Signal-Induced Site-Specific Phosphorylation Targets IKB X to the Ubiquitin-Proteasome Pathway

Home , Hyperphosphorylation, Phosphorylation

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Signal-induced site-specific phosphorylation targets IKBx to the ubiquitin-proteasome pathway

Zhijian Chen, ~ Jeremiah Hagler, 2 Vito J. Palombella, 1 Franeesco Melandri, 1 David Seherer, 3 Dean Ballard, 3 and Tom Maniatis 2 ~Myogenics, Inc., Cambridge, Massachusetts 02139 USA; 2Harvard University, Department of Molecular and Cellular Biology, Cambridge, Massachusetts 02138 USA~ 3Howard Hughes Medical Institute, Department of Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232 USA

The transcription factor NF-KB is sequestered in the cytoplasm by the inhibitor protein IKBc~. Extracellular inducers of NF-KB activate signal transduction pathways that result in the phosphorylation and subsequent degradation of IKBa. At present, the link between phosphorylation of IKB~x and its degradation is not understood. In this report we provide evidence that phosphorylation of serine residues 32 and 36 of IKBcx targets the protein to the ubiquitin-proteasome pathway. IKBa is ubiquitinated in vivo and in vitro following phosphorylation, and mutations that abolish phosphorylation and degradation of IKBa in vivo prevent ubiquitination in vitro. Ubiquitinated IgBa remains associated with NF-KB, and the bound IKBa is degraded by the 26S proteasome. Thus, ubiquitination provides a mechanistic link between phosphorylation and degradation of IKBr [Key Words: Phosphorylation; transcription factor; NF-KB; IKBe~; ubiquitin; Rel; proteasome] Received May 2, 1995; revised version accepted June 5, 1995.

NF-KB and other members of the Rel family of transcrip- An alternative pathway for regulating the activity of tional activator proteins regulate the expression of the NF-KB involves proteolytic processing of the p105 pre- type I human immunodeficiency virus (HIV) as well as a cursor of p50 subunit of NF-KB {Blank et al. 1991; Fan large number of cellular genes that play essential roles in and Maniatis 1991; Mellits et al. 1993; Mercurio et al. immune and inflammatory responses (for review, see 1993; Donald et al. 1995}. The p105 precursor contains Grilli et al. 1993; Baeuerle and Henkel 1994; Siebenlist pS0 at its amino terminus and an IKB-like sequence with et al. 1994; Thanos and Maniatis 1995). The transcrip- ankyrin repeats at its carboxyl terminus. Unprocessed tional activities of Rel proteins are highly regulated in p105 can associate with p65 and other members of the most cell types by a mechanism that involves specific Rel family to form inactive heterodimeric complexes association between heterodimeric Rel complexes and a that are sequestered in the cytoplasm (Capobianco et al. family of monomeric inhibitor proteins designated IKB 1992; Liou et al. 19921 Neumann et al. 19921 Rice et al. (Baeuerle and Baltimore 1988; for review, see Beg and 1992; Mercurio et al. 19931. Processing of p105 results in Baldwin 1993; Gilmore and Morin 1993). Members of degradation of the IKB-like carboxyl terminus and the this inhibitor family share a structural domain com- production of the transcriptionally active p50/Rel pro- prised of five to six ankyrin-like repeats. The best-char- tein heterodimer. acterized IKB protein, IKBa, binds to the p50 {NF-KBlJ/ Recently, p105 processing was shown to require the p65 {RelA} heterodimer of NF-KB and masks the nuclear ubiquitin-proteasome pathway {Palombella et al. 1994}. localization signals of these proteins (Beg et al. 1992; This pathway requires ATP and the covalent conjugation Ganchi et al. 1992; Henkel et al. 1992; Zabel et al. 1993}. of target proteins with multiple ubiquitin molecules (for When cells are exposed to a variety of NF-KB inducers such review, see Goldberg 19921 Hershko and Ciechanover as lipopolysaccharide {LPS}, phorbol esters, tumor necrosis 1992; Jentsch 1992}. Ubiquitination occurs in a three- factor-~ {TNRx}, and mterleukin-1 (IL-IJ, IKB~ is rapidly step process. In the first step, ubiquitin is activated by a phosphorylated and degraded, and NF-KB translocates to ubiquitin-activating enzyme (E 1}, and in the second step, the nucleus where it activates gene expression (Beg et al. the activated ubiquitin is transferred to a ubiquitin car- 1993~ Brown et al. 1993~ Cordle et al. 1993; Henkel et al. rier protein (E2). In the final step, ubiquitin-protein li- 19931 Rice and Ernst 19931 Sun et al. 1993, 1994a). gase (E3} catalyzes the covalent attachment of ubiquitin

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Signal-induced ubiquitination of IKB~ to the target protein. Additional ubiquitins are then dent phosphorylation of IKBOL targets the cytoplasmic in- thought to be added by a processive mechanism to form hibitor to the ubiquitin-proteasome pathway. the multiubiquitin chain (for recent review, see Ciecha- nover 1994). The multiubiquitinated proteins are then rapidly degraded by the 26S proteasome. Results The 26S proteasome consists of a 20S multicatalytic Inducible phosphorylation and ubiquitination protease complex and additional regulatory subunits of IKBa in vivo that are required for the recognition and degradation of multiubiquitinated proteins (for review, see Rechsteiner To determine whether IKBOL is ubiquitinated upon phos- et al. 1993; Peters 1994). The NFKB1 p105 protein is phorylation in vivo, we sought conditions that result in ubiquitinated in vitro, and ubiquitination is required for the stabilization of the hyperphosphorylated form of in vitro processing by purified 26S proteasome (Palom- IKBoL that is rapidly degraded during cellular activation bella et al. 1994). In addition, p105 processing in vitro (for review, see Siebenlist et al. 1994). Previous studies and in vivo is blocked by peptide aldehyde inhibitors of demonstrated that the proteasome inhibitor MG132 (Z- the proteasome. The degradation of IKBOL is also blocked Leu-Leu-Leu-H) blocks TNFot-induced degradation of by such inhibitors, but this process has not yet been IKB~ and leads to the accumulation of phosphorylated shown to require ubiquitination (Finco et al. 1994; IKB~ (Palombella et al. 1994). However, only a small frac- Miyamoto et al. 1994; Palombella et al. 1994; Traenck- tion of endogenous IKBOL remains phosphorylated under ner et al. 1994; Alkalay et al. 1995; DiDonato et al. 1995; these conditions, attributable presumably to the action Lin et al. 1995). of endogenous phosphatases. Calyculin A and okadaic Although the signal transduction pathways leading to acid are phosphatase inhibitors that induce NF-KB by the activation of NF-KB are not well understood, these maintaining phosphorylation and degradation of Iv,Box pathways culminate in the phosphorylation of IKB, p 105, (Thevenin et al. 1990; Menon et al. 1993; Lin et al. 1995). and p65 (Beg et al. 1993; Brown et al. 1993; Mellits et al. We therefore attempted to accumulate phosphorylated 1993; Naumann and Scheidereit 1994; Sun et al. 1994b; IKBa in the Jurkat T-cell line using the combination of Donald et al. 1995). Initially, phosphorylation of IKBa MG132 and calyculin A. Jurkat cells were treated with was thought to promote its dissociation from NF-KB and 40 p,M MG132 alone (Fig. 1A, lane 2), with 0.3 ~M caly- its subsequent degradation (for discussion, see Beg and culin A alone (lane 3), or with both inhibitors (lane 4), Baldwin 1993; Beg et al. 1993). This conclusion was con- and the phosphorylation of IKBa analyzed in a Western sistent with the observation that tosyl-Phe-chlorometh- blot using a polyclonal antibody against the carboxyl ter- ylketone (TPCK) and other alkylating agents block the minus of IKBo~. Treatment with MG132 alone did not degradation of IKBo~ and the activation of NF-KB (Henkel affect the level of unphosphorylated IKBcx under these et al. 1993; Mellits et al. 1993). However, more recent conditions [although IKBe~ is known to be basally phos- studies have shown that these inhibitors actually pre- phorylated (for example, Brown et al. 1995), we will refer vent the signal-dependent phosphorylation of IKBo~ and to unstimulated IKB~ as unphosphorylated]. Treatment have no direct effect on proteasome function (Finco et al. with calyculin A alone resulted in the phosphorylation 1994; Miyamoto et al. 1994; Palombella et al. 1994; and degradation of IKBo~. In contrast, phosphorylated Traenckner et al. 1994; Alkalay et al. 1995; DiDonato et IKBoL accumulated when cells were treated with both in- al. 1995; Lin et al. 1995). In contrast, the presence of hibitors. The calpain inhibitor MG102 (40 ~M), which proteasome inhibitors leads to the accumulation of phos- completely inhibits calpain activity but does not inhibit phorylated IKB~ bound to NF-KB (Finco et al. 1994; the proteasome at this concentration, did not lead to Miyamoto et al. 1994; Palombella et al. 1994; Traenck- accumulation of phosphorylated IKB~ in the presence of ner et al. 1994; Alkalay et al. 1995; DiDonato et al. 1995; calyculin A (data not shown). These results indicate that Lin et al. 1995). These findings suggest that phosphory- calyculin A induces the phosphorylation-dependent deg- lation leads to the degradation of IKB~ by the protea- radation of IKB~ and that the proteasome is required for some, without inducing its dissociation from NF-KB. this process. Recently, serine residues 32 and 36 in IKBe~ have been To determine whether ubiquitination of IKBe~ occurs shown to be required for IKBo~ phosphorylation and deg- in vivo, we prepared cell extracts at different times after radation in response to TNF-c,, phorbol 12-myristate 13- treatment of Jurkat cells with calyculin A in the pres- acetate (PMA), and ionomycin (Brockman et al. 1995; ence of MG132 and then analyzed the samples by West- Brown et al. 1995) or the Tax protein of the type 1 human ern blotting using antibodies against InBa. The extracts T-cell leukemia virus (HTLV-1; Brockman et al. 1995). were prepared in the presence of SDS (0.1%) and N-eth- However, the mechanisms by which phosphorylation ylmaleimide (NEM, 5 mM) tO inhibit isopeptidase activ- leads to the degradation of IKB~x are not understood. In ities that may otherwise affect the detection of ubiquit- this paper we show that IKBer is ubiquitinated in vivo and inated proteins. As shown in Figure 1B, a ladder of high in vitro and that ubiquitination is required for degrada- molecular mass proteins accumulated following stimu- tion by the 26S proteasome. In addition, we demonstrate lation with calyculin A/MG132 (lanes 4--9). The molec- that mutations in IKBoL that prevent its phosphorylation ular mass increments of these ladders were -8.5 kD, and degradation in vivo block ubiquitination in vitro. which is the size of ubiquitin. Ubiquitination of IKBcx Together, these findings indicate that the signal-depen- peaked at 5-15 min following stimulation (lanes 4-6)

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Figure 1. Induced phosphorylation and ubiquitination of IKBa in calyculin A or TNFa-treated cells. (A) Stabilization of the phosphorylated form of IKBc~ by MG132. Jurkat T cells were pretreated with 40 ~M of MG132 {lanes 2,4) or DMSO (a diluent for MG132, lanes 1,3) for 30 min before incubation with 0.3 ~M of calyculin A {lanes 3,4) or DMSO {lanes 1,2) for an additional 30 rain. Cytoplasmic extracts were prepared, fractionated by SDS-PAGE, and analyzed by Western blot using a rabbit polyclonal antibody against the carboxyl terminus of IKBc~ {amino acids 297-317, c-21). The phosphorylated form of IKBc~ is designated p-IKBa. {B) Induced ubiquitination of IKBa by calyculin A. Jurkat T cells were pretreated with MG132 (40 ~tM, lanes 2-9) or DMSO {lane 1) for 30 min before addition of calyculin A (0.3 ~M, lanes 4-9) or DMSO {lanes 2,3). Cell extracts were prepared at 5 rain (lanes 2,4), 10 min {lane 5), 15 rain (lane 6), 20 rain {lane 7), 40 rain (lane 8), and 60 min {lanes 3,9) after adding calyculin A or DMSO. The extraction buffers contained RIPA/0.1% SDS plus 5 mM N-ethylmaleimide (NEM). The extracts were then subjected to Western blot analysis as described in A. (C) Jurkat T cells were treated as described in lanes 1 and 4 in A, except that cytoplasmic extracts were first immunoprecipitated with an antibody against the carboxyl terminus of IKBc~ (see Materials and methods). The immunoprecipitates were then eluted with 0.1 M Capso (pH 11.2), and the eluates were separated by SDS-PAGE, followed by Western blotting with a rabbit polyclonal antibody {anti-Ub) that specifically recognizes ubiquitin conjugates. The arrow marked IgG H indicates rabbit immunoglobulin heavy chain in the immunoprecipitates that cross-reacts with the secondary antibody {alkaline phosphatase conjugated anti-rabbit Fc). (D) Induced ubiquitination of IKBa by TNFa. Experiments were carried out as described in B, except that calyculin A was replaced with TNFa (10 ng/ml).

and then decreased by 40-60 min (lanes 8,9), possibly from control and MG 132/calyculin A-treated Jurkat cell because of residual activities of proteasome and isopep- extracts with an IKB~ antibody. We then performed a tidases. Treatment of Jurkat cells with MG132 alone did Western blot analysis on the immunoprecipitated pro- not lead to significant accumulation of ubiquitinated teins using a polyclonal antibody against ubiquitin (Fig. IKBcx (lane 2). Longer treatment with MG132 (60 rain 1C). The specificity of these antibodies has been docu- plus 30 rain of pretreatment, lane 3) led to the appear- mented extensively, and the antibodies have been widely ance of very faint bands corresponding to ubiquitinated used to detect ubiquitinated proteins (Haas and Bright IKB~, suggesting that ubiquitination may also be in- 1985; Lowe and Mayer 1990). The anti-ubiquitin anti- volved in basal turnover of IKB~. body detected high molecular mass proteins in the caly- To further demonstrate that calyculin A induces ubiq- culin A/MG132-treated extracts. These molecular uitination of IKBo~ in vivo, we immunoprecipitated I,:BoL masses ranged from 60 to 200 kD, consistent with a typ-

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Signal-induced ubiquitination of IKBa ical pattem seen upon multiubiquitination of proteins. These observations, together with data shown below, Taken together, these results suggest that calyculin A clearly show that the high molecular mass species are induces the phosphorylation-dependent ubiquitination multiubiquitinated IKBot. of IKBa in vivo. The conditions required for ubiquitination of It,Bet in Because calyculin A is not a natural inducer of NF-KB vitro were examined in the experiment of Figure 2C. activation, it is possible that induced ubiquitination of Both IKBcx (lanes 1-6) and FLAG epitope-tagged IKBo, (F- IKBa is an unusual effect of calyculin A. To address this IKBot, lanes 7-12) were tested in this experiment. The possibility, we performed an experiment similar to that latter form of IKBa was examined for comparison to a described in Figure 1B, except that calyculin A is re- series of FLAG-tagged IKBot mutants, which were exam- placed by TNFa (Fig. 1D), a natural inducer of NF-KB. ined previously in vivo (Brockman et al. 1995; see below Strikingly, TNFot not only induces hyperphosphoryla- and Fig. 3). With both the native and FLAG-tagged IKBa tion of IKBa in the presence of MG132 but also induces proteins, phosphorylation and ubiquitination were de- multiubiquitination of IKBoL (lanes 4-9). The kinetics of pendent on the presence of okadaic acid (lanes 2 and 5, 8 induction by TNFoL is similar to the induction by caly- and 11), and both reactions were abolished by the addi- culin A. We conclude that IKBa is rapidly phosphorylated tion of EDTA (lanes 6,12). The upper bands labeled and ubiquitinated following stimulation by inducers of p-IKBa and p-F-IKBe~ are phosphorylated forms of IKBa, as NF-v,B. these bands could be converted to the lower bands after treatment with calf intestine alkaline phosphatase (CIAP, data not shown). The presence of Ubal was nec- In vitro-translated IKBa is ubiquitinated in HeLa essary for multiubiquitination of IKBa (lanes 4,10). With- cell extracts, and the ubiquitinated IKBa remains out Ubal, only small amounts of low molecular mass associated with NF-KB conjugates were observed, probably because of the action Next, we carried out experiments to determine whether of isopeptidases in the extract. Multiubiquitination of the phosphorylation and ubiquitination of IKBa could be IKBcx was also inhibited by methylated ubiquitin (MeUb, induced in vitro by the phosphatase inhibitor okadaic lanes 3,9), which prevents the formation of multiubiq- acid. Like calyculin A, okadaic acid has been shown pre- uitin chains (Hershko et al. 1991). Because of the pres- viously to potently activate NF-KB in vivo (Thevenin et ence of competing endogenous ubiquitin in the HeLa cell al. 1990). Preliminary experiments revealed that incuba- extracts, MeUb functions as a ubiquitin chain termina- tion of HeLa cell cytoplasmic extracts in the presence of tor that leads to the generation of low levels of low mo- MgATP and okadaic acid led to a time-dependent phos- lecular mass conjugates. The fact that the formation of phorylation and ubiquitination of endogenous IKBa (data high molecular mass conjugates is dependent on Ubal not shown). We extended this study to in vitro-translated and inhibited by MeUb provides strong evidence that IKBa so that IKBa mutants could be examined (see be- the high molecular mass conjugates are multiubiquiti- low). We prepared 3SS-labeled IKBa by coupled in vitro nated IKBa. Addition of GST-c-Rel protein did not affect transcription/translation of IKBa mRNA in wheat germ the in vitro ubiquitination reaction in HeLa cell extract extracts. The in vitro-translated IKBa was then incubated (data not shown), presumably because the in vitro-trans- in the HeLa cell cytoplasmic extract in the presence of lated IKBa associates with excess endogenous NF-KB pro- MgATP, ubiquitin, okadaic acid, and ubiquitin aldehyde teins present in the extract (see below). Taken together, (Ubal), an isopeptidase inhibitor that prevents the break- these results show that IKBa can be ubiquitinated in down of ubiquitin conjugates. As shown in Figure 2A, vitro. Moreover, ubiquitination requires the presence of there was a time-dependent accumulation of high mo- an inducing agent, such as okadaic acid. lecular mass species characteristic of multiubiquitinated To determine whether the IKBot ubiquitinated in HeLa IKBoL, with a concomitant decrease in unconjugated iKBa. cell extracts is associated with NF-KB, the reaction mix- The molecular weights of the slowly migrating species ture (Fig. 3, lane 1) was precipitated with an antibody range from 60 to >200 kD, consistent with the addition against RelA {lane 2). Both ubiquitinated and unconju- of from 10 to >20 ubiquitin molecules (8.5 kD) to each gated IKBcx were present in the anti-RelA precipitates, molecule of IKBa (37-41 kD, depending on phosphoryla- indicating that both are associated with NF-KB. The ratio tion states). The unconjugated IKBot present during the of ubiquitinated IKBa to unconjugated IKBa in the im- ubiquitination reaction is a doublet, and as described be- munoprecipitates is similar to that in the reaction mix- low, the upper band is likely phosphorylated IKBa. The ture prior to immunoprecipitation, suggesting that both high molecular mass species can be immunoprecipitated forms of IKBe~ associate with NF-KB with similar affinity. by anti-ubiquitin antisera (Fig. 2B, lane 2). In addition, when glutathione S-transferase-ubiquitin (GST-Ub)was used to substitute for ubiquitin, the high molecular mass Serine residues 32 and 36 are necessary species could be precipitated by glutathione-Sepharose for ubiquitination of IKBa in vitro (data not shown). Furthermore, the high molecular mass species could be converted to lower molecular mass Serine residues 32 and 36 of IKBet are required for the forms upon treatment with isopeptidase T (data not phosphorylation and degradation of IKBot in vivo (Brock- shown), which cleaves lysine-48 linked isopeptide bonds man et al. 1995; Brown et al. 1995). However, a mecha- between ubiquitin molecules (Chen and Pickart 1990). nistic link between phosphorylation and degradation had

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Figure 2. Induced phosphorylation and ubiquitination of IKBa in vitro. (A) Time course of ubiquitination. IKBa was in vitro- translated in wheat germ extract (Promega TNT system) in the presence of [aSS]methionine. The labeled IKBa was then incubated in HeLa extracts at 37~ in the presence of Mg-ATP (2 mM), ubiquitin (1 mg/ml), okadaic acid (3.3 lxM), and Ubal (3 laM). An aliquot of the reaction was then quenched at the indicated times in SDS sample buffer, followed by SDS-PAGE and fluorography. (B) Double immunoprecipitation with IKBa and ubiquitin antibodies. Ubiquitinated IKBa was synthesized as described in A, and the reaction mixture was precipitated with IKBa antibody (lane 1 ). An aliquot of the immune complex was then boiled for 5 rain in the presence of 0.5% SDS, and the liberated ubiquitinated IKBa was then precipitated again with anti-ubiquitin antibody (lane 2). The samples were analyzed by SDS-PAGE followed by fluorography. (C) Conditions required for phosphorylation and ubiquitination of IKBa in vitro. Both IKBa (lanes 1-6) and FLAG-epitope-tagged IKBa (F-IKBa, lanes 7-12) were 3ss- labeled by in vitro translation and used as substrates for ubiquitination. The reaction conditions were as described in A, except for the following: Lanes 3 and 9 received MeUb (1.3 mg/ml) instead of Ub; lanes 4 and 10 lacked Ubal in the reaction; lanes 5 and 11 lacked okadaic acid in the reaction; lanes 6 and 12 received EDTA (40 mM) instead of Mg-ATP. After incubation at 37~ for 90 rain, IKBa and F-IKBa were immunoprecipitated with IKBa antisera (c-21) and then analyzed by SDS- PAGE. In A-C, the bands below IKBa are probably nonspecific partial proteolytic cleavage products of IKBa, as the generation of these bands is not affected by EDTA, MeUb, okadaic acid, or Ubal (see C).

not been established. We therefore tested a series of 1995). The $32E and $36E mutants are serine to glutamic phosphorylation-defective mutants of IKBet in the in acid substitutions, which are designed to mimic the neg- vitro ubiquitination assay. Wild-type and mutant ative charge of phosphorylation (note mobility differ- IKBet proteins tagged at their amino termini by the FLAG ences on SDS--polyacrylamide gel; Fig. 4B). These sub- epitope (Brockman et al. 1995) were produced by in vitro stitutions restore the ability of the mutant IKBet to be translation (Fig. 4A). These same mutants were analyzed degraded in response to inducing agents in vivo (Brock- previously for their effects on the inducible degradation man et al. 1995). The AC mutant lacks 75 amino acids at of IKBot in vivo (Brockman et al. 1995). the carboxyl terminus of IKBa. This mutation removes a The first 36 amino-terminal amino acids have been carboxy-terminal PEST sequence, as well as a putative deleted in the AN mutant, whereas the $32A and $36A sixth ankyrin repeat. The sixth ankyrin repeat has been mutants are serine to alanine substitutions at positions shown to be required to bind NF-KB (Ernst et al. 1995). In 32 and 36, respectively. The S32A/S36A mutant is a dou- addition, deletion of the PEST sequence has been shown ble serine to alanine substitution at positions 32 and 36. to stabilize IKBot in vivo (Miyamoto et al. 1994; Brock- All of these mutant proteins are stable when expressed man et. al., 1995; Brown et al. 1995). PEST sequences in in cells treated with TNFot, PMA/ionomycin, or in the other proteins have been implicated in protein degrada- presence of the HTLV Tax protein. Morever, they retain tion (Rogers et al. 1986). (Note that this AC mutant is their ability to associate with RelA, and are dominant different from another recently described AC mutant, negative inhibitors of NF-KB activation (Brockman et al. which is missing only 41 residues at the carboxyl termi-

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Signal-induced ubiquitination of IKB,,

and C, showed a remarkable correlation between phosphorylation and ubiquitination of these mutants in HeLa extracts (Fig. 4B,D). A clear difference in electrophoretic mobility of the unconjugated IKBa mutants following incubation in the HeLa cell extracts can be observed. For example, after incubation in the okadaic acid-supplemented extracts, the unconjugated wild-type IKBa exhib- its a slightly slower migrating band in addition to the band observed prior to incubation (cf. Fig. 4B, lane 1, and 4D, lane 11). This "mobility shift" phenomenon is not observed in aN (lanes 2,12) and S32A/S36A mutants (lanes 8,18). This slower migrating band is attributable to phosphorylation of IKBa. The $32A and $36A mutant proteins were also phosphorylated during the reaction (lanes 4 and 14, 6 and 16), probably because the adjacent unaltered serine residues ($36 and $32, respectively) can serve as phosphoryl group acceptors. The $32E and $36E mutants both migrate more slowly than $32A, $36A, or S32A/S36A even before the ubiquitin conjugation reac- Figure 3. Association of ubiquitinated IKB~ with NFKB. Con- tion (lanes 5 and 15, 7 and 17), an indication that the ditions for the synthesis of ubiquitinated IKBa were as described negative charges on $32E and $36E decrease the mobility in Fig. 2A except that the reaction was carried out at 37~ for 2 of both proteins. The mobility of the $32E and $36E mu- hr. Ten percent of the reaction mixture was saved for SDS- tants did not change appreciably after the reaction, sug- PAGE analysis [lane 1), and IKBa in the remaining mixture was gesting that additional phosphorylation at the alternate coimmunoprecipitated with RelA using antisera against RelA/ serine residue $36 and $32, respectively, did not change p65 (see Materials and methods). The precipitates were boiled significantly the electrophoretic mobility of IKB~. These in SDS sample buffer, followed by SDS-PAGE and fluorography results demonstrate that phosphorylation of the IKB~ (lane 2). An amount equivalent to -50% of the initial reaction volume was loaded in lane 2, which is fivefold greater than that mutants in HeLa cell extracts correlates with their abil- loaded in lane 1. The bulk of the high molecular mass conju- ity to be ubiquitinated. gates in lane 2 is approximately 100 kD, as opposed to 200 kD The alkylating agent TPCK and the antioxidant pyro- seen in lane 1, and this may be attributable to "trimming" by lidinedithiocarbamate (PDTC) have been widely used to isopeptidases during the immunoprecipitation. inhibit the induced degradation of IKB~ (Beg et al. 1993; Henkel et al. 1993; Sun et al. 1993). These agents act by inhibiting the phosphorylation of IKB~. As shown in Fig- ure 4, C and D, TPCK (50 ~M) also inhibited phosphory- nus, and can associate with RelA/p65; Brown et al. lation and ubiquitination of IKBa in HeLa cell extracts 1995). (lane 9). In contrast, PDTC (50 ~M) did not inhibit either Analysis of these mutants in the in vitro ubiquitina- phosphorylation or ubiquitination of IKBR in this assay tion assay revealed an excellent correlation between (lane 10), probably because PDTC acts upstream of the their ability to be degraded in vivo in response to induc- okadaic acid activation step or, alternatively, generation ers and their ability to be ubiquitinated in vitro (Fig. 4C). of reactive oxygen intermediates (ROI) was not faithfully Specifically, AN (lane 12), $32A (lane 14), $36A (lane 16), reproduced in this system. Taken together, there was an and S32A/S36A (lane 18), which escape signal-dependent excellent correlation between phosphorylation and ubiq- degradation in vivo (Brockman et al. 1995), are also not uitination of IKB~ in vitro. We conclude that both serine ubiquitinated in vitro. In contrast, wild-type IKBc~ (lane residues 32 and 36 are required for ubiquitination of IKB~ 11), $32E (lane 15), and $36E (lane 17) are all ubiquiti- in vitro, most likely through direct phosphorylation of nated in this assay, consistent with their functional phe- these sites (see discussion). notype in vivo (Brockman et al. 1995). The aC mutant protein was ubiquitinated in vitro (lane 13), but its degradation is not enhanced in vivo following stimulation of Ubiquitinated IKBa bound to NF-KB is degraded by the 26S proteasome cells. However, the basal turnover of this mutant is in- distinguishable from that of the wild type (data not To determine whether ubiquitination of IKBa is required shown). Given that this/~C mutant does not associate for degradation by the 26S proteasome in vitro, we com- with RelA, it is likely that its behavior reflects signal- pared the fate of conjugated and unconjugated IKB~ when independent (basal) turnover of free IKBa (albeit at low incubated with purified 26S proteasome. IKBa labeled level) in the cell. It is possible that ubiquitination is also with [3SS]methionine was produced by in vitro transla- involved in the basal turnover of free IKBa and that the tion and incubated in HeLa cell extracts in the presence carboxy-terminal sequence including the PEST region is or absence of EDTA. (EDTA blocks ubiquitination and not required for ubiquitination of free IKB~. therefore serves as a control; see Fig. 2C). The IKBa/ A shorter exposure of the film shown in Figure 4, A NF-KB complex formed in vitro was then immunopre-

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Figure 4. Mutations that prevent the phosphorylation of IKB~ in vivo also abolish ubiquitination in vitro. (A) In vitro- translated IKBc~ mutants. IKBc~ mutants were translated in wheat germ extracts (Promega TNT system) in the presence of [3SS]methionine. The translation products were analyzed by SDS-PAGE and fluorography. {B) A shorter exposure of A showing the IKBc~ mutants before ubiquitination reactions. (C) Ubiquitination assays. IKB~ mutants shown in A were incubated in HeLa cell extracts at 37~ for 1 hr under conditions described in Fig. 2A. The reaction mixtures were then subjected to SDS- PAGE and fluorography. In lanes 9 and 10, TPCK (50 ~M) and PDTC (50 ~M) were added to the reactions containing wild-type IKBoL, respectively. {D) A shorter exposure of C, showing the phosphorylation proper- ties of the remaining unconjugated IKBa mutants, p-IKB~ is the phosphorylated form of IKB~ or IKB~ bearing glutamic acid substitutions at position 32 or 36.

cipitated with an anti-RelA antibody [see above and Fig. nated substrates. Similarly, no degradation of the conju- 3}. Both unconjugated and ubiquitinated IKB~ were iso- gates occurred in the absence of the 26S proteasome lated as a complex with NF-KB. The immunoprecipitated {solid triangle}. Importantly, unconjugated IKB~ was not IKBot proteins were then separated by SDS-PAGE as degraded by the 26S proteasome {open diamond). These shown in the fluorograph of Figure 5A {lanes 1,2). Quan- results clearly demonstrate that ubiquitination of IKBa titation of the data by Phosphorlmager analysis showed targets the protein for degradation by the 26S protea- that ubiquitin-conjugated IKBa contained 67% of the to- some. Thus, it appears that IKB~ is not only phosphory- tal radioactivity, and the remaining 33% was unconju- lated and ubiquitinated while associated with NF-KB, gated IKB~. When both conjugated and unconjugated but ubiquitinated IKB~ also serves as a substrate for the IKBtx were incubated with purified 26S proteasomes (23 26S proteasome when complexed to NF-KB. riM) in the presence of Mg and ATP, a significant {47%) reduction in the level of conjugated IKBc~ was observed Discussion (cf. lanes 2 and 4), whereas the amount of unconjugated IKBa did not significantly change (cf. lanes 1 and 3). The The transcription factor NF-KB is activated in response level of unconjugated IKBoL in lane 4 is slightly higher to a large number of distinct extracellular signals, all of than that in lane 2, probably because of isopeptidase ac- which result in the phosphorylation of IKB proteins (for tivities associated with the 26S proteasome (Eytan et al. review, see Siebenlist et al. 1994). Recently, serine resi- 1993). To directly measure the degradation of ubiquiti- dues 32 and 36 were shown to be required for phospho- nated IKBa by the 26S proteasomes, the degradation rylation and degradation of IKBoq and activation of NF-KB products were separated from undegraded IKB~ by tri- (Brockman et al. 1995; Brown et al. 1995). In this paper chloroacetic acid (TCA) precipitation, and the TCA-sol- we have shown that IKBa is ubiquitinated in response to uble radioactivity was determined. The percentage of stimulation both in vivo and in vitro, and that phospho- conjugate degradation, taking into account the unconju- rylation of IKBa is required for ubiquitination. In addi- gated IKB~ present in the conjugate sample, is plotted in tion, we show that ubiquitinated IKBot is degraded by the Figure 5B. Ubiquitinated IKBot is efficiently degraded by 26S proteasome in vitro. The latter observation is con- the 26S proteasome (17 nM, open square). Approximately sistent with previous observations showing that protea- 19% of the substrate was degraded within 5 min, and by some inhibitors block the degradation of IKBa in vivo 1 hr, 51% of the conjugates was degraded. Inclusion of (Finco et al. 1994; Miyamoto et al. 1994; Palombella et EDTA in the degradation reaction abolished degradation al. 1994; Traenckner et al. 1994; Alkalay et al. 1995; of the conjugates (solid square), indicating that the 26S DiDonato et al. 1995; Lin et al. 1995). Although these proteasome-catalyzed degradation is Mg-ATP depen- inhibitors were shown to act on purified proteasomes, dent. The 20S proteasome, which functions as the pro- they can also inhibit other proteases, such as calpains teolytic core of the 26S complex, did not degrade ubiq- and cathepsins. A series of peptide-aldehyde inhibitors uitinated IKB~ {open triangle). This is consistent with with different potencies (ICsos) against the purified pro- the role of the 26S proteasome in degrading ubiquiti- teasome in vitro (Rock et al. 1994) were tested for their

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Signal-induced ubiquitination of IKBa

Figure 5. Degradation of ubiquitinated Iv:Bet by the 26S proteasome. (A} In vitro-translated aSS-labeled IKBcx was incubated in HeLa extracts at 37~ for 2 hr under ubiquitination conditions (see Materials and methodsl, except that either Mg-ATP {lanes 2,4) or EDTA {lanes 1,3) was added to the reaction. The reaction mixtures were then immunoprecipitated by an antibody against RelA under conditions that allow coprecipitation of conjugated {lanes 2,4J or unconjugated {lanes 1,31 IKBa. The immunoprecipitates were then used directly for the degradation assay by the 26S proteasome. Lanes 1 and 2 are minus {- ) 26S; lanes 3 and 4 are plus {+ ) 26S i23 nM). The degradation reactions were carried out at 37~ for 1 hr in the presence of Mg-ATP, and the reaction mixtures were separated by SDS-PAGE, followed by fluorography. IBI Ubiquitinated IKBa in the immunoprecipitates described in A {lane 2) was incubated with 26S proteasome (17 nvtJ at 37~ in the presence of Mg-ATP {EJ). At indicated time points, an aliquot of the reaction was precipitated by 10% TCA. The TCA-soluble radioactivity was then determined by liquid scintillation counting. Similarly, degradation of unconjugated {freeJ IKBa was also determined {O ). In other reactions, 40 mM EDTA was added (11), 26S proteasome was omitted {&J, and 20S proteasome {49 nMJ was added (A1 instead of the 26S proteasome.

ability to inhibit NF-KB in vivo (Palombella et al. 1994; phoserine mimetics {but not alanine} at serine 32 or 36 Read et al. 19951. The rank order potencies of these com- are competent for degradation in vivo (Brockman et al. pounds in vitro and in vivo were in excellent agreement. 1995}. Third, the electrophoretic mobility of mutants In contrast, other calpain and cathepsin inhibitors, even containing mimetics at these serine sites coincides with at high concentrations, did not block NF-KB activation. that of the hyperphosphorylated form of endogenous Thus, it seems likely that the antagonistic effects of IKBet in activated cells {Fig. 4; data not shown}. Fourth, these agents on NF-KB activation derive from their in- disruption of all other potential phosphorylation sites in hibitory activity on the proteasome. Moreover, in related the amino terminus of IKBa has no effect on the function studies it was found that lactacystin, a highly specific of IKB(x {Brockman et al. 19951 Brown et al. 1995). Fifth, inhibitor of the proteasome (Fenteany et al. 19951, also removal of the carboxy-terminal PEST domain of IKBa prevents the processing of p105, the degradation of I~Bo~, fails to prevent inducible hyperphosphorylation in vivo and the activation of NF-KB in vivo {l. Hagler, O.J. Rando, {Brown et al. 1995). Taken together, we propose that G. Fenteany, S.L. Schreiber, and T. Maniatis, unpubl.}. In phosphorylation of serine residues 32 and 36 targets IKBa addition, a new class of synthetic proteasome inhibitors, to the ubiquitin-proteasome pathway. which do not affect any other known cellular proteases, The amino terminus of IKBa, which is not required for also blocks IKBc~ degradation and NF-KB activation {V. its association with NF-KB, is highly susceptible to pro- Palombella and Z. Chen, unpubl.). tease cleavage, and this susceptibility is unaffected by In this paper we show that deletion of the amino-ter- binding to the p65 subunit of NF-KB (laffray et al. 1995). minal 36 amino acids of IKBa, or serine to alanine sub- Thus, the amino terminus of I,:Bet appears to be exposed stitutions at either position 32 or 36, block in vitro ubiq- in the NF-KB complex and can therefore be recognized by uitination. Although direct biochemical proof for phos- an IKB kinase and presumably ubiquitination enzymes. phorylation of I~d3et at serine residues 32 and 36 is still In contrast, the central region of IKBa, which contains a lacking, many independent lines of evidence indicate tandem array of ankyrin repeats, is protease resistant and that these two residues are most likely the sites of phos- connected to the acidic carboxy-terminal domain con- phorylation. First, peptide mapping localizes inducible taining a PEST sequence (]affray et al. 1995}. Recent mu- phosphorylation to the amino terminus of IKBa {Brown tational studies have suggested that this PES'I sequence et al. 1995). Second, mutants of IKBa containing phos- may be required for signal-induced degradation of IKB~

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Chen et al.

(Miyamoto et al. 1994; Brockman et al. 1995; Brown et junction with chaperones, strips IKBcx away from NF-KB al. 1995}. The PEST sequences on the cyclin CLN3 pro- and then unfolds and degrades free IKBcx. tein contain phosphorylation sites, and these sites have We have shown that in vitro-translated IKBc~ is phos- been shown to be required for CLN3 degradation during phorylated and ubiquitinated in HeLa cell extracts in the the cell cycle (Yaglom et al. 1995). It should be noted that presence of the phosphatase inhibitor okadaic acid. Pre- the AC mutant tested in this study lacks the PEST se- vious studies have shown that IKBa can be inactivated in quence as well as additional carboxy-terminal sequences vitro by sphingomyelinase or ceramide iMachleidt et al. that are required for binding to NF-KB (Brockman et al. 1994), LPS (Ishikawa et al. 1995), or ~ protein kinase C 1995). Although the in vitro-translated AC protein is (~PKC) [Diaz-Meco et al. 1994). In contrast, the behavior ubiquitinated in vitro, the significance of this observa- of the in vitro system described here suggests a low level tion with respect to the regulated degradation of NF-KB- of constitutive IKB~xphosphorylation that would ordinar- bound IKB~ remains unclear. Notwithstanding this un- ily not be detected because of the presence of endoge- certainty, these findings suggest that the PEST se- nous phosphatases. However, in the presence of okadaic quences of IKB~x are not required for ubiquitination of acid, the constitutively phosphorylated IKBoL accumu- free IKB~. lates. Investigation of the amino acid sequence requirements Phosphorylation-dependent ubiquitination of IKB~ for the ubiquitin-proteasome-dependent degradation of could occur via two mechanisms, which are not mutu- other proteins has not revealed a common recognition ally exclusive. First, the phosphorylation of IKB~ may element (for review, see Ciechanover 1994). For example, enhance its affinity for constitutive ubiquitination en- a "destruction box" sequence has been shown to be re- zymes {E2s and E3s). Alternatively, one or more of the quired for the ubiquitin-mediated degradation of mitotic enzymes involved in ubiquitination may be activated by cyclins (Glotzer et al. 1991; Luca et al. 1991). In contrast, the same signal transduction cascade. An example of a region containing PEST sequences and multiple phos- such regulation is the activation of a cyclin ubiquitin phorylation sites is required for the degradation of CLN3 protein ligase (E3J by cdc2 (Hershko et al. 1994). (Yaglom et al. 1995). In another example, degradation of There are now several examples in which the ubiq- the transcription factor MAT~2 requires two different uitin-proteasome pathway plays an essential role in the regions of the protein, and these sequences have not been regulation of transcription factor levels. These examples found in other proteins (Hochstrasser et al. 1991). These include the degradation of yeast MATa2 (Hochstrasser et and other examples strongly suggest that different pro- al. 1991) and GCN4 (Komitzer et al. 1994} proteins, and tein substrates are recognized for degradation by the the mammalian c-Jun {Treier et al. 1994) and p53 pro- ubiquitin-proteasome pathway by distinct mechanisms teins [Schefhler et al. 1993). The example of NFKB 1/p 105 (Ciechanover 1994). The recognition of specific sub- is exceptional in that the proteasome selectively de- strates may involve the use of specific ubiquitin protein grades the carboxyl terminus of an inactive precursor ligases (Hershko et al. 1994). protein, leaving the amino terminus intact {Palombella Remarkably, neither phosphorylation nor ubiquitina- et al. 1994). The complete degradation of IKB~ leads to a tion results in the dissociation of IKB~ and NF-KB, and rapid and transient activation of NF-KB. The transient we have shown that the 26S proteasome recognizes and nature of the activation is a consequence of the positive degrades ubiquitinated IKBe~ in the ternary NF-KB com- autoregulation of the IKB~ gene by the activated NF-KB plex. The three-dimensional structure of an NF-KB pS0 and the subsequent restoration of the cytoplasmic IKB~ homodimer bound to DNA has been determined re- pool (Sun et al. 1993). In contrast, when the degradation cently (Ghosh et al. 1995; Muller et al. 1995). In addition, of another IKB protein, IKBB, is induced by LPS and IL-1, specific amino acids in the highly conserved Rel homol- the activation of NF-KB persists {Thompson et al. 1995). ogy domain have been shown to be required for interac- The degradation of IKBB, like that of IKBa, is inhibited by tions between the Drosophila Dorsal and Cactus pro- TPCK, which seems to block the activities of one or teins, homologs of NF-KB and IKB, respectively (Lehming more IKB kinases. IKBB contains an amino-terminal se- et al. 1995). The location of these amino acids in the quence strikingly similar to the Ser-32/Ser-36-1ike re- three-dimensional structure of the Rel homology do- gion of IKBa {Thompson et al. 1995). Thus, it seems main suggests that IKB may fit within a deep groove likely that the degradation of IKBB also involves the formed between the dimerization domain of the two sub- ubiquitin-proteasome pathway. units (Ghosh et al. 1995; Lehming et al. 1995; Muller et Because of the central role played by NF-KB and other al. 1995). Thus, IKBc~ must be phosphorylated and ubiq- Rel family members in the immune and inflammatory uitinated on the surface away from the dimerization do- responses, their activation would be an attractive target main, as the modified IKBe~ remains bound to NF-KB. for the development of pharmacological inhibitors. For How, then, is the IKBoL degraded as part of the NF-KB example, the genes encoding the cell adhesion molecules complex? An interesting possibility is suggested by the expressed on the surface of the vascular endothelium recent observations that molecular chaperones are re- require NF-KB for their induced expression by TNF~x and quired for the degradation of certain ubiquitinated sub- other inflammatory cytokines (for review, see Collins et strates by the proteasome (D.H. Lee, M. Sherman, and al. 1995). Recent studies have shown that the protea- A.L. Goldberg, pers. comm.). Perhaps, the 26S proteasome inhibitor MG132 blocks the induction of the leu- some binds to the ubiquitin chains on IKB~ and, in con- kocyte adhesion molecules E-selectin, VCAM-1, and

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ICAM-1 (Read et al. 1995). The functional consequence RelA-agarose was used for immunoprecipitation, the step in- of this inhibition was the prevention of lymphocyte at- volving addition of protein A-trisacryl was omitted. After a tachment to TNFa-treated endothelial monolayers. The brief centrifugation, the resin was washed four times with finding that ubiquitination is required for the protea- RIPA/0.1% SDS and then boiled in SDS sample buffer. Coimmunoprecipitations were carried out similar to the im- some-dependent degradation of IKBa provides additional, munoprecipitation described above, except that buffer A/0.2% and possibly more specific, targets for inhibition of the NP-40 (see above) instead of RIPA/0.1% SDS was used for an- inflammatory response. tibody incubations. The resin was then washed with buffer B (10 mM HEPES at pH 7.4, 1 mM EDTA, 10 mM KC1, 50 mM NaF, 50 Materials and methods mM glycerol-2-phosphate, 1 mM sodium orthovanadate, 0.1 rag/ Materials ml of PMSF, 0.2% NP-40, and 90 mM NaCI). The washed resin was either boiled in SDS sample buffer, eluted with 0.1 M Capso The proteasome inhibitor MG132 (Z-Leu-Leu-Leu-H) has been (pH 11.2), or used directly for assays (see below). Western blot described before (Palombella et al. 1994; Rock et al. 1994). Ca- analysis was performed according to Fan and Maniatis {1991). lyculin A and okadaic acid were purchased from GIBCO BRL. Antibodies against IKB~ (c-21, sc-371) and RelA/p65 (sc-109), as Ubiquitination assay well as the agarose conjugates of the RelA antibody (sc-109AC) In vitro-translated aSS-labeled IKBa was incubated with HeLa were purchased from Santa Cruz Biotechnology. Affinity-puri- extract (4.5 mg/ml) in the presence of an ATP regenerating sys- fied antibody specific for conjugated ubiquitin was provided by tem 150 mM Tris at pH 7.6, 5 mM MgC12, 2 mM ATP, 10 mM Dr. Cecile Pickart (State University of New York, Buffalo}. creatine phosphate, 3.5 U/ml of creatine kinase, 0.6 U/ml in- Ubiquitin was purchased from Sigma, and MeUb was prepared organic pyrophosphatase), together with ubiquitin (1 mg/ml), according to Hershko and Heller (1985}. Fluorescamine analysis okadaic acid {3 }~M), and Ubal {3 laM). The reactions were incu- showed that >95% of the lysine residues on MeUb was blocked. bated at 37~ for 1 hr unless otherwise indicated. After termi- Ubal was prepared according to Mayer and Wilkinson (1989). nating the reaction with SDS sample buffer, the reaction mix- 20S and 26S proteasomes were purified according to published ture was subjected to SDS-PAGE (9%) and fluorography. methods (Hough et al. 1987; Ganoth et al. 1988). Isolation of ubiquitin-IKBcx conjugates Plasmids, in vitro translation, and cell culture Ubiquitinated IKBcx was synthesized in a 300-~1 reaction mix- The IKBct mutants are described by Brockman et al. (1995}. ture containing 60 03 of in vitro-translated ass-labeled IxBa, 1.5 These mutants were subcloned into pBluescript [SK( + ), Strate- mg of HeLa extract, and other components of the ubiquitination gene] or pSP72 (Promega) for in vitro translation. Wild-type and reaction. The control reaction contained 40 mM EDTA instead mutant IKBtx proteins were produced and labeled with [3SS]me- of Mg-ATP in the mixture. After 2 hr of incubation at 37~ thionine by in vitro translation in TNT wheat germ extracts ubiquitinated IKBa and unconjugated IKBa were coimmunopre- {Promega} using RNA transcribed from NotI linearized plas- cipitated with RelA using 30 lal of anti-RelA agarose conjugates raids. The translation products were used directly in ubiquiti- (0.25 ixg/~l of antibody). Following incubation at 4~ for 1 hr, nation assays (see below). Jurkat ceils (ATCC) were cultured in the resin was washed three times with buffer B and once with RPMI 1640 medium supplemented with 10% fetal calf serum. buffer D (50 mM Tris at pH 7.6, 0.5 mM DTT}. The resin was For metabolic labeling with [35S]methionine/cysteine, 200 ~Ci/ then resuspended in buffer D and used directly in the conjugate ml of EXPRE3SSasS (Dupont NEN) was used in the labeling degradation assay. media lacking methionine and cysteine. Conjugate degradation assay Preparation of cell extracts Ubiquitinated IKBet suspension {~2000 cpm) was incubated Preparation of HeLa cytoplasmic extracts iS 100) was described with 17 nM of 26S proteasome in an ATP-regenerating system earlier (Fan and Maniatis 1991). These extracts were further (see above). At the desired time points, the reaction was concentrated by ammonium sulfate (80%) precipitation, fol- quenched by addition of 125 ~1 of 4% BSA and 575 ~1 of 12% lowed by extensive dialysis in 20 mM Tris (pH 7.6), 0.5 mM TCA. After removal of the TCA precipitates by centrifugation, DTT. The extracts were stored in aliquots at -80~ 600 ~1 of the supernatants was counted in a scintillation lurkat cell cytoplasmic extracts were prepared by lysing the counter. The results are expressed as percentage of the conju- ceils in a hypotonic buffer (buffer A) containing 10 mM HEPES gates that are degraded to TCA-soluble counts. IpH 7.4}, 1 mM EDTA, 10 mM KC1, 1 mM DTT, phosphatase inhibitors (50 mM NaF, 50 mM glycerol-2-phosphate, 1 mM so- Acknowledgments dium orthovanadate, 0.1 ~M okadaic acid), and protease inhib- We are grateful to R. Stein and other scientists at MyoGenics, itors {0.1 mg/ml of PMSF, 10 ~g/ml of leupeptin, 10 ~g/ml of Inc., for encouragement and support, and to A. Goldberg and D. aprotinin). Following incubation on ice for 15 rain, 0.2% NP-40 Finley for helpful advice and critical reading of the manuscript. was added to the lysate, and the mixture was placed on ice for We are also grateful to L. Parent for excellent technical assis- another 5 rain. After centrifugation at 16,000g for 5 rain at 4~ tance. We thank C. Pickart for the ubiquitin antibody. This the supematant (cytoplasmic extract) was stored at -80~ work was supported by MyoGenics, Inc., and by grants from the National Institutes of Health (T.M. and D.B.). J.H. was sup- Imrnunoprecipitation and Western blot analysis ported by a postdoctoral fellowship from the American Cancer Immunoprecipitation was carried out in RIPA buffer 150 mM Society {PF-4053). D.B. is an investigator of the Howard Hughes Tris-HCl at pH 8.0, 150 mM NaC1, 1% NP-40, 0.5% deoxy- Medical Institute. cholate) plus 0.1% SDS. Antibodies were incubated with cyto- The publication costs of this article were defrayed in part by plasmic extracts at 4~ for 1 hr. Protein A-trisacryl (Pierce) payment of page charges. This article must therefore be hereby equilibrated in the same buffer was then added to the mixture, marked "advertisement" in accordance with 18 USC section and the incubation was continued for another hour. When anti- 1734 solely to indicate this fact.

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Chen et al.

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Signal-induced ubiquitination of IKBot

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GENES & DEVELOPMENT 1597 Downloaded from genesdev.cshlp.org on October 4, 2021 - Published by Cold Spring Harbor Laboratory Press

Signal-induced site-specific phosphorylation targets I kappa B alpha to the ubiquitin-proteasome pathway.

Z Chen, J Hagler, V J Palombella, et al.

Genes Dev. 1995, 9: Access the most recent version at doi:10.1101/gad.9.13.1586

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