The Death Domain of Kidney Ankyrin Interacts with Fas and Promotes Fas-Mediated Cell Death in Renal Epithelia MARCELA DEL RIO,* ABUBAKR IMAM,* MARYELY DELEON,* GARY GOMEZ,* JAYA MISHRA, † QING MA, † SAMIR PARIKH, † and PRASAD DEVARAJAN* † *Department of Nephrology, Children’s Hospital at Montefiore, Albert Einstein College of Medicine, Bronx, New York, and † Department of Nephrology and Hypertension, Cincinnati Children’s Hospital Medical Center, University of Cincinnati, Cincinnati, Ohio. Abstract. Ankyrins are a ubiquitously expressed family of conserved proteins that mediate the linkage of integral mem- brane proteins such as transporters and channels with the underlying cytoskeleton. Ankyrins possess a conserved death domain, the functional significance of which has remained puzzling. In this study, the death domain of AnkG190, the isoform of ankyrin expressed in kidney tubules, was used as bait in a yeast two-hybrid screen to identify interacting part- ners. One of these interactions was with the proapoptotic molecule Fas. This was confirmed by coimmunoprecipitation, colocalization, and glutathione S-transferase pull-down assays in cultured renal epithelial (MDCK) cells. Site-directed mu- tagenesis of a conserved arginine (R1496 in AnkG190), pre- viously shown to be critical for the binding of Fas (R234 in Fas) to FADD, abolished the interaction of ankyrin’s death domain with Fas. Overexpression of constructs containing ankyrin’s death domain promoted Fas-mediated apoptosis in MDCK cells. The linkage between ankyrin and Fas was con- firmed in vivo in mouse kidney tubule cells by coimmunopre- cipitation and colocalization. In an established mouse model of renal ischemia-reperfusion injury characterized by apoptotic tubule cell death, the expression of both ankyrin and Fas was markedly induced, and the interaction between these molecules remained intact. The results identify a novel tethering interac- tion between ankyrin and Fas in kidney epithelia and suggest that AnkG190 may play a role as an adapter molecule in renal tubule cell death. Tethering interactions between membrane proteins and the underlying spectrin-based cytoskeleton play key roles in sev- eral cellular activities, including organization of plasma mem- brane domains (1). Ankyrins are a ubiquitously expressed family of conserved proteins that have emerged as critical adapter molecules mediating such linkages because they pos- sess binding sites for spectrin as well as an increasing number of integral membrane proteins (1–5). Three distinct ankyrin genes encode for a variety of alternatively spliced and tissue- specific isoforms. Although the ANK1 and ANK2 gene prod- ucts are largely restricted to red cells and brain, respectively, the ANK3 gene transcribes isoforms that display a general tissue distribution and are hence termed AnkG. These include a 480-kD isoform localized at the axonal initial segment and node of Ranvier (6), a 190/210-kD kidney ankyrin isoform expressed at the plasma membranes of kidney tubule cells (7,8), and truncated isoforms associated with the Golgi appa- ratus (9,10), lysosomes (11), and sarcoplasmic reticulum (12). The majority of ankyrins described to date are modular pro- teins comprising three conserved domains, including an amino- terminal domain containing a varying number of ankyrin re- peats, a spectrin-binding domain, and a death domain located near the carboxyl terminal (1– 8). Several structurally and functionally diverse proteins interact with the repeats domain of ankyrin, including -Na, K-ATPase, anion exchangers, the voltage-dependent sodium channel, sodium/calcium ex- changer, calcium channels, IP3 receptor, ryanodine receptor, clathrin, tubulin, and cell adhesion molecules such as CD44 and the L1 family (1–5). The “death domain” was initially reported as a region of sequence homology within the intracellular portions of the proapoptotic receptors Fas and TNFR1 (13,14). These domains were involved in protein-protein interactions, enabling the sub- sequent identification of several additional key death domain- containing proapoptotic proteins such as FADD, TRADD, and RIP (15–18). Database searches have since identified over a dozen proteins that possess the death domain, including red cell ankyrin (14,19). The death domain within kidney ankyrin (8) displays an even greater similarity to those within proapop- totic molecules than that of red cell ankyrin. However, the functional significance of ankyrin’s death domain has hitherto remained puzzling (3). In this study, we show that kidney ankyrin’s death domain interacts with the death domain of Fas and promotes Fas-mediated apoptosis in renal epithelial cells both in vitro and in vivo. MDR and AI contributed equally to this work. Received August 7, 2003. Accepted October 16, 2003. Correspondence to Dr. Prasad Devarajan, Nephrology and Hypertension, MLC 7022, Cincinnati Children’s Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229-3039. Phone: 513-636-4531; Fax: 513-636-7407; E- mail: prasad.devarajan@cchmc.org. 1046-6673/1501-0041 Journal of the American Society of Nephrology Copyright © 2003 by the American Society of Nephrology DOI: 10.1097/01.ASN.0000104840.04124.5C J Am Soc Nephrol 15: 41–51, 2004