The Death Domain Kinase RIP Mediates the TNF-Induced NF-␬Bsignal

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Immunity, Vol. 8, 297–303, March, 1998, Copyright 1998 by Cell Press The Death Domain Kinase RIP Mediates the TNF-Induced NF-␬BSignal

Michelle A. Kelliher,*§ Stefan Grimm,* The RIP death domain also binds RAIDD (RIP-associ- Yasumasa Ishida,* Frank Kuo,‡ ated ICH1/CED3-homologous protein with a death do- Ben Z. Stanger,* and Philip Leder† main) (Duan and Dixit, 1997). Hence, RIP may directly *Department of Genetics link the TNFR1 complex to the ICE/caspase cascade. Harvard Medical School RIP also interacts via separate domain(s) with TRAF2, † Howard Hughes Medical Institute known to mediate the tumor necrosis factor receptor 2 ‡ Department of Pathology (TNFR2)–NF-␬B signal (Hsu et al., 1996b). Finally, RIP Brigham and Women’s Hospital interacts in vitro with the recently isolated death recep- Boston, Massachusetts 02115 tor 3 (DR3 or WSL1 or Apo-3), a lymphoid-specific receptor also capable of inducing both apoptosis and NF-␬B activation (Chinnaiyan et al., 1996; Kitson et al., 1996; Marsters et al., 1996). Summary The 74 kDa RIP protein consists of three domains: an N-terminal kinase domain, an ␣-helical intermediate The death domain serine/threonine kinase RIP inter- domain, and a C-terminal death domain (Stanger et al., acts with the death receptors Fas and tumor necrosis 1995). RIP is unique among the known death domain receptor 1 (TNFR1). In vitro, RIP stimulates apoptosis, proteins in that it contains an active serine/threonine SAPK/JNK, and NF-␬B activation. To define the physi- kinase domain capable of autophosphorylation (Hsu et ologic role(s) that RIP plays in regulating apoptosis in al., 1996b; Ting et al., 1996). Overexpression of the RIP vivo, we introduced a rip null mutation in mice through protein induces NF-␬B activation (Hsu et al., 1996b; Ting homologous recombination. RIP-deficient mice ap- et al., 1996), stress activated protein kinase/c-Jun N pear normal at birth but fail to thrive, displaying exten- terminal kinase (SAPK/JNK) activation (Liu et al., 1996), sive apoptosis in both the lymphoid and adipose tissue and apoptosis (Stanger et al., 1995; Grimm et al., 1996; and dying at 1–3 days of age. In contrast to a normal Hsu et al., 1996b). In addition, dominant-negative forms of RIP and TRAF2 have been shown to specifically inhibit thymic anti-Fas response, ripϪ/Ϫ cells are highly sensi- tumor necrosis factor ␣ (TNF␣)–induced NF-␬B activa- tive to TNF␣-induced cell death. Sensitivity to TNF␣- tion and SAPK/JNK activation (Hsu et al., 1996b; Liu et mediated cell death in ripϪ/Ϫ cells is accompanied by al., 1996). Although the role of the SAPK/JNK pathway a failure to activate the transcription factor NF-␬B. in apoptosis remains unclear, activation of the transcription factor NF-␬B has recently been associated with Introduction protection from TNF-induced cell death (Beg and Balti- more, 1996; Liu et al., 1996; Van Antwerp et al., 1996; Fas and tumor necrosis factor receptor 1 (TNFR1) are Wang et al., 1996). Thus, the TNFR1 complex is capable cytokine receptors that activate cell death pathways of transducing signals that either activate or suppress through an intracellular homology region called the cell death. These disparate signals may be generated death domain (Nagata, 1997). Fas ligand rapidly induces through modulation of RIP. Deletion analysis of the RIP protein has in fact re- cell death by interacting with Fas, which engages FADD vealed that distinct TNFR1 signals are transduced by the (Fas-associated death domain)/FLICE (FADD-like ICE) kinase, intermediate, and death domains. Since TNF- proteins (Chinnaiyan et al., 1995; Muzio et al., 1996). induced NF-␬B activation requires serine phosphoryla- TNFR1 activation results in stimulation of the cell death tion of the NF-␬B inhibitor protein (I␬B) (Beg and Bald- pathway (Tartaglia et al., 1993) or induction of a protec- win, 1993), it is possible that RIP may activate the I␬B tive pathway mediated by activation of the nuclear tran- kinase pathway. Yet, RIP mutants that only contain a scription factor ␬B (NF-␬B), or both (Hsu et al., 1995). kinase domain fail to activate NF-␬B, but retain an ability Originally identified in a yeast two-hybrid screen for to bind TRAF2 (Hsu et al., 1996b). Similarly, a kinase- Fas-interacting proteins, RIP is a serine/threonine ki- defective RIP restores the TNF-induced NF-␬B signal nase that contains a death domain (Stanger et al., 1995; in a RIP-deficient clone (Ting et al., 1996). However, Hsu et al., 1996b). In mammalian cells, RIP interacts expression of the RIP intermediate domain results in weakly with the death receptors Fas and TNFR1 (Hsu both NF-␬B activation and TRAF2 interaction. All RIP et al., 1996b). Ligand-induced trimerization of the TNFR1 mutants containing the C-terminal death domain are results in the recruitment of the death domain adapter capable of inducing cell death when overexpressed, protein TRADD (TNFR1-associated death domain pro- demonstrating that the death domain is both necessary tein) (Hsu et al., 1995). TRADD is also known to interact and sufficient for apoptosis. Thus, the kinase and inter- with FADD and with the RING finger–containing protein mediate domains of RIP are involved in activation of NF- TNFR receptor–associated factor 2 (TRAF2) (Hsu et al., ␬B and TRAF2 binding, whereas the RIP death domain 1996a). RIP interacts via its death domain with TRADD, associates with TRADD and other death domain pro- and this interaction is TNF dependent (Hsu et al., 1996b). teins to elicit the apoptotic response. The exact contribution of RIP to the Fas/TNFR1 pathways has been difficult to ascertain because of the com- § To whom correspondence should be addressed (e-mail: kelliher@ plexity and apparent functional redundancy in the mam- rascal.med.harvard.edu). malian cell death pathway. To elucidate the precise Immunity 298

Figure 1. Generation of ripϪ/Ϫ Mice (Top) Targeting of the rip gene. A map of the targeting vector is shown along with the wild- type and mutant loci. Restriction enzyme sites: As, Asp718 ; H, HindIII; E, EcoRI; and C, ClaI. Homologous recombination of wild type DNA with pPNT-RIP results in the inser- tion of pgk-neo gene in exon 2 of the rip gene. (Bottom) Southern blot analysis of DNA prep- arations from tails of 18 day embryos from heterozygote matings. The three genotypic categories are indicated as wild type (ϩ/ϩ), heterozygote (ϩ/-), and homozygote (Ϫ/Ϫ). Digestion of genomic DNA with Asp718 fol- lowed by hybridization with the probe de- picted above the wild-type locus generates a 21 kb fragment for the wild-type locus and a 17 kb fragment for the targeted locus.

functional role of RIP in mediating these signal transduc- than their littermates, weighing one-third to one-half tion pathways in vivo, we have generated RIP-deficient their normal weight. These pups, although feeding, mice. Mice lacking the RIP death domain kinase appear failed to gain weight and died. No ripϪ/Ϫ pups survived normal at birth, but become runted and die postnatally. beyond 3 days after birth. At autopsy, edema of the Hence, this study reveals a novel developmental role neck and axillary and inguinal areas was observed in for RIP beyond that of TNF signaling. In addition to the all of the ripϪ/Ϫ animals. Examination of ripϪ/Ϫ animals postnatal lethality, RIP-deficient mice exhibit extensive revealed that all internal organs appeared grossly normal. apoptosis in the lymphoid and adipose tissues, support- ing the notion that RIP functions in vivo to suppress cell ripϪ/Ϫ Mice Exhibit Immune and Adipose death. Tissue Defects Since RIP has been implicated in Fas ligand– and TNF- Results and Discussion mediated cell death, the lymphoid lineages in RIP-deficient mice were examined by flow cytometry. To the Targeted Disruption of rip extent that B cell development could be analyzed at To disrupt the murine rip gene in embryonic stem (ES) embryonic day 18, ripϪ/Ϫ mice contained similar numbers cells by homologous recombination, we designed a tar- of B220ϩ cells in bone marrow as control littermates geting construct that would disrupt exon 2 of the rip (Figure 2B). Flow cytometric analysis of thymus from gene 50 bp after the ATG initiation codon (Figure 1, top). some embryonic day 18 RIP-deficient mice revealed To detect homologous recombinants by Southern blot, normal CD4 and CD8 thymocyte populations (Figure genomic DNA isolated from transfected ES cells was 2A), indicating that thymocyte maturation can proceed digested with Asp 718 and hybridized with a flanking normally in the absence of RIP. Yet in other ripϪ/Ϫ mice probe (Figure 1, top and bottom). This assay generated at embryonic day 18, dramatic decreases in thymocyte a 21 kb wild-type band and a 17 kb mutant band. ES viability were observed. Thymic sections from these cells with a disrupted rip allele were injected into blasto- ripϪ/Ϫ mice contained numerous cells with pyknotic nu- cysts to generate chimeric animals. Male mice from a clei, where nuclear fragmentation appeared to have clone that gave greater than 90% chimerism transmitted taken place (Figure 3B). Numerous apoptotic bodies the recombinant allele to their progeny. Heterozygous were observed throughout the thymus in both the cortex animals were indistinguishable from wild-type animals and the medulla. The excessive thymic cell death was and subsequently were interbred. observed in all ripϪ/Ϫ mice examined, evident in some ripϪ/Ϫ mice at embryonic day 18 and in others at birth. Early Postnatal Lethality in ripϪ/Ϫ Mice In addition to the thymic effects, RIP-deficient mice No gross phenotypic abnormalities were observed in also displayed abnormal lymph node development. In the heterozygous rip mice but, of a total of 50 genotyped contrast to littermate controls, all of the developing ripϪ/Ϫ pups, no homozygotes were obtained, suggesting that lymph nodes lacked normal architecture, appearing ne- RIP is essential for survival. Timed matings were subse- crotic, edematous, and infiltrated with granulocytes(Fig- quently used to obtain embryos at different stages of ures 3C and 3D). Granulocytic infiltration and tissue development. Up to late gestation, wild-type, hetero- damage was also observed in the subcutaneous tissue, zygous, and homozygous animals were present in the specifically in the dermis and thermogenic fat (data not expected Mendelian ratios. Two to 3 days after birth, shown and Figures 3E and 3F). approximately one quarter of the pups appeared smaller Consistent with the tissue inflammation, ripϪ/Ϫ mice RIP Mediates the TNF-Induced NF-␬B Signal 299

those of control littermates. However, differential blood analysis of ripϪ/Ϫ mice revealed an increased percentage of myeloid cells (70% in ripϪ/Ϫ vs. 20%–25% in ripϩ/ϩ) and a concomitant decreasein peripheral blood lympho- cytes. The increased percentage of myeloid cells in the peripheral blood most likely reflects the granulocytic infiltration of lymphoid and subcutaneous tissues. In the absence of RIP, mice exhibit postnatal lethality and extensive cell death in the lymphoid and adipose lineages, revealing that the primary function of RIP in these tissues is to provide a survival signal. Furthermore, the postnatal lethality and adipose tissue effects reveal role(s) for the RIP kinase in mediating other, as yet unde- fined, signaling pathways.

RIP Does Not Mediate Fas-Induced Apoptosis To explore the role of RIP in Fas-mediated signaling, heterozygote rip mice were mated, and at embryonic day 18 the thymus was removed from all embryos. To determine the percentage of thymocytes susceptible to Fas killing, half of the thymus was stained with anti-CD4 and anti-CD8 antibodies and analyzed by flow cytometry. The remaining thymocytes were stimulated with Figure 2. Analysis of Hematopoietic Cells in ripϪ/Ϫ Mice anti-mouse Fas antibody, and 18 hr later the percentage Flow cytometric analysis of hematopoietic cells in the thymus and of dead cells was determined by trypan blue staining. bone marrow from ripϩ/ϩ and ripϪ/Ϫ neonatal littermates. (A) Viable Ϫ/Ϫ cells residing in the lymphoid gate were analyzed. (B) Total white Although the cell death induction in rip thymocytes blood cells were analyzed. The percentage of total cells in a particu- appeared somewhat less efficient as compared to that lar region is indicated. of ϩ/ϩ or ϩ/Ϫ thymocytes, this reflects the decreased percentage of viable CD4ϩCD8ϩ cells in the ripϪ/Ϫ mice. Viable CD4ϩCD8ϩ thymocytes from RIP-deficient mice exhibited an immature periportal myeloid cell hyperpla- were as susceptible to killing induced by anti-Fas as sia and an increase in the splenic red pulp (data not wild-type and heterozygote controls (Table 1). Similar shown). The total red blood cell and white blood cell results were obtained when RIP-deficient embryonic fi- counts of ripϪ/Ϫ mice were not significantly different from broblasts (EFs) were tested for their susceptibility to

Figure 3. ripϪ/Ϫ Mice Exhibit Immune andAdi- pose Abnormalities Sections through embryonic day 18 thymi from ripϩ/ϩ (A) and ripϪ/Ϫ (B) mice. Numerous pyknotic nuclei are observed in rip Ϫ/Ϫ thymus (arrow in [B]). Sections through the developing cervical lymph nodes from ripϩ/ϩ (C) and ripϪ/Ϫ (D) neonates.Arrow, region of granulocytic infiltration. Section through thermogenic brown fat of ripϩ/ϩ (E) and ripϪ/Ϫ (F) mice. Immunity 300

Table 1. Fas-Mediated Apoptosis in rip ϩ/ϩ, ϩ/Ϫ, and Ϫ/Ϫ Thymocytes Genotype % Dead % CD4ϩCD8ϩ % Dead/%CD4ϩCD8ϩ ϩ/ϩ 69.5 Ϯ 9.8 91.5 Ϯ 3.0 0.76 Ϯ 0.09 ϩ/Ϫ 73.4 Ϯ 10.4 90.0 Ϯ 4.0 0.81 Ϯ 0.11 Ϫ/Ϫ 56.3 Ϯ 24.3 68.9 Ϯ 30.0 0.83 Ϯ 0.21

Embryonic day 18 thymus was stimulated with 1 ␮g/ml anti-mouse Fas antibody (Jo2) in the presence of 100 ng/ml cycloheximide for 18 hr. Percentage of dead cells was determined by trypan blue staining. Four wild-type (ϩ/ϩ), seven heterozygote (ϩ/Ϫ), and seven rip-deficient (Ϫ/Ϫ) animals were tested. anti-Fas killing (data not shown). Therefore, in the ab- 1996; Van Antwerp et al., 1996; Wang et al., 1996). We sence of RIP, Fas-induced apoptosis remains unaf- reasoned that the TNF sensitivity observed in rip Ϫ/Ϫ cells fected, demonstrating that RIP is not required for Fas may reflect an inability to activate NF-␬B. To determine signaling. whether TNF-induced NF-␬B activation occurs in the absence of RIP, nuclear extracts obtained from embry- ripϪ/Ϫ Cells Are Sensitive to TNF␣-Induced onic day 18 fetal liver-derived Abelson-transformed Cell Death preB cell lines treated with hTNF␣ were analyzed by The rapid recruitment of RIP to the TNFR1 upon ligand electrophoretic mobility shift assay using an NF-␬B-spe- stimulation (Hsu et al., 1996b) prompted us to investi- cific probe. No evidence of NF-␬B activation was ob- gate whether ripϪ/Ϫ cells differed in their response(s) to served in TNF-treated ripϪ/Ϫ cells, whereas an increase TNF␣ . Since human TNF␣ (hTNF␣) binds exclusively to in NF-␬B binding activity was evident in TNF-treated mouse TNFR1 and not to TNFR2 (Lewis et al., 1991), we ripϩ/ϩ cells (Figure 5A). As expected, the NF-␬B activity were able to distinguish between effects mediated by observed in wild-type cells was reactive to antisera spe- the two mouse TNF receptors. A 4 hr treatment of ripϪ/Ϫ cific for p65 (data not shown). No differences in binding fibroblasts with human TNF␣ resulted in a dramatic de- activity were observed when the nuclear extracts de- crease in cell viability (Figure 4). In contrast, no substan- rived from the ripϩ/ϩ and ripϪ/Ϫ cells were tested with an tial effect on viability of ripϩ/ϩ EFs was noticed at any octamer-1–specific probe (Figure 5B). Failure to activate time after TNF␣ treatment (Figure 4). Thus, the absence NF-␬B was not due to down-regulation of the NF-␬B of RIP sensitizes fibroblasts to TNF␣-induced cell death, suggesting that the normal function of RIP in the TNFR1 pathway may be to mediate a survival signal. ripϪ/Ϫ Cells Fail to Activate NF-␬B in Response to TNF␣ Several recent studies have demonstrated that activation of the transcription factor NF-␬B prevents TNF- induced cell death (Beg and Baltimore, 1996; Liu et al.,

Figure 5. Gel Shift Analysis of ripϩ/ϩ and ripϪ/Ϫ Cells Treated with Figure 4. ripϪ/Ϫ Fibroblasts Are Susceptible to TNF␣-Induced Cell TNF Death PreB cell lines were left untreated (Ϫ) or stimulated with mouseTNF␣ ripϩ/ϩ and ripϪ/Ϫ tail fibroblasts were treated with hTNF␣ (10 ng/ml) (10ng/ml) or hTNF␣ (10 ng/ml). Nuclear extracts were prepared and for 4, 12, and24 hr. Viable cells remaining after treatment with hTNF␣ analyzed for NF-␬B activation by gel shift with 32P end–labeled NF- are shown as a percentage of viable untreated cells. Standard error ␬B probe (A) or a control Oct-1 probe (B). Activation of NF-␬Bby of the mean was calculated from four independent readings within DOC (C). Cytoplasmic extracts from ripϩ/ϩ and ripϪ/Ϫ cells were left a single experiment. Identical results were obtained with multiple untreated or reacted with DOC and then analyzed by NF-␬B gel independent isolates of embryonic tail fibroblasts. shift. RIP Mediates the TNF-Induced NF-␬B Signal 301

abnormalities. Histologic analysis did reveal necrosis of the thermogenic fat, suggesting potential defect(s) in adipocyte function. The thymic cell death is first apparent in the ripϪ/Ϫ embryos at embryonic day 18 and corre- lates with the emergence of the CD4ϩCD8ϩ cells in the fetal thymus. Yet postnatal lethality and thymic cell death are not features observed in TNFR1-deficient mice (Pfeffer et al., 1993; Rothe et al., 1993). Therefore, RIP may be acting in concert with other TNFR family members, possibly DR3 in the thymus (Marsters et al., 1996). Alternatively, the thymic phenotype observed in RIP- deficient mice may be an indirect effect of the mutation and, for example, the result of corticosteroid release, Figure 6. Normal Cytokine-Induced SAPK/JNK Activation in ripϪ/Ϫ as reported in TRAF3-deficient mice (Xu et al., 1996). It Cells seems unlikely that the RIP thymic cell death results ripϩ/ϩ and ripϪ/Ϫ fibroblasts were incubated with TNF (A) or IL-1 (B) from concentrated steroid levels since the phenotype for the various times indicated or with sorbitol (S) for 15 min. Protein is first evident in utero, when small molecules such as lysates from individual samples were immunoprecipitated with a SAPK/JNK-specific antibody, and kinase activity was measured us- steroids readily diffuse across the placenta. ing a GST-ATF-2 fusion protein as the substrate, as described in Defects in lymph node development are not observed Experimental Procedures. in TNFR1- or TNFR2-deficient mice (Pfeffer et al., 1993; Rothe et al., 1993; Erickson et al., 1994) but are apparent in RIP-deficient mice, lymphotoxin␣ (LT␣)–deficient heterodimer in ripϪ/Ϫ cells. Treatment of cytoplasmic ex- mice (DeTogni et al., 1994), and LT␤-deficient mice (Koni tracts with the detergent DOC, which activates NF-␬B, et al., 1996). LT␣-deficient mice lack all lymph nodes, revealed that ripϩ/ϩ and ripϪ/Ϫ cells expressed compara- whereas LT␤-deficient mice have normal cervical and ble amounts of NF-␬B (Figure 5C). Similar results were mesenteric lymph nodes. RIP-deficient mice, like LT␣- obtained with nuclear extracts derived from human deficient mice, fail to develop any normal lymph node TNF␣-treated ripϪ/Ϫ EFs (data not shown). structures. Taken together, these studies suggest that NF-␬B can be activated by a variety of other agents, RIP might mediate growth signals from additional TNFR including the cytokine interleukin-1 (IL-1) and the bacte- members, possibly the LT␤-receptor, known to bind a rial mitogen lipopolysaccharide (Baeuerle and Henkel, cell surface LT␣–LT␤ heteromer (Crowe et al., 1994), or

1994). However, a dominant-negative RIP has been the putative LT␣3-receptor. shown to inhibit specifically TNF-induced, not IL-1– The phenotype of the RIP-deficient mouse is remark- induced, NF-␬B activation (Hsu et al., 1996b). Consistent ably similar to TRAF2- deficient mouse (Yeh et al., 1997). with these studies, no differences in NF-␬B activity were Both mutant mice exhibit neonatal lethality character- observed when ripϩ/ϩ and ripϪ/Ϫ cells were treated with ized by a wasting syndrome, lymphopenia, and thymic IL-1␣ or lipopolysaccharide, indicating that RIP is a spe- cell death, suggesting that RIP and TRAF2 have similar cific mediator of the TNF-induced NF-␬B pathway (data developmental roles. Yet in the absence of TRAF2, TNF- not shown). induced NF-␬B activation appears for the most part normal, and sensitivity to TNF-induced cell death is accom- ripϪ/Ϫ Cells Exhibit Normal Cytokine-Induced panied by an inability to stimulate SAPK/JNK activity SAPK/JNK Activation (Yeh et al., 1997). In contrast, in the absence of RIP, In addition to TNF-induced NF-␬B activation, RIP has TNFR1 activation fails to result in NF-␬B activation, and also been implicated in the SAPK/JNK response to TNF␣ consequently ripϪ/Ϫ cells undergo TNF␣-induced cell (Liu et al., 1996). To test whether the cytokine-induced death. Together, these studies firmly establish that SAPK/JNK response was affected by mutation of rip, TRAF2 mediates TNF-induced SAPK/JNK activation and ripϩ/ϩ and ripϪ/Ϫ cells were treated with human TNF␣ or that the primary function of RIP in the TNFR1 pathway mouse IL-1␣ for varying periods of time and JNK activity is to transduce the NF-␬B signal. measured by an immune complex kinase assay. In con- Activation of NF-␬B requires the site-specific serine trast to studies in which a dominant-negative RIP inhib- phosphorylation of the NF-␬B inhibitor protein, I␬B(Ver- its TNF-induced JNK activation (Liu et al., 1996), no ma et al., 1995; Baldwin, 1996). Recently, several groups differences in JNK activation were observed when wild- have identified I␬B kinases, designated IKK-␣ and IKK-␤, type and ripϪ/Ϫ cells were treated with TNF␣ or IL-1 which mediate the Ser32/Ser36 phosphorylation of I␬B␣ (Figures 6A and 6B). Taken together, these results sug- and Ser19/Ser23 phosphorylation of I␬B␤ (DiDonato et gest that protection from TNF-induced cell death medi- al., 1997; Mercurio et al., 1997; Regnier et al., 1997; ated by RIP involves NF-␬B and not SAPK/JNK acti- Woronicz et al., 1997; Zandi et al., 1997). TNFR1 activa- vation. tion may stimulate RIP kinase activity, which then phos- In the absence of the RIP death domain–containing phorylates the NF-␬B-inducing kinase, NIK (Malinin et protein, mice exhibit postnatal lethality and immune and al., 1997). Activated NIK in turn phosphorylates IKK-␣ adipose lineage defects. Although ostensibly normal at and IKK-␤, resulting in I␬B␣ phosphorylation and subse- birth, RIP-deficient animals fail to gain weight, and die. quent degradation. Yet, a kinase defective RIP restores Gross and histologic examination of organs that must NF-␬B inducibility in a RIP-deficient, mutagenized Jur- function postnatally (kidneys, liver, lung, and gastroin- kat clone (Ting et al., 1996). Similarly, deletion studies testinal and neuromuscular systems) reveals no obvious map the region required for NF-␬B activation to the Immunity 302

␣-helical, intermediate domain of the RIP protein (Hsu Kinase Assay et al., 1996b; Ting et al., 1996), suggesting that the RIP Fibroblasts (5 ϫ 106)were left untreated or treated with hTNF␣ (15 kinase is not involved in NF-␬B regulation. Both studies, ng/ml), mouseIL-1␣ (4 ng/ml) for various periods or 0.4 M sorbitol for 15 min. Cells were then lysed in an ice-cold lysis buffer (potassium however, involve RIP overexpression, which may cause phosphate/EDTA [pH 7.05], 5 mM EGTA [pH 7.2], 10 mM MgCl2,50 TRAF aggregation and NF-␬B activation. Further study mM ␤-glycerophosphate [pH 7.2], 0.5% NP-40, 0.1%Brij-35 [Sigma], of the I␬B kinase pathway in the RIP-deficient cellshould 1 mM dithiothreitol, 1 mM sodium orthovanadate, 1 mM phenylmeth- reveal the mechanism of RIP-mediated NF-␬B acti- ylsulfonylfluoride, 0.7 ␮g/ml pepstatin, and 0.5 ␮g/ml leupeptin). vation. Cleared lysates were adjusted to equal protein concentrations as determined by the Bio-Rad protein assay. SAPK/JNK were immunoprecipitated using a rabbit polyclonal anti-SAPK/JNK reactive Experimental Procedures against JNK1 (Santa Cruz Biotech) and harvested on protein A-Sepharose beads. The immune complex was washed three times Generation of ripϪ/Ϫ Mice and then the kinase assay performed in 20 ␮l of kinase buffer (20 Recombinant phage containing genomic DNA of the rip locus were mM HEPES [pH 7.2], 10 mM MgCl2, 0.1 mg/ml bovine serum albumin, 32 isolated from a 129 mouse library (Stratagene) by using a mouse 3mM␤-mercaptoethanol), 10 ␮Ci P, and 1.5 ␮g of glutathione rip cDNA probe (Stanger et al., 1995). Three genomic clones were S-transferase (GST)–ATF-2 fusion protein (Gupta et al., 1995) for 15 subcloned into pBluescript IIKS(ϩ) (Stratagene) and mapped using min at 30ЊC. The samples were separated on a 12% sodium dodecyl restriction enzymes. The rip targeting vector was generated with sulfate–polyacrylamide gel and visualized by autoradiography. the pPNT plasmid (Tybulewicz et al., 1991). The upstream genomic fragment was excised from a genomic clone as a 5.5 kb NotI–ApaI Acknowledgments fragment (the NotI site comes from the Bluescript polylinker) and inserted into the NotI/XhoI sites of pPNT. The downstream fragment We thank Fen Zhou for her technical assistance, Cathie Daugherty was excised as a 5.5 kb ApaI–Asp718 fragment and cloned into the for the ES cell injections, and Peter Juo and Dr. John Blenis for the Asp718 site of pPNT. Homologous recombination with this construct GST-ATF-2fusion protein and technical advice.M. A. K. is supported (pPNT-RIP-KO) would result in truncation of the RIP polypeptide at by a Leukemia Society Special Scholar Award. S. G. is supported residue 50, thereby deleting most of the RIP protein (583 of 633 by the AIDS Stipendium of the Deutsche Krebsforschungszentrum, amino acids). pPNT-RIP-KO was linearized at the unique NotI site Heidelberg. and electroporated into TC1 ES cells and selected with G418 and FIAU. The culture, electroporation, and selection of TC1 cells were carried out as described (Deng et al., 1995). G418- and FIAU-resis- Received August 15, 1997; revised January 19, 1998. tant colonies were isolated and genotyped by Southern blot analysis using an external DNA probe (Figure 1A). ES cells heterozygous for References the targeted mutation were microinjected into C57BL/6 blastocysts to obtain germline transmission. Heterozygotes were interbred to Baldwin, A.S. (1996). The NF-kappa B and I kappa B proteins: new obtain homozygotes. discoveries and insights. Annu. Rev. Immunol. 14, 649–681. Baeuerle, P., and Baltimore, D. (1988). 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