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Arf and Rho GAP adapter protein ARAP1 participates in the mobilization of TRAIL-R1/DR4 to the plasma membrane

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Abstract

TRAIL, a ligand of the TNFα family, induces upon binding to its pro-death receptors TRAIL-R1/DR4 and TRAIL-R2/DR5 the apoptosis of cancer cells. Activated receptors incite the formation of the Death-Inducing Signaling Complex followed by the activation of the downstream apoptotic signaling. TRAIL-induced apoptosis is regulated at multiple levels, one of them being the presence and relative number of TRAIL pro- and anti-apoptotic receptors on the cytoplasmic membrane. In a yeast two-hybrid search for proteins that interact with the intracellular part (ICP) of DR4, we picked ARAP1, an adapter protein with ArfGAP and RhoGAP activities. In yeast, DR4(ICP) interacts with the alternatively spliced ARAP1 lacking 11 amino acids from the PH5 domain. Transfected ARAP1 co-precipitates with DR4 and co-localizes with it in the endoplasmic reticulum/Golgi, at the cytoplasmic membrane and in early endosomes of TRAIL-treated cells. ARAP1 knockdown significantly compromises the localization of DR4 at the cell surface of several tumor cell lines and slows down their TRAIL-induced death. ARAP1 overexpressed in HEL cells does not affect their TRAIL-induced apoptosis or the membrane localization of DR4, but it enhances the cell-surface presentation of phosphatidyl serine. Our data indicate that ARAP1 is likely involved in the regulation of the cell-specific trafficking of DR4 and might thus affect the efficacy of TRAIL-induced apoptosis.

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Abbreviations

TNF:

Tumor necrosis factor

TRAIL/Apo2L:

TNF-related apoptosis-inducing ligand

Arf:

ADP-ribosylation factor small GTPase

ARAP1:

ArfGAP, RhoGAP, Ankyrin repeats and pleckstrin homology (PH) domains containing protein

DD:

Death domain

DISC:

Death-inducing signaling complex

ER:

Endoplasmic reticulum

GAP:

GTPase-activating protein

IAP:

Inhibitor of apoptosis

MFI:

Median fluorescence intensity

MMP:

Mitochondrial membrane permeabilization

PARP:

Poly-ADP ribose polymerase

SiRNA:

Small inhibitory RNA

TGN:

Trans-Golgi network

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Acknowledgements

We are grateful to Drs. J. Tschopp, L. LoMuzio, P.A. Randazzo, S. Krugmann and K. Drbal for providing cells and reagents, and J. Dutt for critical reading of the manuscript. The project was supported by the Center for Molecular and Cellular Immunology 1M0506, the FP6 program LSHG-CT-2006-037278 and by the Institutional Grant AV0Z50520514.

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Authors and Affiliations

  1. Laboratory of Cell Signaling and Apoptosis, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Vídeňská 1083, 14220, Praha 4, Czech Republic

    Šárka Šímová, Martin Klíma, Lukas Cermak, Vladimíra Šourková & Ladislav Anděra

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  1. Šárka Šímová
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Correspondence to Ladislav Anděra.

Electronic supplementary material

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Suppl. Table 1

Sequences of the primers used for the generation of DR4 mutants and for RT-PCR or real-time PCR to detect the relative abundance of the splice variant ARAP1Δexon30. (EPS 403 kb)

Suppl. Fig. 1

ARAP1Dexon30 is the main splice variant expressed in human tumor-derived cell lines. (A) cDNAs prepared from different primary cells and cell lines were analyzed for their relative expression of ARAP1 splice variants (FL and Dexon30) by quantitative real-time PCR as described in Suppl. Materials and Methods. cDNAs prepared from HEK293 cells transfected either with ARAP1FL or ARAP1Δexon30 were used as a controls. Average means and standard errors from 3 independent experiments are shown. (B) Apa1 cleavage of exon 30 within the PCR-amplified 3′ end of ARAP1 (3350–4495; arrow) yields two fragments of 417 bp and 729 bp (asterisks); the PCR product (1146 bp) of ARAP1-CΔexon30 is not cleaved. 1,2—uncleaved and Apa I-digested PCR product of ARAP1-FL; 3,4—uncleaved and Apa I-digested PCR product of ARAP1 amplified from NCTC cDNA. Gene Ruler 1 kb DNA Ladder (Fermentas) was used as a size marker. (EPS 368 kb)

Suppl. Fig. 2

(A) Alignment of the first two a-helices in the death domains of the TNFR death receptors. Amino acids F377 and D378 in DR4/DR5 that were in DR4 mutated to proline and leucine, respectively, are shown in the box. First and second a-helices in Fas DD are underlined. (B) NCTC cells were treated either with TRAIL (200 ng/ml) for 20 and 50 min or pre-incubated with zVAD (20 μM final concentration) for 30 min prior to the TRAIL treatment. Cell lysates were precipitated with anti-DR4 monoclonal antibody and analyzed by Western blotting with the appropriate antibodies (Ips, immunoprecipitations; CLs, cell lysates). Filled arrow marks ARAP1(FL) and empty arrow processed form of ARAP1. (EPS 2497 kb)

Suppl. Fig. 3

Graphical representation of the relative cell surface expression of selected receptors on Saos-2, MG-63 and HCT116 cells. Relative representations of the averaged fluorescence medians for Saos-2, MG-63 and HCT116 cells from 4 independent experiments, together with the standard errors and statistical significance, are shown (* = P < 0.05). (EPS 487 kb)

Suppl. Fig. 4

Suppression of ARAP1 expression slows down TRAIL-induced apoptosis of MG-63 cells. MG-63 cells were grown in 24-well plates and transfected either with luciferase or with ARAP1 siRNA. Fifty-two hours after transfection, some of the cells transfected with luciferase siRNA were pre-incubated with anti-DR4 or anti-DR6 monoclonal antibodies antibody (at a final conc. of 10 mg/ml for 1 h) before the TRAIL treatment. Cells were treated with TRAIL (200 ng/ml) plus cycloheximide (10 ug/ml) for 0, 90, 120, 150 or 180 min, harvested and washed with ice-cold PBS. Cells were then fixed with ice-cold methanol, stained with FITC-conjugated anti-M30 antibody according to the manufacturer’s protocol and analyzed by flow cytometry. The abscissa above the histograms with the percentage indicated represents the percentage of M30-positive, apoptotic cells. (EPS 2757 kb)

Suppl. Fig. 5

ARAP1 co-fractionated with DR4 in the light membrane fractions of a sucrose-density gradient. NCTC cells were grown in 100 mm plates and transfected either with luciferase or with ARAP1 siRNA. Fifty-two hours after transfection, cells were lysed by hypotonic lysis and the postnuclear supernatants were subjected to sucrose-density gradient centrifugation. The gradient was split into 10 equal volume fractions from the top to the bottom. One tenth of each fraction together with the pellet was analyzed by Western blotting using anti-DR4 and ARAP-1 antibodies. The light membrane fractions (1–3) contain the plasma membrane. Arrows indicate the migration of DR4. (EPS 518 kb)

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Šímová, Š., Klíma, M., Cermak, L. et al. Arf and Rho GAP adapter protein ARAP1 participates in the mobilization of TRAIL-R1/DR4 to the plasma membrane. Apoptosis 13, 423–436 (2008). https://doi.org/10.1007/s10495-007-0171-8

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