full NF-kB activation in response to IL1b. The K48-K63 Branch Counteracts CYLD-Mediated K63 Deubiquitylation We explored the molecular mechanism of HUWE1-mediated enhancement of NF-kB signaling and the involvement of branched chain formation in that process. Decoding of the ubiquitin signal is achieved by specific interactions between reader proteins and ubiquitin surfaces created by specific linkages. Because K48 is located near the Ile44 hydrophobic patch, which is frequently used by readers for ubiquitin recognition, the branch at K48 may interfere with recognition of K63 chains. The downstream effector TAB2 and the signal attenuators CYLD and A20 are considered to be major readers for TRAF6-conjugated K63 chains. Therefore, we first asked whether branched chains assembled by TRAF6 and HUWE1 affect deubiquitylation. We confirmed that K63-chain disassembly by CYLD and A20 was not affected by R54A mutation (Figure S6A). Next, we subjected branched chains conjugated on TRAF6 by sequential reaction of TRAF6-UBC13/UEV1A and HUWE1-UBCH7 to deubiquitylation assays. We found that K48-K63 branched chains were less susceptible to CYLD-mediated K63 disassembly in a HUWE1 dose-dependent manner (Figure 6A). Because the K63-cleavage activity of A20 is activated by IKK phosphorylation and is antagonized by either mixed or branched M1 chains (Wertz et al., 2015), we also tested the impact of K48-K63 branching on A20 activity. We found that K48-K63 branching did not interfere with A20- mediated disassembly of K63 linkages, as assessed by deubiquitylation products of short chains (Figure 6B). From the previously published co-crystal structures of K63- linked diubiquitin in complex with CYLD (PDB: 3WXG) (Sato et al., 2015), the K48 residue in the distal ubiquitin is buried in the surface of the CYLD USP domain (Figure S6B), as reported previously (Komander et al., 2008). On the other hand, the proximal K48 was not involved in the interaction (Figure S6B). Therefore, we asked whether K48 branching at distal ubiquitin would affect disassembly of K63 chains by CYLD. For this purpose, we used as a model substrate a tetra-ubiquitin in which the distal ubiquitin in a K63-linked diubiquitin was modified at K48 (K48/ 63/48-Ub4) (Figure S6C). We found that cleavage of the K63 linkage by CYLD was significantly inhibited in the presence of the distal K48 linkage (Figure 6C). To determine the effect of K48 branching at the proximal ubiquitin on CYLD-mediated deubiquitylation, we synthesized triubiquitin branched at K48 and K63 (Figure S6D). C-terminally tagged ubiquitin and lysine-less (K0) ubiquitin were used as the acceptor and donor, respectively, and were incubated with a mixture of K63-specific UBC13-UEV1A and K48-specific UBE2K. A ubiquitin polymer corresponding to the size of K48- K63 branched tri-ubiquitin (25 kda) was generated by this reaction. Mass-spectrometric analysis confirmed that this band, indeed, represented K48-K63 branched tri-ubiquitin (Figure S6D). PRM analyses, the data were normalized against the amount of total ubiquitin chains in each experiment and presented as the change relative to siscramble. Error bars indicate means ± SD (n = 2). HUWE1 knockdown efficiency is shown in the right panel. (B) HUWE1 is a positive regulator of NF-kB-mediated gene induction. Untreated U2OS_shUb cells were transfected with two independent HUWE1 sirnas, TRAF6 sirna, or a scrambled sirna for 3 days and then treated with IL1b for 1 hr; RNAs were isolated for qrt-pcr. Data were normalized against b-actin. Error bars indicate means ± SEM (n = 3). (C) HUWE1 is a positive regulator of NF-kB activity. 293F cells were transfected with the indicated sirnas for 1 day. The cells were then transfected with NF-kB reporter plasmids and the indicated expression vectors and incubated for an additional hr before cell lysis for luciferase assay. Error bars indicate means ± SEM (n = 3). (D) HUWE1 positively regulates IL1b-induced IkBa phosphorylation. Untreated U2OS_shUb cells were transfected with the indicated sirnas for 72 hr and then treated with IL1b for the indicated periods. Whole-cell lysates were analyzed by western blotting. (E) Interaction of TRAF6 with TAB2/TAK1 complex and modification with K63-polyubiquitin are impaired by HUWE1 knockdown. 293F cells transfected with the indicated sirnas for 24 hr were transfected with FLAG-TRAF6 and HA-Ub(R54A) for an additional 2 days. Cell lysates were immunoprecipitated with anti-flag antibody and then analyzed by immunoblotting or PRM quantification of branched linkages. Right panel shows means ± SEM (n = 3). IP, immunoprecipitation; WCL, whole-cell lysate. See also Figure S5. Molecular Cell 64, , October 20,

13 Figure 6. The K48-K63 Branch Counteracts CYLD-Mediated K63 Deubiquitylation (A and B) GST-TRAF6 (0.5 mg) bound to glutathione beads was incubated first with Ubc13-Uev1a and subsequently with or without HUWE1 HECT (0.1 mgor1.0mg (++)) in an in vitro ubiquitylation assay. After washing the beads, the resultant K63 chains or K48-K63 branched chains conjugated to 0.1 mg TRAF6 were subjected to deubiquitylation assay with (A) 0.05 mg CYLD or (B) 0.5 mg A20 phosphorylated by IKKb for the indicated periods. The K63 chains were blotted using anti-k63ub antibody. (C) Distal K48 branching inhibits cleavage of the K63 linkage by CYLD. K63-diUb (0.25 mg) or K48/63/48-tetraUb (0.5 mg) was incubated with recombinant CYLD (10 ng) at 37 C for the indicated periods. Ubiquitin and ubiquitin chains were stained with SYPRO Ruby. Relative intensities of the bands are plotted in the lower panel. (D) The K48 branch does not affect recognition of K63-diubiquitin by TAB2. K48-K63 branched triubiquitin, as well as K48- or K63-linked diubiquitin, was synthesized using donor and acceptor ubiquitin and E2 enzymes UBC13-UEV1A and/or UBE2K, as detailed in Figure S6D. Pull-down assay was performed using GST-tagged TAB2-NZF as bait. Bound ubiquitin was blotted using anti-ub antibody. TAB2-NZF was stained with SYPRO Ruby (lower panel). (legend continued on next page) 262 Molecular Cell 64, , October 20, 2016

14 Using this tri-ubiquitin as a model substrate, we found that K48 branching at the proximal ubiquitin moiety of the K63-linked diubiquitin did not affect cleavage by CYLD (Figure S6E). Together, these results suggest that cleavage of K63 linkages by CYLD is inhibited by K48 branching at the distal ubiquitin. We also analyzed co-crystal structures of K63-linked diubiquitin in complex with the TAB2-NZF domain (PDB: 2WWZ) (Kulathu et al., 2009; Sato et al., 2009). TAB2 binds near the K48 residue in the proximal ubiquitin; consequently, branching may affect the TAB2 interaction, whereas the distal K48 is unlikely to affect it (Figure S6F). Therefore, we subjected a synthesized K63-diubiquitin branched at proximal K48, along with K48- or K63-linked di-ubiquitin (lanes 1 and 2 in Figure 6D), to a pull-down assay with the NZF domain of TAB2. K48-K63-branched tri-ubiquitin interacted with TAB2-NZF at a level similar to that of K63-linked di-ubiquitin (Figure 6D, lanes 4 6). These results suggest that K48 branching does not interfere with recognition of K63 chains by TAB2. To determine whether endogenous HUWE1 and CYLD act antagonistically in cells, we performed double-knockdown analyses of these two regulators. We reasoned that, if HUWE1 antagonizes CYLD activity during NF-kB activation, then double knockdown of CYLD should rescue the effect of HUWE1 knockdown in NF-kB signaling. To test this idea, we first analyzed complex assembly of TRAF6 with TAB2/TAK1 and TRAF6 ubiquitylation. Consistent with the data shown in Figure 5E, interaction of TRAF6 with TAB2/TAK1, as well as K63 polyubiquitylation, was suppressed by knockdown of HUWE1 (Figure 6E). Importantly, double knockdown of CYLD+HUWE1 abolished the effect of the HUWE1 single knockdown. We observed a similar effect with immunoprecipitation of TAK1 (Figure S6G). We also confirmed the antagonistic function of HUWE1 and CYLD in functional analyses of NF-kB activity in luciferase assays (Figure 6F) and target gene expression (Figure 6G). Indeed, the data revealed that repression of NF-kB activity by HUWE1 knockdown was abolished by double knockdown of CYLD+HUWE1. These results suggest that ubiquitin chain branching differentially affects the readout of the ubiquitin code by downstream regulators: recognition of the K63 linkage by TAB2 (resulting in downstream NF-kB activation) is not impaired, whereas cleavage of the K63 linkage by CYLD (resulting in signal attenuation) is suppressed by K48 branching. Based on these in vitro and in vivo findings, it is likely that HUWE1 enhances NF-kB activity, at least in part, by antagonizing CYLD through K48 branching of K63 linkages. DISCUSSION Branched Ubiquitin Chains Are Abundant in Cells Although recent studies reported important roles for heterogeneous chains such as mixed and multiple chains, the nature of the branched ubiquitin chains, including their abundance, has remained largely unclear. In this study, we established a strategy for quantifying branched ubiquitin linkages by focusing on the K48 and K63 branches, two of the most abundant and best characterized chain types. Our quantitative experiments revealed that K48-K63 branched linkages are abundant in mammalian cells (Figure 7). Even in the absence of MG132 treatment, approximately one fifth of all K63 linkages were actually K48- K63 branched linkages, rendering such branched chains relatively common. Global levels of K48-K63 branched linkages increase after proteasomal inhibition, indicating that they may be involved in the degradation of certain substrates. Our strategy for quantifying branched chains has several advantages. First, it enables unambiguous identification of the branched linkage. Second, it uses PRM, enabling highly sensitive and accurate quantification. Third, it can distinguish branched and unbranched K48/K63 linkages. Our data suggest that K48- K63 branched linkages respond differently than unbranched K48 or K63 linkages, i.e., they have different meanings within the ubiquitin code. Fourth, our method is directly applicable to functional analysis of other E3s/E2s/DUBs/UBDs, although it should be empirically determined whether the R54A mutation retains the function of the E3s/E2s/DUBs/UBDs of interest. Moreover, it remains to be seen how extensively the branched chain is conserved among eukaryotes and whether its abundance changes in tissue- or developmental-stage-specific manner. Ubiquitin Code Crosstalk through Branching of Ubiquitin Chains Like their cellular abundance, the functional significance of branched chains is poorly understood. In this study, we demonstrated crosstalk between specific ubiquitin codes via branching of ubiquitylation. Although the K48 linkage on its own acts as a mark for proteasomal degradation, K48 branching in K63 chains serves as a signal stabilizer to enhance the downstream cascade in NF-kB signaling. We propose that ubiquitin chain branching constitutes a new component of the ubiquitin code, rather than the simple addition of two distinct components, by differentially altering recognition by reader proteins. It is possible that the branched K48 linkage also serves as a degradation signal at a later stage. Taken together with previous reports (Kim et al., 2007; Meyer and Rape, 2014), these observations suggest that the branched chains regulate biological pathways via multiple mechanisms and are functionally distinct from mixed or multiple chains. Because there are 28 potential types of branched linkage, further characterization of other branched linkages is an important priority for future studies. Mechanism of Branched Chain Assembly and Reading In this study, we identified HUWE1 as a novel enzyme physiologically involved in formation of branched chains. Although HUWE1 (E) The effect of HUWE1 knockdown on association of TRAF6 with TAB2/TAK1 is abolished by CYLD double knockdown. 293F cells transfected with the indicated sirnas were transfected with FLAG-TRAF6 (lanes 2 5) and HA-Ub (lanes 1 5). The cells were subjected to anti-flag immunoprecipitation (IP). WCL, whole-cell lysate. (F and G) The effect of HUWE1 knockdown on NF-kB activity (F) and target gene induction (G) is abolished by CYLD double knockdown. (F) 293F cells were transfected with the indicated sirnas and plasmids, as in Figure 5C, and subjected to luciferase assay. Error bars indicate means ± SEM (n = 6). (G) Untreated U2OS_shUb cells were transfected with the indicated sirnas, and qrt-pcr was performed as in Figure 5B. Error bars indicate means ± SEM (n = 3). See also Figure S6. Molecular Cell 64, , October 20,

15 ubiquitin moieties within K63 chains after recruitment to K63 chains on TRAF6 and attach new ubiquitin moieties to K48 residues. Moreover, our sequential analysis revealed that preformed K63 chains are required for branched chain formation by HUWE1. This observation indicates that ubiquitin chain branching by TRAF6-HUWE1 proceeds in one direction: the pre-formed K63 linkage is subsequently recognized by HUWE1 and is branched at K48. Our findings reveal a unique mechanism for readout of the branched ubiquitin code: the K48-K63 branch does not interfere with recognition by TAB2 but, instead, restricts cleavage by CYLD, thus enhancing NF-kB activity (Figure 7). Our results indicate the robustness of ubiquitin code readout by TAB2, which is designed to be resistant to branching of the chain. On the contrary, the activity of CYLD is sensitive to branching. The K63 residue at distal ubiquitin is exposed from CYLD s surface, enabling cleavage of internal K63 linkages (Komander et al., 2008), whereas the K48 residue of the distal ubiquitin is occluded by the surface of CYLD, thereby restricting recognition of branched chains. A recent study reported that M1-linked chains, either mixed or branched with K63 chains, inhibit A20-mediated disassembly of K63 chains to enhance tumor necrosis factor a (TNF-a) signaling (Wertz et al., 2015). We found that K48-K63 branches did not interfere with A20-mediated K63 deubiquitylation, indicating that the structural determinants of CYLD substrate specificity are unique. Based on these findings, it is likely that the recognition of specific linkages by UBDs, E2s/E3s, and DUBs are differentially regulated by the presence of particular branch types in the chain. These features add new diversity to the ubiquitin code. Figure 7. The K48-K63 Branched Ubiquitin Chain Is an Abundant Modification and a Positive Regulator of NF-kB Signal Mass-spectrometry-based quantification revealed that the K48-K63 branched ubiquitin chain is an abundant modification in cells. During IL1b signaling, the E3 ubiquitin ligase HUWE1 assembles K48-K63 branched ubiquitin chains in cooperation with TRAF6, which enhances NF-kB activation by inhibiting CYLD-mediated K63 chain disassembly while preserving recognition by TAB2 for the downstream cascade. We propose that ubiquitin chain branching constitutes a new component of the ubiquitin code, adding additional diversity to ubiquitin biology. mainly assembles K48 chains by itself, it also adds K48 branches to K63 chains pre-formed by TRAF6. Our analysis suggests a sequential mechanism for branched chain assembly: HUWE1 recognizes TRAF6 pre-modified by K63 chains through its ubiquitin-interacting UBA-UIM domain, generating K48 branches on K63 chains to yield K48-K63 branched linkages. Because HECTtype E3s noncovalently interact with acceptor ubiquitin via the HECT domain to elongate ubiquitin chains (Maspero et al., 2011), it is likely that HUWE1 HECT can interact with internal Biological Role of Branched Chains in NF-kB Activation The biological significance of the K48-K63 branched ubiquitin chain was demonstrated by its involvement in NF-kB signal pathway. We found that K48-K63 chains were assembled on the TRAF6 complex in an IL1b-dependent manner. Although directly analyzing the roles of branched chain in cells is challenging, because it is not possible to use conventional lysineto-arginine mutants, we were able to obtain important insights through the knockdown of HUWE1, which assembles branched chains in cooperation with TRAF6. We found that HUWE1-generated K48-K63 branched linkage protects K63 chains from CYLDmediated deubiquitylation and that HUWE1 functionally antagonizes CYLD during NF-kB activation in cells. Although we could not exclude the possibility that HUWE1 also exerts such a function by regulating other pathways, it is most likely that HUWE1 regulates NF-kB signaling, at least in part, through TRAF6 and branched chain formation. Interestingly, our results show that the K48-K63 branch does not interfere with A20 activity. Given that A20 is induced by NF-kB, resulting in the termination of NF-kB signaling (Harhaj and Dixit, 2011), it is possible that the ability of A20 to disassemble K48-K63 branched chains contributes to its signalattenuating role. Moreover, the mechanism underlying the HUWE1-mediated increase in the level of M1 chains is currently unknown. Given that M1 chains are assembled onto preformed K63 chains in the IL1b signaling pathway (Emmerich et al., 2013), HUWE1-mediated stabilization of K63 chains 264 Molecular Cell 64, , October 20, 2016