ilpip, a novel anti-apoptotic protein that enhances xiap- mediated

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ILPIP, a novel anti-apoptotic protein that enhances XIAP-mediated activation of JNK1 and protection against apoptosis.

M. Germana Sanna1, Jean da Silva Correia1, Ying Luo2, Betty Chuang2, Lorien M. Paulson1, Binh Nguyen1, Quinn L. Deveraux3, and Richard J. Ulevitch1*.

1 The Scripps Research Institute, Department of Immunology, 10550 N. Torrey Pines La Jolla, CA 92037. USA; 2 Rigel Inc., 240 E. Grand Ave, South San Francisco, CA 94080; 3 The Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, CA 92121. USA;

*To whom correspondence should be addressed:

Richard J. Ulevitch

The Scripps Research Institute

10550 N. Torrey Pines Road

La Jolla, CA 92037.

Tel. (858) 784-8219

Fax (858) 784-8239

E-mail: [email protected]

Running title: ILPIP, a new XIAP-interacting protein

Copyright 2002 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on June 4, 2002 as Manuscript M203312200 by guest on A

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SUMMARY We have previously described a new aspect of the Inhibitor of Apoptosis family of

proteins (IAP) anti-apoptotic activity that involves the TAK1/JNK1 signal transduction

pathway (1,2). Our findings suggest the existence of a novel mechanism that regulates

IAPs’ anti-apoptotic activity that is separate from caspase inhibition but instead involves

TAK1-mediated activation of JNK1.

In a search for proteins involved in the XIAP/TAK1/JNK1 signaling pathway we

isolated by yeast two-hybrid screening a novel XIAP-interacting protein that we called

ILPIP (hILP-Interacting Protein). Whereas ILPIP moderately activates JNK family

members when expressed alone, it strongly enhances XIAP-mediated activation of JNK1,

JNK2 and JNK3. The expression of a catalytically inactive mutant of TAK1 blocked

XIAP/ILPIP synergistic activation of JNK1 thereby implicating TAK1 in this signaling

pathway. ILPIP moderately protects against ICE- or Fas-induced apoptosis and

significantly potentiates the anti-apoptotic activity of XIAP. In vivo co-precipitation

experiments show that both ILPIP and XIAP interact with TAK1 and TRAF6. Finally,

expression of ILPIP did not affect the ability of XIAP to inhibit caspase activation,

further supporting the idea that XIAP protection against apoptosis is achieved by two

separate mechanisms: one requiring JNK1 activation and a second involving caspase

inhibition.

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INTRODUCTION Apoptosis is an active process in which an individual cell responding to internal and/or

external cues commits suicide. Apoptosis is involved in many diverse homeostatic

processes in multicellular organisms, both during development and in the mature

organism, and is increasingly being recognized as a biological process of critical

importance, not only in normal physiology but also in the pathogenesis of diseases such

as cancer and neurodegenerative diseases (3-5). Apoptosis is genetically determined and

controlled through the expression of an increasing number of genes, many of which are

conserved in nematodes, mammals and viruses (6).

Caspases, a family of cysteine proteases, are the most extensively studied

activators of apoptosis. Among the anti-apoptotic gene products is the IAP (Inhibitor of

Apoptosis) family of proteins. Initially discovered in baculovirus, where they were shown

to be involved in suppressing the host cell death response to viral infection (7,8), IAP

homologues were then isolated in Drosophila, C. elegans, yeast and mammalian

organisms. To date, seven members of the IAP family have been identified in mammalian

cells, with XIAP being the most extensively studied member of the family (9-16). IAPs

have been shown to protect against a wide spectrum of apoptotic triggers. The diversity

of stimuli against which the IAPs suppress apoptosis is greater than that observed for any

other family of apoptotic inhibitors. At least one suggested mechanism of IAP apoptotic

suppression appears to be through direct caspase inhibition. Several of the human IAP

family proteins have been reported to directly bind and inhibit specific members of the

caspase family (17).


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XIAP has been shown to participate in the BMP signaling pathway by binding

with both the BMP receptor and the adaptor molecule TAB1, which is a co-activator of

TAK1, thus linking the BMP receptors to TAB1-TAK1 and therefore participating in the

bone morphogenic protein-signaling pathway involved in mesoderm induction and

patterning in early Xenopus embryos (18). XIAP also stimulates NF-kB via the TAK1

signaling pathway (19). Consistent with these findings, we have recently described an

alternative mechanism for the IAP anti-apoptotic protection which is distinct from

caspase inhibition and involves activation of the MAP kinase JNK1 through the

TAB1/TAK1 complex (1,2,20).

XIAP involvement in signal transduction pathways is still poorly characterized.

Few proteins have been shown to interact with XIAP. A recently described XIAP-

interacting protein is Smac/DIABLO, which promotes caspase activation by binding and

inhibiting the IAPs (21,22). XAF1 has also been reported to bind to XIAP and inhibit its

anti-apoptotic effect apparently by triggering the redistribution of XIAP from the cytosol

to the nucleus (11). However, none of these interactions has been correlated with the

XIAP-mediated activation of JNK1. This led us to search for new XIAP-interacting

proteins and to investigate their role in the XIAP/TAK1/JNK1 signaling cascade.


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EXPERIMENTAL PROCEDURES

Yeast two-hybrid screening and isolation of ILPIP.

A cDNA fragment encoding human XIAP was amplified by PCR and inserted into the

Xho1 site of pBD-GAL4 Cam (Stratagene, LA Jolla, CA) to generate pBD-GAL4

Cam/XIAP. After verification of the sequence, Saccharomyces cerevisiae CG1945 cells

(CLONTECH, Palo Alto, CA) were sequentially transformed with pBD-GAL4

Cam/XIAP and human fetal brain library (MATCHMACHER, CLONTECH; 108 cfu/ml)

in pACT using a lithium acetate transformation protocol. Selection was done by growth

on SD medium lacking histidine, uracil and tryptophan (CLONTECH). Twenty clones

exhibiting activation of the lacZ reporter gene were identified among 3X106

transformants by the β-galactosidase assay. A few clones showing a strong reproducible

interaction with XIAP were chosen. Plasmids were isolated from positive yeast colonies

by a glass bead phenol-chlorophorm extraction protocol (CLONTECH). Escherichia coli

DH5α bacteria cells were transformed with the plasmids and bacteria containing the

pACT vector were selected on ampicillin resistant plates. The pACT plasmids were

isolated from E. coli and restriction-mapped (XhoI), and the sequence of the insert

determined. The partial cDNA two-hybrid clone was used to design a probe to screen a

human liver Uni-ZAP cDNA lambda library (Stratagene) and 5’-RACE techniques.

Phage plaques were isolated and screened to verify the cDNA size by PCR using the T3

and T7 primers. The phagemid pBLUESCRIPT vector carrying the cDNA of nine

individual clones was isolated by in vivo excision from the Uni-ZAP vector according to

the manufacturer’s instructions (Stratagene). The isolated cDNAs were sequenced and

sequences were analyzed using the GCG Sequence Analysis software package (Madison,


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WI). The full length ILPIP gene encoding an approximately 2.4 kb cDNA was subcloned

by BamHI-XhoI restriction sites into pcDNA3 vector encoding an HA-Tag at the N-

terminus. The sequence contains an initiator methionine, a stop codon and a poly-

adenylation tail and provide evidence for a novel gene which we have named ILPIP.

Northern Blot was performed using the partial ILPIP cDNA isolated from the two-hybrid

system following standard procedures.

Plasmids.

Plasmids encoding JNK1, JNK2, JNK3, p38, ERK2, β-gal-ICE, XIAP, TAB1, ASK1

(KM, Lysine 709 to Methionine), used in this study have been previously described (1).

TAK1 was expressed in pCMV6. TRAF6 and TRAF6∆ (aa 1-287 deleted at the N-

terminus, therefore expressing only the TRAF domain) were expressed in pRK5. The

capacity of TAK1 (KW; Lysine 63 to Tryptophane) to act as dominant negative was

determined previously (23). ILPIPα and ILPIPβ were expressed in pcDNA3 with an HA

or FLAG tag.

Transfection and cell culture.

Human embryonic kidney cells (293T) were grown at 37°C in 5% CO2 in

Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum, 2 mM

glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin. For transfection, each well

of a six-well plate was seeded with 7x105 cells. Cells were transfected 18 hours later

using Lipofectamine Plus reagent (Gibco) for 3 hours and incubated for 18 hours before

lysis. MCF7-Fas cells were grown in RPMI 1640 containing 10% FBS, 200 µg/ml G418


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and 100 µg/ml hygromycin, and grown at 37°C in 5% CO2. For transfection each well of

a six-well plate was seeded with 2.5x105 cells and 24 hours after plating were transfected

using Lipofectamine Plus reagent (Gibco). 24 hours after transfection cells were treated

with anti-Fas antibody (150 ng/ml). After 16 hours, cells were fixed and stained as

described below.

Stable transfectants were obtained as follows: human embryonic kidney cells

(293T) were transfected with pBMN-Z-I-Blasto, pBMN-TAK1(K63W)-I-Blasto, or

pBMN-ASK1(KW)-I-Blasto by calcium phosphate precipitation and selected in medium

containing blasticidine S (10µg/ml) (23).

Cell lysis and kinase assay.

Cell lysis was performed for 30 min at 4°C with lysis buffer (25 mM Hepes [pH

7.6], 1% Triton X-100, 137 mM NaCl, 3 mM ß-glycerophosphate, 3 mM

ethylendiaminetetraacetic acid, 0.1 mM sodium orthovanadate, 1 mM

phenylmethylsulfonylfluride). Expression of MAP kinase proteins was quantified by

densitometry after Western blot analysis and equivalent amounts were

immunoprecipitated at 4°C for 2 hours. The immunoprecipitates were washed twice with

lysis buffer, and twice with kinase buffer (see below) before performing the kinase assay.

HA-and Myc-tagged proteins were immunoprecipitated using 20 µl of agarose-Protein A

(Pierce) pre-incubated with anti-HA antibody or anti-Myc antibody (5 µg, Roche and

Upstate Biotechnology, respectively) and FLAG-tagged proteins with 20 µl agarose

conjugated with the M2 anti-FLAG monoclonal antibody (Sigma).


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In vitro kinase assays was performed as previously described (Sanna et al. 1998)

with the difference that a 30-minute incubation time was used for the detection of JNK2

and JNK3 kinase activity. UV activation of JNK1 was carried out in 293T cells as

previously reported (Sanna et al. 1998).

Detection of apoptotic cells.

β-galactosidase staining

Cells were transfected with the indicated plasmids together with a β-gal

expressing vector and stained with X-gal (reagent for β-gal expression) to allow

visualization of transfected cells and morphology observation. Quantification of apoptotic

cells was determined at the microscope by counting over five fields for each sample.

Apoptotic cells appear to be smaller, rounder and show condensed and misshapen nuclei

compared to viable cells, which are flat, well spread, and with easily discernible nuclei.

Protein expression for all the transfected constructs was assessed by Western blot on

duplicate lysates of original transfections used for the apoptosis assays.

AnnexinV-PE/FACS analysis

Cell were transfected with the indicated plasmids together with GFP vector

(Clontech Laboratories) to allow quantitation of transfection efficiency. AnnexinV-PE

staining was performed as suggested by the manufacturer (PharMingen). Briefly,

adherent cells were detached from the plates and centrifuged for 5 min. at 1000 rpm.

After removing the supernatant, cells were washed with AnnexinV binding buffer,

centrifuged again and supernatant decanted by inversion of the tube. Cells were


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resuspended and AnnexinV-PE conjugate (5 ìl) added to each sample, incubated for 10

min. in the dark and then analyzed by FACS within 1 h.

Death by apoptosis was quantified both by X-gal staining of cells and AnnexinV-

PE-FACS analysis for each experiments. The results obtained using the two different

techniques were comparable and therefore only the x-gal data are shown.

Co-immunoprecipitations and immunoblot assays.

Cells were washed extensively and lysed in 200 ìl lysis buffer containing 50 mM

Hepes, 100 mM NaCl, 2 mM EDTA, 10 % glycerol, 1 % Nonidet P-40, 14 mM pepstatin

A, 100 mM leupeptin, 3 mM benzamidine, 1 mM PMSF, 1 mM sodium pyrophosphate,

10 mM sodium orthovanadate, 100 U/ml aprotinin, 100 mM sodium fluoride. After

incubation for 30 min on ice, cell lysates were centrifuged (14’000 rpm, 10 min, 4 °C)

and the supernatants were recovered. Cell lysates were pre-cleared 3 times for 20 min at 4

°C with 20 ìl of protein A-Sepharose beads, and mixed with specified antibodies for 3 h

at 4 °C under constant agitation. Immune complexes were allowed to bind to 20 ìl

protein A-Sepharose beads overnight, beads were washed 3 times with lysis buffer and

the washed beads resuspended in 30 µl of Laemmli buffer and boiled for 10 min.

Immunoprecipitates were separated on 12 % SDS-PAGE and transferred to nitrocellulose

membranes. Filters were blocked with 5 % nonfat milk in blocking buffer (TBS, 50 mM

Tris.-HCl, pH 7.5; 150 mM NaCl; 0.1 % Tween 20), and incubated with the specified

antibody for 2 h and with peroxidase-conjugated secondary antibody for 1 h at ambient

temperature. Specific bands were revealed using the ECL Plus system (Amersham).


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In vitro binding assays.

In vitro translation of TAK1 was performed using standard procedures (Promega). XIAP-

GST protein was expressed from a pGEX vector (Pharmacia) and purified as suggested

by the manufacturer and JNK1-HIS protein was purchased from Santa Cruz. Gluthatione

or Ni-NTA conjugated beads (from Sigma and Qiagen respectively) were used to

precipitate XIAP-GST and JNK1-HIS. Binding assays were performed in lysis buffer and

TAK1 interaction with XIAP or JNK1 was detected by Western blot using an anti-TAK1

antibody (Santa Cruz).

Caspase activation in cytosolic extracts.

Cytosolic extracts from transfected 293T (100 mm dishes) were prepared

essentially as described (24), with several modifications (25). Briefly, cells were washed

once with ice-cold buffer A and pelleted by centrifugation. Packed cell pellets were

suspended in 1-2 volumes of buffer A, incubated on ice for 20 minutes and then disrupted

by 15-30 passages through a 26-gauge needle. Cell extracts were clarified by

centrifugation at 16,000 g for 10 min. and the resulting supernatants were used for “cell-

free” assays. For initiating caspase activation, 10 µM horse heart cytochrome c (Sigma)

together with 1 mM dATP was added and the assays were incubated at 30 °C for 10

minutes. 1 µl (10 µg total protein) was measured for caspase activity by monitoring the

release of AFC DEVD-containing synthetic peptides using continuous-reading

instruments as described (26). Fluorogenic 7-amino-4-trifluoromethyl coumarin (AFC)

caspase substrate (Ac-DEVD-AFC) was purchased from Sigma.


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RESULTS

XIAP bait identifies ILPIP in a two-hybrid screening.

As a first step in the characterization of the XIAP/JNK1/TAK1/TAB1 interactions, we

used a XIAP-Gal4 DNA binding domain fusion construct as bait in the two-hybrid

system to screen a human fetal brain cDNA-Gal4 activation domain function library

(Matchmaker, Clontech). Among the selected primary transformants, positive yeast

colonies were independently identified and isolated. Plasmids were isolated from positive

yeast cells and the sequence of the insert was determined. This process lead to the

identification of a 700 bp cDNA sequence. This partial cDNA two-hybrid clone was used

to design a probe to screen a human liver Uni-ZAP cDNA lambda library (Stratagene).

The library screen yielded the parent 2.4-kb cDNA containing an initiator methionine, a

stop codon and a poly-adenylation tail that encoded for a 418 amino acid protein that we

have named ILPIPα (Fig. 1A). The predicted ILPIP amino acid sequence revealed that

ILPIP is identical to ALS2CR2, a novel gene identified within the ALS2 (juvenile

amyotrophic lateral sclerosis) critical region (27). At the protein level, the strongest

homologies were found with serologically defined breast cancer antigen NY-BR-96 (~20

% identity, 46 % homology) and with SPAK a member of the Ste20/SPS1family of

kinases and other Ste20-like kinases (Fig. 1B, up to ~20% overall amino acid identity,

~45% similarity). ILPIP has a putative protein kinase domain (aa 58-369; Fig. 1C) with a

hypothetical ATP-binding site located at the N-terminus of the protein and several Serine,

Threonine or Tyrosine active and phosphorylation sites towards the C-terminus (Fig. 1C).

In the same screening, a shorter isoform encoding a protein of 280 amino acids was

isolated (Fig. 1A). The shorter isoform was named ILPIPβ and originates from a


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methionine at amino-acid 139 of the ILPIPα due to an insertion in the 5’ region of the

gene (data not shown) which introduces two stop codons before the initial ILPIPα

methionine. The existence of the ILPIPα and β isoforms was confirmed by RT-PCR on

RNA extracted from several different cell lines. Two PCR products were obtained and

sequence analysis matched the original sequences for ILPIPα and β. Northern blot

analysis revealed that ILPIP is expressed in normal tissues as a single 2.4 kb transcript

with higher levels of expression in muscle, liver and heart (Fig. 2). ILPIP was also

expressed as a 2.4 kb band in human embryonic kidney cells (293T) (Fig. 2).

ILPIP moderately activates JNK family members and strongly enhances the XIAP-

mediated activation of JNK1, JNK2 and JNK3.

We have previously shown that XIAP-mediated activation of JNK1 is necessary

for its anti-apoptotic (1,2). Thus we addressed the question whether expression of ILPIP

alone or ILPIP and XIAP together would have an effect on MAP kinase activation. 293T

cells were co-transfected with vectors encoding for JNK1, JNK2 or JNK3 in the absence

or presence of increasing concentrations of plasmids encoding for ILPIP or XIAP, alone

or in combination. Expression of MAP kinase proteins was quantified by densitometry

after Western blot analysis and equivalent amounts were immunoprecipitated at 4°C for 2

hours. Kinase activity was measured using ATF-2 as substrate. As previously reported

XIAP expression increased JNK1 activity. Expression of ILPIPα or ILPIPβ also resulted

in a slight increase in JNK activity. However, co-expression of ILPIPα with XIAP, and

to a lesser degree ILPIPβ, resulted in a marked synergistic activation of JNK1 that was

comparable to UV activation (Fig. 3A). Cooperative activation of JNK2 and JNK3 was


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also observed, although to a lesser extent (Fig. 3B). Expression of ILPIPα or ILPIPα plus

XIAP had no effect on p38 or ERK2 activation (data not shown). Therefore ILPIP acts

synergistically with XIAP in specifically activating JNK family members.

ILPIP moderately protects against ICE- or Fas-induced apoptosis and significantly

potentiates the anti-apoptotic activity of XIAP.

XIAP is known to protect against a variety of apoptotic stimuli. Since XIAP and

ILPIP synergistically activated JNK1 and XIAP-mediated activation of JNK1 is

important for the protection against apoptosis we investigated whether ILPIP and XIAP

were also cooperatively protecting against apoptosis. 293T cells were transfected with a

plasmid encoding for ICE-ß-galactosidase in the presence of increasing amounts of XIAP

or ILPIP DNA, alone or in combination. A GFP expressing vector was used as negative

control. Each apoptotic assay was quantified both with X-gal staining of cells and

AnnexinV-PE-FACS analysis. The results obtained using the two different techniques

were comparable and therefore only the data from representative experiments performed

with X-gal are shown. Expression of ILPIP was able to partially inhibit ICE-induced

apoptosis (Fig. 4A), although this effect was not as pronounced as XIAP’s. However,

ILPIP remarkably enhanced XIAP protection against ICE-induced apoptosis (up to 90%

of viable cells), suggesting that ILPIP and XIAP cooperatively protect against ICE-

induced apoptosis. Similar results were obtained in MCF7/Fas cells when apoptosis was

induced by treating the cells for 18h with anti-Fas antibody (Fig 4B). ILPIPα partially

protected against Fas-induced apoptosis and significantly potentiated the protective effect

of XIAP.


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XIAP and ILPIPαα synergistic activation of JNK1 involvesTAK1.

We have previously reported that XIAP-mediated activation of JNK1 involves the

MAP3 kinase TAK1. In order to investigate if the synergistic effect of ILPIP on XIAP-

mediated JNK1 activation was also dependent on TAK1, 293T stably transfected cells

expressing either LacZ control vector or catalytically inactive TAK1 (TAK1 (KW)) were

transfected with vectors encoding for XIAP or ILPIPα, alone or in combination, and

JNK1 activation was determined. 293T cells stably expressing a catalytically inactive

mutant of ASK1 (ASK1 (KM)) were also used as a control of specificity. XIAP-,

ILPIPα- or XIAP/ILPIPα-mediated JNK1 activation was inhibited in the presence of

TAK1 (KW) (Fig 5B), whereas LacZ or ASK1 (KM) had no effect (Fig. 5A, 5C). These

data suggest that activation of JNK1 by ILPIPα or XIAP and the synergistic activation of

JNK by ILPIP and XIAP is dependent upon TAK1.

XIAP and ILPIP interact with TAK1 and TRAF6.

Since TAK1 appears to transmit the signal between the XIAP, ILPIPα and JNK1,

we investigated the possibility that XIAP and ILPIPα would physically interact with

TAK1. In an in vivo binding assay, vectors encoding XIAP-FLAG or ILPIPα-FLAG

were co-transfected with wt TAK1-HA or AKT-HA as a control of specificity in 293T

cells. Cell extracts were immunoprecipitated using anti-FLAG antibody and analyzed by

Western blot with an anti-HA antibody (Fig. 6A). Cell extracts were also directly

subjected to immunoblot analysis to check for protein expression. TAK1 was found to

co-precipitate with XIAP and ILPIPα suggesting that an interaction exists between these


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proteins. Results were confirmed by reverse co-precipitation using anti-HA antibody

(data not shown).

It has been previously reported that TAK1, TRAF6, TAB1 and TAB2 associate in a

complex (28-30). Therefore we addressed the question whether XIAP and ILPIPα would

also bind to TRAF6, TAB1 and TAB2. Vectors encoding TRAF6-Myc or a deletion

mutant of TRAF6 (TRAF6∆−Myc) were co-transfected with ILPIPα-FLAG or XIAP-

FLAG in 293T cells. Cell extracts were immunoprecipitated using anti-Myc antibody and

analyzed by Western blot with an anti-FLAG antibody (Fig. 6B). TRAF6 was found to

co-precipitate with XIAP and ILPIPα therefore suggesting that an interaction exists

between these proteins. Interestingly, TRAF6∆ also co-precipitate with XIAP and

ILPIPα indicating that the interactions occur through the TRAF domain of TRAF6. Cell

extracts were also directly subjected to immunoblot analysis to confirm expression of the

respective proteins. Results were confirmed by reverse co-precipitation using anti-FLAG

antibody (data not shown). As a control of specificity TRAF2 was also assayed for

coprecipitation with ILPIP and found to be negative (data not shown).

In order to determine if the adaptor molecules TAB1 and TAB2 were also part of

this complex, vectors encoding TAB1 or TAB2 were co-transfected with XIAP-HA

ILPIPα-HA in 293T cells. Cell extracts were immunoprecipitated using anti-TAB1 or

anti-TAB2 antibody and analyzed by Western blot with an anti-HA antibody. Co-

precipitation of XIAP with TAB1 was confirmed as previously published (18).

Interestingly binding between XIAP and TAB2 was also detected. Surprisingly, ILPIPα

did not interact either with TAB1 or TAB2 suggesting that ILPIPα interaction with

TAB1 and TAB2 is achieved through XIAP and TAK1 (Fig. 6C).


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Since ILPIPα was cloned as a XIAP-interacting protein in a yeast two-hybrid

screening we also addressed the question whether XIAP and ILPIPα were able to co-

precipitate in an in vivo binding assay. Vectors encoding ILPIPα-FLAG and XIAP-Myc

were co-transfected in 293T cells and cell extracts were immunoprecipitated using anti-

Myc antibody and analyzed by Western blot with an anti-FLAG antibody. Surprisingly,

no interaction was detected (Fig. 6B). Similar results were obtained using HA tagged-

XIAP thus excluding the possibility that the lack of interaction observed could have been

due to the low expression of XIAP-Myc. This result may be explained by the fact that the

interaction between XIAP and ILPIPα is transient and unstable and thus undetectable

with this method.

To determine if ILPIP could directly interact with XIAP, we used in vitro

translated ILPIPα protein and recombinant GST-XIAP in in vitro binding assays. In these

studies, GST protein was also used as negative control. XIAP-GST or GST proteins were

incubated with glutathione-conjugated beads. ILPIPα was added to the reactions and

bound proteins were separated on SDS gels and analyzed by Western blots using an anti-

HA antibody. ILPIPα was found to associate with XIAP but not with GST (Fig. 6B, C).

Thus, these data are consistent with those observed in our yeast two-hybrid studies which

suggested that XIAP and ILPIP interact directly. The totality of these results supports the

idea that XIAP, ILPIPα, TAK1, TRAF6, TAB1 and TAB2 are likely to co-exist in a

complex.


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ILPIPα α does not affect XIAP inhibition of caspase activation.

XIAP has been reported to be a strong inhibitor of caspase activity (25,31).

However, we have previously shown that XIAP protection against apoptosis requires

JNK1 and TAK1 activation and does not affect the ability of XIAP to inhibit caspase

activity. Since ILPIPα dramatically enhances the XIAP-mediated activation of JNKs,

passes through TAK1 and significantly potentiates the anti-apoptotic activity of XIAP

against ICE- or Fas-induced apoptosis, we investigated whether expression of ILPIPα

would influence the ability of XIAP to inhibit caspase activity.

293T cells were transfected with control vector or vectors encoding XIAP or

ILPIPα, alone or in combination, and the effect on caspase activity detected by

measuring cleavage of short fluorigenic peptides (24,25)) in a cell free system where

exogenously added cytochrome c induces proteolytic activation of caspase-9 and

subsequently caspase-3 in cytosolic extracts. As expected, expression of XIAP strongly

suppressed cytochrome-c induced caspase activation, while expression of ILPIPα alone

had no effect. Co-expression of ILPIPα with XIAP did not appear to inhibit nor enhance

XIAP’s ability to block caspase activation. In order to rule out the possibility that the

effect of ILPIPα on XIAP inhibition of caspases activity was too subtle to be detected

when caspase activity is near completely inihibited by XIAP, we diluted the XIAP and

XIAP/ILPIP containing extracts 10 fold with the control extracts. Under these conditions

cytochrome-c mediated activation of caspases is significantly increased above

background, however ILPIPα did not affect the inhibition mediated by XIAP. Combined,

these data suggest that ILPIPα cooperatively enhances XIAP protection against apoptosis

by a mechanism that is independent of inhibition of caspase activity.


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DISCUSSION

XIAP has been shown to protect against a wide spectrum of apoptotic triggers. A

suggested mechanism of IAP apoptotic suppression appears to be through direct caspase

inhibition, in fact several of the human IAP family proteins have been reported to directly

bind and inhibit specific members of the caspase family. Our findings suggest the

existence of an alternative mechanism regulating XIAP’s anti-apoptotic activity that is

separate from caspase inhibition and involves the TAB1/TAB2/TAK1/JNK1 signaling

cascade. In an attempt to characterize the pathway connecting XIAP to JNK1 we

performed a yeast two-hybrid screening and isolated ILPIP, a novel XIAP-interacting

protein. The characterization of ILPIP functional properties highlighted some interesting

features. First, ILPIP expression slightly activates JNK and strongly enhances JNK1

activation when co-expressed with XIAP. Activation of MAP kinases has been reported

to regulate the activities of many transcription factors and regulatory molecules and is

required for the regulation of inflammatory responses, cell proliferation and apoptosis

(32). In particular, the involvement of the JNK family in apoptotic cell death has been

most actively studied. JNK activation is observed in apoptosis induced by a variety of

stimuli in different cell types. However, the consequence of its activation has been

contradictory resulting in protection from apoptosis in some cases and induction of

apoptosis in others (32-36). Despite these apparent contradictions, there is a growing

consensus that correlation between activation of JNK and protection or induction of

apoptosis is stimuli and/or cell-type dependent (37-40). With this in mind, we

investigated if XIAP/ILPIP synergistic activation of JNK1 was correlated with the ability

of XIAP to protect against apoptosis. Interestingly, ILPIP moderately protects against


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ICE- or Fas-induced apoptosis and significantly potentiates the anti-apoptotic activity of

XIAP therefore further supporting the idea that XIAP-mediated activation of JNK1

promotes cell survival.

XIAP and ILPIP synergistic activation of JNK1 involves TAK1 as demonstrated

by inhibition of JNK1 activity using a catalytically inactive form of TAK1. It has been

previously reported that XIAP participates in the BMP signaling pathway by binding with

both the BMP receptor, the adaptor molecule TAB1 (2,18) and with TAK1 (2). The

findings that ILPIP also co-precipitates with TAK1, that both XIAP and ILPIP bind to

TRAF6, and that XIAP also binds to TAB2, further support the idea that these molecules

behave in a functional complex.

Surprisingly, we were unable to detect association between XIAP and ILPIP in

cells suggesting that such an interaction may be transient. That could be explained by the

possibility of ILPIP being a kinase, as predicted by the homology with Serine/Threonine

kinases and by the presence of a putative kinase domain. This possibility is currently

under investigation in our laboratory. A direct interaction between XIAP and ILPIPα was

demonstrated in an in vitro binding assay thus supporting the original interaction showed

by the yeast two-hybrid system.

Finally, expression of ILPIP did not affect XIAP inhibition of caspase activation

further supporting the idea that XIAP protection against apoptosis is achieved by two

separate mechanisms: one requiring JNK1 activation and a second involving caspase

inhibition. The interaction between XIAP, ILPIP and TRAF6 may also suggest that XIAP

might be involved in the IL-1 inflammatory response which TRAF6 has been previously

shown to regulate (41).


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XIAP has been reported to interact with a few other proteins all of which act as

negative regulators. Among these is SMAC/DIABLO, which is a nuclear-encoded,

mitochondrially localized protein that is released in response to apoptotic stimuli and acts

as a negative regulator of XIAP anti-apoptotic function (21,22). Similarly Omi/HtrA2, a

serine protease localized in the mithocondria, inhibits XIAP’s-protective effect by

binding to its BIR-3 domain (Ref.) A third negative regulator of XIAP is XAF1, which is

thought to exert its effect through sequestering XIAP in the nucleus (42). Importantly, we

show here that ILPIP is the first protein able to potentiate the anti-apoptotic effect of

XIAP instead of antagonizing it.

Taken together our results describe a novel XIAP-interacting protein that acts as a

co-factor enhancing XIAP-mediated activation of JNK1 and XIAP’s caspase-independent

protection against apoptosis.

ACKNOWLEDGMENTS

We thank Dr. C. Fearns for critical reading of the manuscript. This work was supported

by Grants GM36796; GM28485; AI15136.


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FOOTNOTES

Abbreviations in this paper are: ILPIP, hILP Interacting Protein; XIAP, X-chromosome-

linked IAP; JNK, amino-terminal c-jun kinase; IAP, Inhibitor of Apoptosis; TAK1, TGF-

beta-activated kinase-1; ICE, interleukin-1 beta converting enzyme; TRAF6, TNF

receptor-associated factor 6; TNF, tumor necrosis factor; BMP, bone morphogenetic

protein; NF-κB, nuclear factor kappaB; MAPK, mitogen-activated protein kinase;

SMAC, mitochondria-derived activator of caspases; DIABLO, direct IAP binding protein

with low pI; TAB1/TAB2, TAK1 binding protein; BIR, baculovirus IAP repeats; XAF1,

XIAP-associated factor 1.


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FIGURE LEGENDS

Fig. 1. ILPIP predicted sequences and homologies.

(A) Nucleotide and amino acid sequence of ILPIP. ILPIP cDNA was cloned as described

in the text. Starting methionines for ILPIPα and ILPIPβ are highlighted in bold at amino

acid number 1 and 139, respectively. The ILPIPα and ILPIPβ sequences have been

submitted to GenBank under accession number AY093697.

(B) Predicted amino acid homology of ILPIPα with known proteins.

By protein sequence homology ILPIP is related to serologically defined breast cancer

antigen NY-BR-96. Both ILPIP and NY-BR-96 exhibit significant homology to SPAK

and other Ste20/SPS1 related kinases. The alignment was generated by DNASTAR

megalign program using the clustal method.

(C) Schematic representation of the ILPIPα domains. The predicted amino acid sequence

of ILPIPα encodes for a protein 418 aa long. ILPIPα has a “putative” protein kinase

domain (aa 58-369). The ATP-binding site is located at the N-terminus of the protein.

Several putative serine, threonine or tyrosine active and phosphorylation sites are shown.

Fig. 2. Tissue distribution and expression pattern of ILPIP mRNA.

Northern blot analysis of ILPIP. 32P radiolabeled DNA fragment of ILPIP was hybridized

at high stringency to a nitrocellulose membrane bearing 2 µg poly(A) RNA/lane isolated

from normal human tissues (Clontech). A single 2.4 kb transcript was detected using the

ILPIP probe with higher levels of expression in muscle, liver and heart. ILPIP was also

expressed as a 2.4 kb band in human embryonic kidney cells. GAPDH was used to check

equal loading of RNA. The size of the transcripts is shown on the left.


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Fig. 3. ILPIP moderately activates JNK family members and strongly enhances the

XIAP-mediated activation of JNK1, JNK2 and JNK3.

Effect of XIAP, ILPIPα or ILPIPβ individual or synergistic expression on JNK kinases

activation. 293T cells were transfected with vectors encoding for JNK1 (A), JNK2 or

JNK3 (B) (200 ng each) in the absence or presence of increasing concentrations of XIAP,

ILPIPα or ILPIPβ (200 or 800 ng). The amount of transfected DNA was kept constant in

each sample by adding control pcDNA3 vector. In vitro kinase assay was performed

using ATF-2 as substrate. Kinase activity was quantitated by PhosphorImager analysis

and is expressed as fold induction relative to the basal level of phosphorylation of each

JNK. UV was used as positive control for each JNK activation (data not shown for JNK2

and JNK3). Western blots showing expression levels of JNK1, XIAP, ILPIPα or ILPIPβ

are shown for each experiment. Expression of ILPIPα or ILPIPβ corresponds to proteins

of 52 or 35 kDa, respectively. Asterisks indicate the presence of an unspecific band that

appears when the anti-HA antibody is used.

Fig. 4. ILPIP moderately protects against ICE- or Fas-induced apoptosis and significantly

potentiates the anti-apoptotic activity of XIAP.

(A) Effect of XIAP, ILPIPα or ILPIPβ individual or synergistic expression on ICE-

induced apoptosis. 293T cells were transfected with plasmids encoding for ICE-ß-

galactosidase alone (200 ng) or ICE together with increasing concentrations of DNA

expressing XIAP or ILPIP alone (200, 800 ng) or in combination (200 ng of XIAP and

200 and 800 ng of ILPIPα or ILPIPβ). % apoptosis indicates the number of apoptotic


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cells among the β-galactosidase-positive cells. Data represent the mean ± SE of at least

three experiments, each run in duplicate and scored blind.

(B) Effect of XIAP or ILPIPα individual or synergistic expression on Fas-induced

apoptosis. MCF7-Fas cells were co-transfected with control vector pcDNA3 alone or

plasmids encoding for XIAP or ILPIPα alone (200, 800 ng) or in combination (200 ng of

XIAP and 800 ng of ILPIPα). DNA expressing ß-galactosidase (200 ng) was also

transfected to allow visualization of transfected cells and quantitation of apoptotic cells.

Transfected samples were treated for 16 h with anti-Fas antibody (150 ng/ml) and

analyzed as described above.

Fig. 5. XIAP/ILPIPα synergistic activation of JNK1 is mediated by TAK1.

Effects of LacZ, TAK1 (KW) or ASK1 (KM) on XIAP and ILPIPα synergistic activation

of JNK1. Plasmids encoding wt JNK1 (100 ng) and increasing amounts of XIAP or

ILPIPα alone or in combination (200 and 800 ng) were transfected in 293T cells stably

expressing LacZ control gene (A), TAK1 (KW) (B) or ASK1 (KM) (C). In vitro kinase

assay was performed on immunoprecipitated JNK1 using ATF-2 as substrate and kinase

activity quantitated by PhosphorImager. UV stimulation is also shown. Western blots

show equal expression of JNK1. Similarly there was equal expression of XIAP or ILPIPα

(data not shown).

Fig. 6. XIAP and ILPIP interact with TAK1 and TRAF6.

(A) In vivo interaction of XIAP or ILPIPα with TAK1. Vectors encoding XIAP-FLAG,

or ILPIPα-FLAG were co-transfected with wt TAK1-HA or AKT–HA in 293T cells. Cell


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extracts were immunoprecipitated using anti-FLAG antibody conjugated beads. Co-

precipitated TAK1 or AKT was detected by Western blot analysis with an anti-HA

antibody. Cell extracts were directly subjected to immunoblot analysis to check for

protein expression.

(B) Interaction of XIAP or ILPIPα with TRAF6. Vectors encoding XIAP-FLAG, or

ILPIPα-FLAG were co-transfected with wt TRAF6-Myc or deletion mutant TRAF6-Myc

(TRAF6∆−Myc) in 293T cells. Cell extracts were immunoprecipitated using anti-Myc

antibody. Co-precipitated XIAP or ILPIPα was detected by Western blot analysis with an

anti-FLAG antibody. Vectors encoding for XIAP-Myc and ILPIPα−FLAG were also

transfected in 293T cells to check for co-precipitation between XIAP and ILPIPα. Cell

extracts were immunoprecipitated using anti-Myc antibody and co-precipitated

ILPIPα was detected by western blot analysis with an anti-FLAG antibody.

(C) Interaction of XIAP or ILPIPα with TAB1 and TAB2. Vectors encoding XIAP-HA

or ILPIPα-HA were co-transfected with TAB1 or TAB2 in 293T cells. Cell extracts were

subjected to immunoprecipitation with anti-TAB1 or anti TAB2 antibody and co-

precipitated XIAP or ILPIPα were detected by Western blot analysis using anti-HA

antibody. Cells extracts were subjected to immunoblot analysis to check protein

expression.

(D) In vitro interaction of XIAP with ILPIPα. GST-XIAP or GST recombinant proteins

were incubated with gluthatione conjugated beads and in vitro translated ILPIPα protein

was added. Co-precipitation of ILPIPα was detected by Western blot using an anti-HA

antibody. Input proteins were detected by Western blot using anti-GST antibodies.


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Fig. 7. ILPIPα does not affect XIAP inhibition of caspase activation.

Effect of ILPIPα on XIAP inhibition of caspase activity. 293T cells were seeded in 100

mm dishes and transfected with plasmids encoding for XIAP or ILPIPα alone (3 µg) or

in combination. Empty vector was also transfected alone as a control (6µg). Cell extracts

were prepared and cytochrome c was added to induce proteolytic processing of pro-

caspase-3. Caspase activity was measured by monitoring the release of AFC DEVD-

containing synthetic peptides. The last two columns represent the results obtained using

cell extracts expressing XIAP alone or with ILPIPα, diluted ten fold.


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A

CCACGGCCCCGGTCGCGGCCTCGCCGCCCTCCCGCGCCCCGCGCCGGGAGCGGGCCTAGAGCGCTCGCCTCGC

CCCTCCGCGAGCAGGGGCTCTGGCGCCCGCCCCTGTCCGCACCGCTGCAGCCTGAAGAGAGTCGCTGGCCGTGGTCGCCGCTAGGTAGGA TATATCTGCATCTTGAAAGGAAGATAAAACAAAAGCCTTCTTTGGAATAGATGGATTTTTGTCACTTTCTGTGTGAAAGTGATTCACTAA ATGTCTCTTTTGGATTGCTTCTGCACTTCAAGAACACAAGTTGAATCACTCAGACCTGAAAAACAGTCTGAAACCAGTATCCATCAATAC

1 M S L L D C F C T S R T Q V E S L R P E K Q S E T S I H Q Y TTGGTTGATGAGCCAACCCTTTCCTGGTCACGTCCATCCACTAGAGCCAGTGAAGTACTATGTTCCACCAACGTTTCTCACTATGAGCTC

31 L V D E P T L S W S R P S T R A S E V L C S T N V S H Y E L CAAGTAGAAATAGGAAGAGGATTTGACAACTTGACTTCTGTCCATCTTGCACGGCATACTCCCACGGGAACACTGGTAACTATAAAAATT

61 Q V E I G R G F D N L T S V H L A R H T P T G T L V T I K I ACAAATCTGGAAAACTGCAATGAAGAACGCCTGAAAGCTTTACAGAAAGCCGTGATTCTATCCCACTTTTTCCGGCATCCCAATATTACA

91 T N L E N C N E E R L K A L Q K A V I L S H F F R H P N I T ACTTATTGGACAGTTTTCACTGTTGGCAGCTGGCTTTGGGTTATTTCTCCATTTATGGCCTATGGTTCAGCAAGTCAACTCTTGAGGACC

121 T Y W T V F T V G S W L W V I S P F M A Y G S A S Q L L R T TATTTTCCTGAAGGAATGAGTGAAACTTTAATAAGAAACATTCTCTTTGGAGCCGTGAGAGGGTTGAACTATCTGCACCAAAATGGCTGT

151 Y F P E G M S E T L I R N I L F G A V R G L N Y L H Q N G C ATTCACAGGAGTATTAAAGCCAGCCATATCCTCATTTCTGGTGATGGCCTAGTGACCCTCTCTGGCCTTTCCCATCTGCATAGTTTGGTT

181 I H R S I K A S H I L I S G D G L V T L S G L S H L H S L V AAGCATGGACAGAGGCATAGGGCTGTGTATGATTTCCCACAGTTCAGCACATCAGTGCAGCCGTGGCTGAGTCCAGAACTACTGAGACAG

211 K H G Q R H R A V Y D F P Q F S T S V Q P W L S P E L L R Q GATTTACATGGGTATAATGTGAAGTCAGATATTTACAGTGTTGGGATTACAGCATGTGAATTAGCCAGTGGGCAGGTGCCTTTCCAGGAC

241 D L H G Y N V K S D I Y S V G I T A C E L A S G Q V P F Q D ATGCATAGAACTCAGATGCTGTTACAGAAACTGAAAGGTCCTCCTTATAGCCCATTGGATATCAGTATTTTCCCTCAATCAGAATCCAGA

271 M H R T Q M L L Q K L K G P P Y S P L D I S I F P Q S E S R ATGAAAAATTCCCAGTCAGGTGTAGACTCTGGGATTGGAGAAAGTGTGCTTGTCTCCAGTGGAACTCACACAGTAAATAGTGACCGATTA

301 M K N S Q S G V D S G I G E S V L V S S G T H T V N S D R L CACACACCATCCTCAAAAACTTTCTCTCCTGCCTTCTTTAGCTTGGTACAGCTCTGTTTGCAACAAGATCCTGAGAAAAGGCCATCAGCA

331 H T P S S K T F S P A F F S L V Q L C L Q Q D P E K R P S A AGCAGTTTATTGTCCCATGTTTTCTTCAAACAGATGAAAGAAGAAAGCCAGGATTCAATACTTTCACTGTTGCCTCCTGCTTATAACAAG

361 S S L L S H V F F K Q M K E E S Q D S I L S L L P P A Y N K CCATCAATATCATTGCCTCCAGTGTTACCTTGGACTGAGCCAGAATGTGATTTTCCTGATGAAAAAGACTCATACTGGGAATTCTAGGGC

391 P S I S L P P V L P W T E P E C D F P D E K D S Y W E F * TGCCAAATCATTTTATGTCCTATATACTTGACACTTTCTCCTTGCTGCTTTTTTTTCTGTATTTCTAGGTACAAATACCAGAATTATACT TGAAAATACAGTTGGTGCACTGGAGAATCTATTATTTAAAACCACTCTGTTCAAAGGGGCACCAGTTTGTAGTCCCTCTGTTTCGCACAG AGTACTATGACAAGGAAACATCAGAATTACTAATCTAGCTAGTGTCATTTATTCTGGAATTTTTTTCTAAGCTGTGACTAACTCTTTTTA TCTCTCAATATAATTTTTGAGCCAGTTAATTTTTTTCAGTATTTTGCTGTCCCTTGGGAATGGGCCCTCAGAGGACAGTGCTTCCAAGTA CATCTTCTCCCAGATTCTCTGGCCTTTTTAATGAGCTATTGTTAAACCAACAGGCTAGTTTATCTTACATCAGACCCTTTTCTGGTAGAG GGAAAATGTGTGTGCTTTCCCTTTTTCTTCTGTTAATACTTATGGTAACACCTAACTGAGCCTCACTCACATTAAATGATTCACTTGAAA TATTAAAAAAAAAAAAAAAAAAAAAAAAAAA


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M S L L D C F C T S R T Q V E S L R P E K - - - Q S E T S I H Q Y L V D E P T L S W S R P S T R A S E V L C S - - - - - - - - - - - T N V S 1 ILPIP

M S F L V - - - S K P E R I R R W V S E K F I V E G L R D L E L F G E Q P P G D T R R K S E S I A S F S K Q E V M S S F L P E G G 1 NY-BR-96M A E - - - - - P S G S P V H V Q L P Q Q A A P V T A A A A A A P A A A T A A P A P A A P A A P A P A P A P A P A A Q A V G W P I C R D A - 1 SPAK

H Y E L Q V E I G R G F D N L T S V H L A R H T P T G T L V T I K I T N L E N C N E E R L K A L Q K A V I L S H F F R H P N I T T Y W T V F 57 ILPIPC Y E L L T V I G K G F E D L M T V N L A R Y K P T G E Y V T V R R I N L E A C S N E M V T F L Q G E L H V S K L F N H P N I V P Y R A T F 68 NY-BR-96- Y E L Q E V I G S G A T A V V Q A A L - - C K P R Q E R V A I K R I N L E K C Q T S M D E L L K E I Q A M S Q - C S H P N V V T Y Y T S F 65 SPAK

T V G S W L W V I S P F M A Y G S A S Q L L R T Y F P E G - - - - - - M S E T L I R N I L F G A V R G L N Y L H Q N G C I H R S I K A S H I 127 ILPIPI A D N E L W V V T S F M A Y G S A K D L I C T H F M D G - - - - - - M N E L A I A Y I L Q G V L K A L D Y I H H M G Y V H R S V K A S H I 138 NY-BR-96V V K D E L W L V M K L L S G G S M L D I I K Y I V N R G E H K N G V L E E A I I A T I L K E V L E G L D Y L H R N G Q I H R D L K A G N I 131 SPAK

L I S G D G L V T L S G L S H L H S L V K H G Q - - R H R A V Y D F P Q F S T S V Q P W L S P E L L R Q D L H G Y N V K S D I Y S V G I T A 191 ILPIPL I S V D G K V Y L S G L R S N L S M I S H G Q - - R Q R V V H D F P K Y S V K V L P W L S P E V L Q Q N L Q G Y D A K S D I Y S V G I T A 202 NY-BR-96L L G E D G S V Q I A D F G V S A F L A T G G D V T R N K V R K T F - - - - V G T P C W M A P E V M E Q - V R G Y D F K A D M W S F G I T A 201 SPAK

C E L A S G Q V P F Q D M H R T Q M L L Q K L K G P P Y S P L D I S I F P Q S E S R M K N S Q S G V D S G I G E S V L V S S G T H T V N S D 259 ILPIPC E L A N G H V P F K D M P A T Q M L L E K L N G T V P C L L D T S T I P A E E L T M S P S R S V A N S G L S D S - L T T S T P R P S N G D 270 NY-BR-96I E L A T G A A P Y H K Y P P M K V L M L T L Q N D P P T - L E T G V - - E D K E M M K K Y G K S F R K L L S L C L Q K D P S K R P T A A E 266 SPAK

R L H T P S - S K T F S P A F F S L V Q L C L Q Q D P E - - - - - - - - - K R P S A S S L L S H V - - - - - - - - - - - - - - - - - - - - - 329 ILPIPS P S H P Y - H R T F S P H F H H F V E Q C L Q R N P D - - - - - - - - - A R P S A S T L L N H S - - - - - - - - - - - - - - - - - - - - - 339 NY-BR-96L L K C K F F Q K A K N R E Y - - L I E K L L T R T P D I A Q R A K K V R R V P G S S G H L H K T E D G D W E W S D D E M D E K S E E G K A 333 SPAK

- F F K Q M K E E S Q D S I L S L L P P A Y N K P - - S I S L P P V L P W T E P E - - - - - - - - - - - - - - - C - - - - - D F P D E K D S 368 ILPIP- F F K Q I K R R A S E A L P E L L R P V - - T P - - I T N F E G S Q S Q D H S G - - - - - - - - - - - - - - - I F G L V T N L E E L E V D 378 NY-BR-96A F S Q E K S R R V K E E N P E I A V S A S T I P E Q I Q S L S V H D S Q G P P N A N E D Y R E A S S C A V N L V L R L R N S R K E L N D I 401 SPAK

Y W E F

415 ILPIP

D W E F

428 NY-BR-96

R F E F T P G R D T A D G V S Q E L F S A G L V D G H D V V I V A A N L Q K I V D D P K A L K T L T F K L A S G C D G S E I P D E V K L I G 471 SPAK

418 ILPIP

431 NY-BR-96

F A Q L S V S 541 SPAK

B

T N D A S


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N

1 418

ATP-bindingsite

S-T kinase active site

Y kinase active site

Y kinaseP site

C

58 369

Protein Kinase Domain

N

1 418

ATP-binding S-T kinase Y kinase

C

58 369

Protein Kinase Domain

C by guest on A

pril 2, 2018http://w

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.jbc.org/D

ownloaded from

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GAPDH

ILPIP

Kb

9.57.5

4.4

2.4

1.3

Hea

rtB

rain

Plac

enta

Live

rM

uscl

eK

idne

yPa

ncre

as

Lung

PolyA mRNA

293TotalRNA

5 µµg

10 µµ

g


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A

XIAPILPIPααILPIPββ

JNK1

---

200--

800--

-200

-

-800

-

--

200

--

800

200200

-

200800

-

200-

200

200-

800

UV---

800200

-

800-

200

ATF-2

FoldActivation

1.0 4.2 9.1 2.3 3.5 1.8 2.5 16.0 35.0 6.4 17.0 36.0 25.0 19.5

BJNK1 JNK3 JNK2

FoldActivation

1.0 3.2 7.0 31.0 1.0 2.3 6.6 13.0 1.0 2.6 9.0 15.2

ATF-2

--

200-

200200

200800

--

200-

200200

200800

--

200-

200200

200800

XIAPILPIPαα

WB anti-FLAG

WB anti-Myc

WB anti-HA

WB anti-FLAG

WB anti-Myc

WB anti-HA

JNK1

ILPIPααILPIPββ

XIAP

ILPIPαα

JNKs

XIAP

*


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0

10

20

30

40

50

60Vec

tor

GFP 8

00

ILPIP

αα80

0

ICE-transfected 293T cells

XIAP 200

% A

po

pto

sis

XIAP 8

00

XIAP 2

00

ILPIP

α α 200

ILPIP

ββ80

0

ILPIP

ββ20

0

ILPIP

αα80

0

ILPIP

α α 200

ILPIP

ββ80

0

ILPIP

ββ20

0

A

B

0

10

20

30

40

50

60

70

XIAP 200

Vecto

rXIA

P 200

XIAP 8

00

GFP 8

00

ILPIP

α α 200

ILPIP

α α 800

ILPIP

α α 800

αα-Fas treated cells

% A

po

pto

sis


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ATF-2

FoldActivation

1.0 3.0 9.1 50.0 2.2 3.4 5.3 13.0

JNK1

A

XIAPILPIPαα

LacZ

--

200-

800-

-200

-800

UV --

200200

200800

WB anti-FLAG

ATF-2

B

XIAPILPIPαα

TAK1 (KW)

--

200-

800-

-200

-800

UV --

200200

200800

JNK1

FoldActivation

1.0 1.0 1.2 49.0 1.0 1.0 1.4 1.8

WB anti-FLAG

C

XIAPILPIPαα

ASK1 (KM)

--

200-

800-

-800

UV --

200200

200800

-200

FoldActivation

1.0 5.5 11.0 51.0 3.2 3.5 10.0 13.0

JNK1WB anti-FLAG

ATF-2


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IP: anti-FLAG

A

IP: anti-FLAG

ILPIPαα-FLAG

XIAP-FLAG

TAK1-HA

AKT-HA

+- +- -- + --- -+ -+ - +-+ +- -+ - --- -- +- + +

IP: anti-HA

XIAP

ILPIPαα

AKTTAK1

TAK1

61

49

61

49

61

49

WB

Anti-HA

Anti-FLAG

Anti-HA

IP: anti-Myc

IP: anti-Myc

IP: anti-FLAG

XIAP

ILPIPαα

XIAP

TRAF6∆∆

XIAP

ILPIPαα

TRAF6

61

61

61

49

49

49

36

80

TRAF6-Myc -- - -TRAF6∆∆-Myc -- - -

ILPIPαα-FLAG +- - +-XIAP-FLAG - - -

XIAP-Myc -- ++

++ + -- --- - ++ +

+- - +- --- + -- +

-- - -- -

B

WB

Anti-FLAG

Anti-Myc

Anti-FLAG


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C

D

XIAP-GST

ILPIPαα-HA

+-+

-++

GST

XIAP

GST

IP: anti-GST anti-HAWB

anti-GSTInput

ILPIPαα

ILPIPαα-HAXIAP-HA

TAB1

TAB2

-+ +- +- - -+- -- -+ + --- ++ -+ - --- -- -- - +

IP: anti-TAB1or

anti-TAB2

Input

InputTAB2TAB1

XIAPWB

anti-HA

anti-TAB1 or

anti-TAB2

anti-HA

+--+

-+-+

XIAPILPIPαα


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0

20

40

60

80

100%

Cas

pas

eac

tivi

ty

Cyto-c

XIAPILPIPαα

+ +++++++++

+ ++

- -- -

--

--


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Binh Nguyen, Quinn L. Deveraux and Richard J. UlevitchM. Germana Sanna, Jean da Silva Correia, Ying Luo, Betty Chuang, Lorien M. Paulson,

JNK1 and protection against apoptosisILPIP, a novel anti-apoptotic protein that enhances XIAP-mediated activation of

published online June 4, 2002J. Biol. Chem.

10.1074/jbc.M203312200Access the most updated version of this article at doi:

Alerts:

When a correction for this article is posted•

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http://www.jbc.org/cgi/alerts?alertType=correction&addAlert=correction&correction_criteria_value=early/2002/06/04/jbc&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/early/2002/06/04/jbc.M203312200.citation

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ilpip, a novel anti-apoptotic protein that enhances xiap- mediated

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