Identification of a cell protein (FIP-3) as a modulator of NF-kappaB activity and as a target of an adenovirus inhibitor of tumor necrosis factor alpha-induced apoptosis - PubMed (original) (raw)

Identification of a cell protein (FIP-3) as a modulator of NF-kappaB activity and as a target of an adenovirus inhibitor of tumor necrosis factor alpha-induced apoptosis

Y Li et al. Proc Natl Acad Sci U S A. 1999.

Abstract

FIP-3 (14.7K interacting protein) was discovered during a search for cell proteins that could interact with an adenovirus protein (Ad E3-14.7K) that had been shown to prevent tumor necrosis factor (TNF)-alpha-induced cytolysis. FIP-3, which contains leucine zippers and a zinc finger domain, inhibits both basal and induced transcriptional activity of NF-kappaB and causes a late-appearing apoptosis with unique morphologic manifestations. Ad E3-14.7K can partially reverse apoptotic death induced by FIP-3. FIP-3 also was shown to bind to other cell proteins, RIP and NIK, which previously had been described as essential components of TNF-alpha-induced NF-kappaB activation. In addition, FIP-3 inhibited activation of NF-kappaB induced by TNF-alpha, the TNFR-1 receptor, RIP, NIK, and IKKbeta, as well as basal levels of endogenous NF-kappaB in 293 cells. Because the activation of NF-kappaB has been shown to inhibit apoptosis, FIP-3 appears both to activate a cell-death pathway and to inhibit an NF-kappaB-dependent survival mechanism.

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Figures

Figure 1

Polypeptide sequence of FIP-3 derived from cDNA sequence. The figure shows the polypeptide sequence of FIP-3, which was deduced from the sequence of the yeast two-hybrid clone starting at amino acid 180 plus the 5′ rapid amplification of cDNA ends/PCR-derived sequences. Gene-specific primers (5′-CTCCTCGGCCTGCTGGAGCTGCTG 3′) and adapter primers (5′-CCATCCTAATACGACTCACTATAGGGC 3′) present in rapid amplification of cDNA ends–ready heart cDNA templates (CLONTECH) were used to identify the missing 5′ sequence (18). Three continuously underlined regions are three putative leucine-zipper regions. A zinc finger domain is shown in bold at the C terminus of the protein with the canonical C and H residues underlined, and putative nuclear localization signals are double-underlined. The sequence of FIP-2 is juxtaposed with the homologous amino acids, indicated by conventional symbols to indicate identity (27%) or degrees of similarities (38%).

Figure 2

Interaction of FIP-3 and Ad E3–14.7K proteins. Immunohistochemical studies of mouse C3HA cells, which constitutively express Ad E3–14.7K protein, were performed after transient transfection with a T7-tagged FIP-3 (FIP-3Δ179, 1 μg)-expressing plasmid. The relationship of these two proteins in the cells was shown by the use of anti-T7 mAbs (A) or anti-14.7K polyclonal antibodies (B). The immunoreactivities were visualized by confocal microscopy after staining with fluorescein-conjugated anti-mouse antibodies (A) or Texas red-conjugated anti-rabbit sera (B). The single asterisks in B denote cells that were expressing E3–14.7K in a diffuse distribution pattern in the cytoplasm in the absence of FIP-3. The double asterisks refer to cells coexpressing FIP-3 and E3–14.7K. The dense perinuclear bead-like structures were stained heavily with antibodies directed against the FIP-3 and E3–14.7K proteins after transient transfection of the cells with a T7-FIP-3 expressing plasmid. (Bar = 10 μm.) Direct in vitro and in vivo interactions between FIP-3 and E3–14.7K also were demonstrated as described (36). 35S-met-labeled FIP-3 from an in vitro translation reaction was incubated with either bacterial-expressed GST or GST-14.7K protein immobilized on glutathione beads. After extensive washes, labeled FIP-3 retained on the beads was analyzed by SDS/PAGE and autoradiography (C). Human 293 cells were seeded on 100-mm dishes and were transfected by using the LipofectAmine technique with 2 μg each of the following plasmids (D). Lanes: 1, FLAG-FIP-2Δ134 plus T7-FIP3; 2, FLAG-14.7 plus T7-FIP3; 3, FLAG-14.7 plus T7-FIP-3, treated with 20 ng/ml TNF-α for 20 min before harvesting the cells. Twenty-four hours after transfection, cell lysates were prepared and treated with 2 μl of anti-FLAG antibody followed by incubation with protein A beads. After several washes, the protein complex bound to the beads was analyzed by SDS/PAGE. Immunoprecipitated T7-tagged FIP-3 protein was identified by Western blotting with horseradish peroxidase-conjugated anti-T7 antibody (Novagen).

Figure 3

Overexpression of FIP3 induces a unique morphology and intracytoplasmic inclusion bodies. Studies of FIP-3 morphology by using a GFP marker are shown in A. Human 293 cells on 6-well plates were transfected with 2 μg of pcDNA-T7-FIP3 and 1 μg of pGFP plasmid DNA. In B, cells transfected with 2 μg of pcDNA-T7 and 1 μg of pGFP plasmid as normal controls are shown. Forty hours after transfection, cells were examined and photographed under a fluorescent microscope with a fluorescein isothiocyanate filter. The cells with the intracellular round structures, stained green in the original and typical of the FIP-3 phenotype, are indicated by arrows (A). Another cell in an advanced stage of disintegration is designated by an arrowhead. (Bar = 10 μm.)

Figure 4

Induction of apoptosis by FIP-3 and prevention of cell death by Ad E3–14.7K. Human 293 cells were seeded onto 100-mm dishes and were transfected by using the LipofectAmine protocol with plasmids as indicated below. In addition, each plate contained 0.3 μg of the GFP plasmid for monitoring transfection efficiency. Forty hours after transfection, the transfected cells were lysed (A). Low molecular weight nuclear DNAs were isolated and analyzed on 2% agarose gel as described in Materials and Methods. Lanes: 1, 4.7 μg of pcDNA-T7; 2, 1.5 μg of pcDNA-T7-FIP3 plus 3.2 μg pcDNA T7. In B, 293 cells were transfected similarly, but apoptosis was assayed and quantified by an ELISA technique as described in Materials and Methods. Lanes: 1, 4.5 μg of pcDNA T7; 2, 1.5 μg of pcDNA FIP-3; 3, 1.5 μg of pcDNA FIP-3 and 3 μg of pcDNA E3–14.7K; 4, 1.5 μg of pcDNA FIP-3Δ1–179; 5, 2.0 μg of pcDNA TR55. The total amount of transfected DNA in each lane was adjusted to 4.5 μg by the addition of pcDNA T7.

Figure 5

Ad E3 14.7K affects the intracellular localization of FIP-3. 293 cells were transfected with plasmids expressing various combinations of pcDNA-T7-FIP-3 and pcDNA-FLAG-14.7K. The nuclear and cytoplasmic fractions were prepared by Dounce homogenization in hypotonic buffer, and each was analyzed for FIP-3 protein 24 hours later by Western blots using antibody to the T7 tag conjugated with horseradish peroxidase (Novagen) at a dilution of 1:2,000 for 1.5 hours at room temperature. The blots were washed 3–4 times in PBS with 0.1% Tween 20 and were developed with enhanced chemiluminescence reagents (Boehringer Mannheim). Lanes: 1 and 2, negative controls that contain 5 μg of pcDNA-T7 and 3 μg of pcDNA-14.7K, respectively; 3–7, 1 μg of pcDNA-FIP-3; 4–7, increasing amounts of pcDNA-14.7K; 4, 1 μg; 5, 2 μg; 6, 3 μg; 7, 4 μg. All transfections were normalized to contain 5 μg of DNA by the addition of pcDNA-T7.

Figure 6

Association of FIP-3 with RIP and NIK in 293 cells. In A, 293 cells were transfected with the following plasmids for coimmunoprecipitation and Western blot analysis. Lanes: 1, 2 μg of pcDNA-FLAG-RIP plus 2 μg of pcDNA-T7-FIP1; 2, 2 μg of pcDNA-FLAG-FIP-2Δ134 plus 2 μg of pcDNA-T7-FIP3; 3, 2 μg of pcDNA-FLAG-RIP plus 2 μg of pcDNA-T7-FIP3; 4, same as lane 3 plus TNF-α (20 ng/ml for 20 min before harvest). Cell lysate preparation, immunoprecipitation with anti-FLAG antibody, and Western blots with anti T7 antibody were done as described in Fig. 2_D_. In B, cytoplasmic extracts of 293 cells transiently transfected in 100 mm dishes with 8–10 μg of plasmids containing various RIP domains were added to anti-FLAG M2 affinity gels for 2 hours at 4°C. After washing the gels three times, 35S-FIP-3 made in reticulocyte lysates was added for an additional 2 hours, and the washing was repeated. The labeled proteins were eluted from the gel with SDS sample buffer, were separated by PAGE, and were detected by autoradiography. Lanes: 1, the RIP kinase domain (KD); 2, the intermediate domain (ID); 3, the death domain (DD); 4, intact RIP; lane 5, the labeled FIP-3 added directly to the SDS/PAGE. In C, 293 cell lysate preparation, immunoprecipitation with anti-HIS antibody, and Western blots with anti-FLAG antibody were done as described in A and Fig. 2_D_. Lanes: 1, the extracts of cells cotransfected with FLAG-NIK and HIS-FIP-3; 2 , the FLAG-NIK alone. The position of the NIK protein is shown. The lower two bands are nonspecific. In D, 35S-FIP-3 was added for 4 hours to extracts derived from Escherichia coli transfected with GST-NIK or GST in pGEX plasmids. The GST proteins were immobilized on glutathione Sepharose 4B before the addition of the radioactive extract, were washed five times, and were eluted for SDS/PAGE. Lanes: 1, 35S-FIP-3 added directly to SDS/PAGE; 2, the GST protein; 3, the GST-NIK protein.

Figure 7

Inhibition of TNF-α, TNFR-1, RIP, NIK, or IKK-β-induced transcriptional activation of NF-κB by FIP-3. 293 cells on 6-well plates were transfected with one of the following plasmids: 1.0 μg pcDNA-T7; 0.3 μg of pcDNA-TR55; 0.1 μg of pcDNA-FLAG-RIP; 0.5 μg of pcDNA-NIK; or 0.4 μg of pcDNA-IKKβ. One microgram of pcDNA-T7-FIP3 was added to one of each paired samples as designated. The amount of DNA in each transfection was normalized to 2.5 μg with control plasmid pcDNA-T7. In addition, each well contained the following plasmids: 0.2 μg pGreen Lantern and 0.1 μg pAd-CMV-LacZ to monitor transfection efficiency, 1 μg of baculovirus p35, and 0.2 μg of the NF-κB-dependent luciferase reporter construct pIgκ-Luc. Eighteen hours after transfection, cells were harvested, and luciferase activity was assayed by using the Luciferase Reporter Assay Kit (Boehringer Mannheim) following the manufacturer’s protocol. In addition, TNF-α (15 ng/ml) was added at 18 hours to paired samples previously transfected with pcDNA-T7 or FIP-3 plus the additional indicator plasmids. These cells were harvested at 24 hours and were processed for luciferase activity. The experiment was repeated at least three times, and similar results were obtained. Data shown here represented the luciferase activities from one of these experiments.

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Identification of a cell protein (FIP-3) as a modulator of NF-kappaB activity and as a target of an adenovirus inhibitor of tumor necrosis factor alpha-induced apoptosis - PubMed (original) (raw)