Supplementary Information s75

Supplementary Information

Materials and Methods

Plasmids and reagents. Full-length human A20 was subcloned into the pJDM vector or pMSCVhygro (Clontech) with an N-terminal FLAG tag or into the pST239 vector

(Genentech). Deletion mutants were generated by standard PCR methods and point mutants were generated using a Quikchange Kit as directed by the manufacturer

(Stratagene). Mammalian expression constructs for myc-RIP, FLAG-TRAF2, and

FLAG-TRAF6 were gifts from Dr. David Goeddel (Tularik, Inc., CA, USA). HA-Ub was a gift from Dr. Dirk Bohmann (University of Rochester, NY, USA). K48-only and

K63-only Ub with N-terminal 3xHA tags were synthesized by MCLAB and subcloned into pcDNA3.1 (Invitrogen). Antibody reagents were purchased from the indicated vendors: FLAG M2 monoclonal and affinity gel (Sigma); c-myc 9E10 monoclonal

(Roche); c-myc 9E10 affinity matrix and HA monoclonal (Covance); Ni-NTA agarose

(Qiagen); -tubulin and actin monoclonal (ICN Biomedicals); TRAF2 (H-249) and

TNFR1 (H-271) polyclonals and ubiquitin (P4D1) monoclonal (Santa Cruz Biotech); biotin-HRP (Cell Signaling Tech); anti-human A20 and anti-human and anti-murine RIP monoclonals (Pharmingen); RIP polyclonal (Zymed); goat anti-murine TNFR1(R&D).

Monoclonal antibodies against C-terminal human A20 (clone 1B9.6) were produced at the University of Michigan and monoclonal anti-TNFR1 antibodies (clone 985) were produced at Genentech. Hamster anti-murine TNFR1 (55R286) was a gift from Dr.

Kathy Sheehan (Washington University, MO, USA) and hUBC13 antibody was a gift from Dr. Wei Xiao (University of Saskatchewan, Canada). Recombinant human and murine TNF- were produced at Genentech.

1 Cell culture, transfections, and luciferase reporter assays. HEK293T cells1 and

MEFs2 were cultured as described. HeLaS3 cells were cultured in 50:50 medium with

10% fetal bovine serum, 1% Pen/Strep, 1x GHT, and 1x L-Glutamine. HEK293T cells were transfected with Geneporter2 Transfection Reagent (Gene Therapy Systems) and

MEFs were transfected with Lipofectamine 2000 (Invitrogen) as recommended by the respective manufacturers. Alternatively, MEFs were infected with recombinant virus obtained from A20-pMSCVhygro-transfected 293 Phoenix packaging cells. Transfection with NF--driven luciferase and Renilla luciferase constructs and analysis of reporter activity have been described1. To prevent myc-RIP-induced apoptosis, HEK293T cells were co-transfected 1:1 with a CrmA mammalian expression construct.

Immunoprecipitations and immunoblotting. Unless otherwise noted, HEK293T cells were used for transfections and analysis of protein interactions. In some cases (Fig. S6,

2e, 3c), cells were treated prior to collection with 25µM MG-132 (Calbiochem) and MG-

132 was included in the lysis buffer. Cells were washed with PBS and lysed for 30 minutes at 4˚ C in a buffer containing 120 mM NaCl, 50 mM HEPES pH 7.2, 1 mM

EDTA, 0.1% NP-40, and Complete protease inhibitor cocktail (Roche). Lysates were cleared by centrifugation and proteins were immunoprecipitated 2 hours to overnight with anti-FLAG or anti-myc affinity gel at 4˚ C. Immunoprecipitates were washed three times with lysis buffer, proteins were reduced and alkylated, separated by SDS-PAGE, and transferred onto PVDF membranes (Invitrogen) following standard procedures. Since components of the TNFR1 signaling complex are degraded in lipid-insoluble rafts3, A20-

2 mediated RIP degradation was also assessed using the lysis buffer described above with either 1% Triton X-100 and 0.5% Brij-78, or 1% Triton X-100, 1% SDS, and 0.5% Brij-

78 added to solubilize lipid raft components. A20 promoted RIP degradation equally well in all lysis buffers tested (results not shown). Endogenous TNFR1 immunoprecipitations were performed as previously described4 with HeLaS3 cells (Fig.

S6) or with MEFs (Fig. 4d, 4e; where 7 80% confluent 15-cm plates were used per lane) using the species-specific TNFR1 monoclonal antibodies described above.

FLAG elutions and protein purifications. For FLAG elutions, HEK293T cells were transfected with the indicated FLAG-tagged constructs as described above. After 24-48 hours, cells were collected in PBS and dounce homogenized in a hypotonic lysis buffer containing 10 mM NaCl, 1.5 mM MgCl2, 10 mM Tris pH 7.5, and Complete protease inhibitor cocktail (Roche). Lysates were cleared by centrifugation and immunoprecipitated with anti-FLAG affinity gel (Sigma) 4 hours to overnight.

Immunoprecipitates were washed once with wash buffer #1 (20 mM HEPES pH 7.9, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 25% glycerol, Complete protease inhibitor cocktail) and four times with wash buffer #2 (20 mM Tris pH 7.4, 20% glycerol, 0.2 mM

EDTA, 300 mM NaCl, 0.1% NP-40, Complete protease inhibitor cocktail) and rotated at least 2 hours in wash buffer #2. Samples were eluted with 300 µg/mL FLAG peptide

(Sigma) according to the manufacturer’s instructions. Polyhedrin promoter driven N- terminal-3xFLAG-tagged RIP expressed in baculovirus-infected Hi5 cells was purified as described1, and hUBC13 and hMMS2 purified from E. coli were prepared as described1. 3xHA epitope-tagged wild-type, K48-only, and K63-only ubiquitin (gene

3 cassettes synthesized by MCLABS) were subcloned into pET21b (Novagen) and transformed in BL21(DE3) competent cells (Stratagene) harboring the plasmid pJY2

(Affiniti Research Products Ltd.) for suppression of lysine misincorporation. To induce protein expression, bacterial cultures were incubated with shaking for 3 – 5 hours at room temperature after addition of 0.4mM IPTG final concentration. The pelleted material was lysed in 25mM TRIS pH7.6, 2mM EDTA, 100ug/ml lysozyme, and incubated for 20 minutes with gentle rotation at room temperature. After mild sonication on ice to decrease the sample viscosity, lysates were pelleted, resuspended in “basic buffer” (TBS plus 0.1% Tween 20) plus 8M urea and dialyzed 2 hours at room temperature in basic buffer. Samples were then dialyzed in the basic buffer containing 4M urea for 2 hrs, with a final dialysis in basic buffer plus 2M urea. Precipitated proteins were pelleted, and the soluble, purified ubiquitins were collected and used for in vitro assays. Recombinant full-length A20, including wild-type, OTU mutant (C103A) ZnF3 mutant (C521A,

C524A) or ZnF4 mutant (C624A, C627A) versions, N-terminal A20 (residues 1-386), C- terminal A20 (residues 370-790) and the catalytic core of murine Usp2 were purified from E. coli following standard procedures (Marston purification). For N-terminal A20 used for in vitro de-ubiquitination assays, a PCR fragment containing residues 1- 371 of

A20 was cloned into pST239 and expressed under the PhoA promotor in 58F3 cells with an N-terminal HQ tag. The C103S mutant was generated using QuikChange

Mutagenesis (Stratagene). The 58F3 cells were grown at 30oC to an optical density of 1.0 at 600nm. The temperature was then lowered to 23oC during the overnight inducton. The cells were lysed using a microfluidizer, the supernatant was applied to a Ni-NTA agarose column (Qiagen) and eluted with a buffer containing 50 mM Tris, pH 8.5, 300 mM NaCl,

4 2 mM ß-mercaptoethanol and 250 mM imidazole. Fractions containing A20 were pooled and loaded onto a superdex-75 gel filtration column (Amersham Biosciences) equilibrated with 50 mM Tris, pH 8.5, 300 mM NaCl, and 5 mM ß-mercaptoethanol.

Polyubiquitin chains composed of K48-only or K63-only ubiquitin were purchased from

Boston Biochem and were purified by gel filtration over a Superdex 75 PC3.2/3.0 column

(Amersham) in a buffer containing 50 mM HEPES pH 8.0, 0.5 mM EDTA, 0.01% Brij-

35, and 3 mM DTT. Fractions containing tri- and tetra-Ub chains were used for in vitro de-ubiquitination assays (Fig. 3a). FLAG-RIP was purified from HEK293T cell lysates for in vitro de-ubiquitination assays by boiling lysates in 1% SDS (v/v) to dissociate any non-covalently bound proteins. Lysates were diluted 1:10 in dissociation dilution buffer containing 1% Triton-X 100, 0.5% deoxycholate, 120 mM NaCl, 50 mM HEPES pH 7.2, and Complete protease inhibitor cocktail (Roche), and FLAG-RIP was immunoprecipitated with anti-FLAG agarose (Sigma).

In vivo ubiquitination and de-ubiquitination assays. HEK293T cells were transfected as described above with the indicated constructs (Fig. 3b, 3e, 3f, 4c), and were pre-treated with 25µM MG-132 to block A20-mediated RIP degradation. Cells were collected, lysed, and lysates were cleared by centrifugation as described above, but with 25µM MG-

132 and 10 mM N-ethylmalemide added to the lysis buffer. To assess ubiquitination of immunoprecipitated substrates, proteins were first dissociated in 1% SDS (v/v) by heating at 90˚ C for 5 minutes. Samples were diluted 10-fold with the dissociation dilution buffer described above. Myc-RIP was immunoprecipitated with anti-myc agarose (Covance) and immunoblotted as indicated (Fig. 3b, 3f, 4c), and endogenous RIP

5 (Fig. 3e) was immunoprecipitated at 4˚ C by rotating 3 hours with a cocktail of 2µg each of all three RIP antibodies followed by a 2-hour incubation with Protein A/G Plus beads

(Santa Cruz). Samples were washed and prepared for Western blot analysis as described above.

In vitro ubiquitination assays. Autoubiquitination assays were performed in 50 µL reaction volumes and contained the following components as indicated: 2µg N-terminal biotinylated Ub (Boston Biochem) or 3xHA-tagged ubiquitin purified from E. coli, 0.2µg

E1 (Calbiochem), 1.0 µg E2 (Boston Biochem) or 0.5 µg each hUBC13 and MMS2, 15

µL (up to 1µg) E3, and 5 µL 10X reaction buffer (300 mM HEPES pH 7.2, 20 mM ATP,

50 mM MgCl2, 2 mM DTT). Reactions were incubated at 30˚ C for 1 hour with agitation at 750 rpm and subsequently prepared for immunoblot analysis using the indicated antibodies as described above. For in vitro RIP ubiquitination by A20, reactions were performed as for autoubiquitination assays except 1µg recombinant RIP was included and

5µg and 3xHA-tagged ubiquitin purified from E. coli (described above) was used instead of biotinylated Ub. Reactions were performed in quadruplicate and after incubation were recombined for a final volume of 200µL. 50µL was reserved for immunoblotting and

SDS (1% v/v) was added to the remaining 150µL, which was heated at 90˚ C for 10 minutes. These samples were diluted to a final volume of 5 mL with a dissociation dilution buffer (see “FLAG elutions and protein purification”) and an antibody cocktail containing 2µg each of all three anti-RIP antibodies were added. Samples were rotated for 5 hours at 4˚ C, 100 µL protein A/G Plus beads (Santa Cruz) were added for an

6 additional two hours, immunoprecipitates were washed three times with the dissociation dilution buffer, and samples were prepared for immunoblot analysis as described above.

In vitro de-ubiquitination assays. Polyubiquitin chains, FLAG-RIP substrate, and A20 proteins were purified as described above. FLAG-RIP immunoprecipitates were washed twice with dissociation buffer, once with PBS, and twice with buffer containing 50 mM

HEPES pH 8.0 and 0.01% Brij-35. Enzymes (up to 1µg) and substrates (up to 1µM) were combined in a buffer containing 50 mM HEPES pH 8.0, 0.01% Brij-35, and 3mM

DTT and were reacted at 37˚ C with agitation at 750 rpm for 150 minutes (free polyubiquitin chains) or for the indicated time (FLAG-RIP substrate). Samples were subsequently prepared for western blot analysis as described above and probed with the indicated antibodies.

RNA interference. TRAF2 siRNA duplexes (5’ CGACAUGAACAUCGCAAGC 3’) with 3’dTdT overhangs were synthesized at Genentech. Scramble II siRNA duplexes were purchased from Dharmacon and hUBC13 siRNA duplexes have been described1.

HEK293T cells were transfected as described above three times at 24-hour intervals.

Prior to the third transfection, cells were split to 80% confluency and were co-transfected with the indicated oligos, myc-RIP, and CrmA constructs, and collected 30 hours thereafter.

7 Supplementary Figures

S1. A multiple alignment of representatives of all versions of the A20 ZnF domains.

The two major groups of the A20 ZnFs determined by K-means clustering are shown to the side using brackets. The individual proteins are designated using their Genbank GI numbers. The multiple ZnF domains in A20 are indicated by their numbers. The sequence conservation pattern is shown below the alignment by means of a consensus for each group. h: hydrophobic residue (ACFILMVWY); a: aromatic residue (FHWY); l: aliphatic residue (VIL); b: bulky residue (LIYERFQKMW); p: polar residue

(CDEHKNQRST); -: acidic residue (DE); s: small residue (GASCVDNPT); u: tiny residue (GAS). The secondary structure (ss) was predicted using the PROF and PHD programs (http://cubic.bioc.columbia.edu/predictprotein/) and is shown above the alignment using the following convention: H: helix (90% threshold); h: helix (70% threshold); e: strand (70% threshold). Unlike the RING finger, the A20 ZnF is predicted to be a mononuclear Zn-chelating domain, although secondary structure prediction is compatible with the A20 ZnF adopting an abbreviated version of the treble clef fold5, which is also seen in the RING finger.

S2. The two distinct A20 domains and their connections to the Ub system. The ordered graph depicts a portion of the domain architecture network that includes the N-

(OTU-DUB) and C-terminal (A20 ZnF) domains of A20. The arrows indicate the order in which the domains are linked together in multidomain proteins. The bidirectional arrow shows that the respective domains are fused in either order in different proteins.

The double arrowhead indicates the insertion of the OTU domain into another

8 deubiquitinating enzyme6. Around the network, cartoon representations of the architectures of various proteins containing the A20 ZnF and OTU domains are shown.

A20, Cezanne (not shown), Trabid, VCIP135, AWP1 and Rabex-5 are human proteins,

F38B7.5 is a C. elegans protein and Yfl044cp is a yeast protein. Cezanne has an architecture similar to the A20 proteins but has only a single C-terminal A20 finger.

Domain abbreviations: An1-ZnF: the AN1-type Zinc finger; Ub: ubiquitin; UBA/CUE: small, - helical ubiquitin binding domain; RAB-GEF: Vps9-type exchange factor for

RAB GTP-ases; LF: Little Finger domain; UBCH: Ub hydrolase; and C2H2-U: a specialized version of the C2H2 ZnF implicated in Ub signaling (L.A., unpublished).

S3, S4. In vitro ubiquitination assays with A20 truncation mutants. A20 proteins were FLAG-eluted from HEK293T cells. Deletional analysis maps the A20 Ub ligase domain to ZnFs 3 and 4.

S5. In vitro ubiquitination assays with A20 point mutants. A20 proteins were FLAG- eluted from HEK293T cells. Point mutations of conserved cysteines in A20 ZnF4

(ZnF4MT), but not ZnF3 (ZnF3MT), reduce in vitro ubiquitination catalyzed by A20.

S6. TNFR1-recruited RIP is ubiquitinated and targeted for proteasomal degradation. HeLaS3 cells were treated with TNF- (TNF Rx) for the indicated time, with or without MG-132 pre-treatment. Endogenous TNFR1 immunoprecipitates were then blotted with an anti-RIP antibody to reveal TNFR1-associated RIP. 1/100 of the total cell lysate was used for straight WB analysis.

9 S7. Proposed mechanism of A20-mediated NF- downregulation. Step 1: proximal signaling proteins are recruited to the activated TNFR1. //: IK complex. Step 2:

TRAF2-mediated K63-linked polyubiquitination (black diamonds) stabilizes association of proximal signaling components, thereby promoting IK complex activation. Step 3:

A20 removes K63-linked Ub chains from RIP, which is a prerequisite for Step 4. Step 4:

A20 polyubiquitinates RIP with K48-linked chains (grey ovals). Step 5: K48- ubiquitinated RIP is targeted for proteasomal degradation, thereby terminating NF-B signaling.

10 ss ...eeee..eeee.eeee..eeeeHhHHHHHHHHHhh.. 9910156 LPPTQTKCKQPNCSFYGHPETNNFCSCCYREELRRRERE\ 8778645 SPSEPKLCVK-GCGFFGSPSNMNLCSKCYRDIRATEEQT| 19923036 AIPPSILCEN-NCGFYGNRANNNLCSKCYREFLEKQKKE| 11121500 HSQAPMLCST-GCGFYGNPRTNGMCSVCYKEHLQRQNSS| 21359918 HSQVPMLCST-GCGFYGNPRTNGMCSVCYKEHLQRQNSS| 4107017 QSSQAQLCRRNGCGFYGNSKFEGMCSMCYKDTVQKKNNS| 18702331 PGPVQRRCQRENCAFYGRAETEHYCSYCYREELRRRREA| 24645315 SNPMQPMCRS-GCGFYGNPATDGLCSVCYKDSLRKKQQP| 21299715 IKQKDLKCRN-GCGFYGNAQWNGLCSKCYRERSLKERHT| 19923036 LGQQDLKCRS-GCGFYGTPQNEGLCSMCFREKFNDKQRK| 7657496 VDQSDLLCKK-GCGYYGNPAWQGFCSKCWREEYHKARQK| 15225894 RLQEPRLCAN-NCGFFGSTATQNLCSKCFRDLQHQEQNS| 25082726 PPEGPKLCTN-NCGFFGSAATMNMCSKCHKDMLFQQEQG| 22328622 MTGEPSLCIR-GCGFFSTSQTKNLCSKCYNDFLKDESAR| 18404102 TPEGHRLCVN-NCGFFGSSATMNLCSNCYGDLCLKQQQQ| 6677605 QTPGPMLCST-GCGFYGNPRTNGMCSVCYKEHLQRQQNS| 11345484 APSLPPRCP---CGFWGSSKTMNLCSKCFADFQKKQPDD| 5454132.4 DRTGTSKCRKAGCVYFGTPENKGFCTLCFIEYRENKHFA/ |A20 90% ...... bC.p.sCuFaGps...shCS.Ca.-...pbp.. 5454132.5 RFQNTIPCLGRECGTLGSTMFEGYCQKCFIEAQNQRFHE | 5454132.1 LSLMDVKCETPNCPFFMSVNTQPLCHECSERRQKNQNKL\ | 5454132.2 SETTAMKCRSPGCPFTLNVQHNGFCERCHNARQLHASHA| | 5454132.3 RHLDPGKCQ--ACLQDVTRTFNGICSTCFKRTTAEASSS| | 5454132.6 QSTSRPKCARASCKNILACRSEELCMECQHPNQRMGPGA| | 5454132.7 EDPPKQRCRAPACDHFGNAKCNGYCNECFQFKQMYG---| | 25005144 LKANDLLCVN-GCGFYGTPQWENRCSKCWRAHQNEMKKC| 15235819 QASEPKLCVK-GCGFFGSPSNMDLCSKCYRGICAEEAQT| 21593800 PPTEPKLCDN-GCGFFGSPSNMNLCSKCYRSLRAEEDQT| 17506733 QAQTAPSCRA-GCGFFGASATEGYCSQCFKNTLKRQQDT| 21166068 QQNTPTPCSK-GCGFFGNPLTENMCSKCFRTKKETTSNN| 8307828 RAPEITLCAN-SCGFPGNPATQNLCQNCFLAATASTSSP/ 90% ...... C...sC....ss...shCppC.p..p...... overall 90%...... C...sCs.h.ss...shCp.Ca.....p....

11 12 13 Supplementary Information References

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