Impairment of Fas-ligand–caveolin-1 interaction inhibits Fas-ligand translocation to rafts and Fas-ligand-induced cell death

Abstract

Fas-ligand/CD178 belongs to the TNF family proteins and can induce apoptosis through death receptor Fas/CD95. The important requirement for Fas-ligand-dependent cell death induction is its localization to rafts, cholesterol- and sphingolipid-enriched micro-domains of membrane, involved in regulation of different signaling complexes. Here, we demonstrate that Fas-ligand physically associates with caveolin-1, the main protein component of rafts. Experiments with cells overexpressing Fas-ligand revealed a FasL N-terminal pre-prolin-rich region, which is essential for the association with caveolin-1. We found that the N-terminal domain of Fas-ligand bears two caveolin-binding sites. The first caveolin-binding site binds the N-terminal domain of caveolin-1, whereas the second one appears to interact with the C-terminal domain of caveolin-1. The deletion of both caveolin-binding sites in Fas-ligand impairs its distribution between cellular membranes, and attenuates a Fas-ligand-induced cytotoxicity. These results demonstrate that the interaction of Fas-ligand and caveolin-1 represents a molecular basis for Fas-ligand translocation to rafts, and the subsequent induction of Fas-ligand-dependent cell death. A possibility of a similar association between other TNF family members and caveolin-1 is discussed.

Advances in Understanding TKS4 and TKS5: Molecular Scaffolds Regulating Cellular Processes from Podosome and Invadopodium Formation to Differentiation and Tissue Homeostasis

“…* The first and second SH3 domains cooperate to form a common "super SH3 platform" and allow the binding of the proline-rich region of the partner protein [33]. GST pull-down assay 4th SH3 domain Ectodomain shedding, cell adhesion, and signaling [35] Cortactin [9] Co-immunoprecipitation, GST pull-down assay, immunofluorescence colocalization Unknown Regulation of actin cytoskeleton [36] CR16 [37] GST pull-down assay Weak interaction with the 2nd, 3rd, and 4th SH3 domains Reorganization of actin cytoskeleton [38] DNM2 [37] GST pull-down assay 3rd SH3 domain Endo-/exocytosis [37] FasL (CD178) [39] Phage display screening 3rd and 4th SH3 domains Apoptosis induction [40] GRB2 [28] Affinity purification-selected reaction monitoring mass spectrometry…”

“…Adaptor protein involved in the regulation of RTK signaling, cycle progression, actin-based cell motility, podosome formation [41] NOXA1 [42,43] Co-immunoprecipitation, GST pull-down assay Unknown ROS generation through NOX1 activation [44] [35] Cortactin [9] Co-immunoprecipitation, GST pull-down assay, immunofluorescence co-localization Unknown Regulation of actin cytoskeleton [36] CR16 [37] GST pull-down assay Weak interaction with the 2nd, 3rd, and 4th SH3 domains Reorganization of actin cytoskeleton [38] DNM2 [37] GST pull-down assay 3rd SH3 domain Endo-/exocytosis [37] FasL (CD178) [39] Phage display screening 3rd and 4th SH3 domains Apoptosis induction [40] NOXA1 [42,43] Co-immunoprecipitation, GST pull-down assay Unknown ROS generation through NOX1 activation [44] N-WASP [37] GST pull-down assay 2nd SH3 domain A scaffold protein regulating actin cytoskeleton reorganization, and actin polymerization during cell motility and invasion [45] OPHN1 [37] GST pull-down assay 3rd SH3 domain Endo-/exocytosis [37] RUK/CIN85 [46] GST pull-down assay Unknown…”

“…Regulation of cell growth, differentiation, proliferation, survival, adhesion, migration, and motility [9,32] SYNJ1 [37] GST pull-down assay 3rd SH3 domain and weak interaction with the 4th SH3 domain Endo-/exocytosis [37] (b) F-actin [29] GST pull-down assay, and mass spectrometry 5th SH3 domain Component of cytoskeleton [55] FasL (CD178) [39] Phage display screening 5th SH3 domain Apoptosis induction [40] FGD1 [56] Co-immunoprecipitation and mass spectrometry, GST pull-down assay, immunofluorescence co-localization 4th and 5th SH3 domains A guanine nucleotide exchange factor for the Rho-GTPase CDC42, assembly of podosomes and invadopodia, control of secretory membrane-trafficking, and cell cycle [56,57] Girdin [58] Co-immunoprecipitation, immunofluorescence co-localization Unknown actin-binding protein regulating actin remodeling and cell polarity, collective migration of neuroblasts, epithelial and cancer cells [59] GRB2 [28,29] Co-immunoprecipitation Polyproline sequences…”

“…Future research focusing on the discovery of novel binding partners is expected to reveal not only the detailed molecular level mechanisms resulting in TKS-related phenotypes in FTHS patients and TKS KO animal models, but also, to shed light on yet unknown functions of these proteins. For example, while both TKS4 and 5 were found to be potential binding partners of the Fas ligand (FasL, CD178) (Table 1), which is a known cell death inducer [39,40], possible functions for TKS4/5 in the regulation of cell death have not yet been investigated. A detailed understanding of the TKS4/5 interactome, bolstered by an in-depth description of the molecular modifications of the TKS4/5 proteins during signaling may facilitate the development of new therapies to correct defects in signaling pathways underlying pathological conditions.…”

A review of the role of cav-1 in neuropathology and neural recovery after ischemic stroke

“…Cav-1 KO disrupted death-induced signaling complex formation following colocalization and interaction between cav-1 and Fas [150]. Recently, Glukhova et al found that the deletion of both caveolin-binding sites (which bind the N-terminal domain and B-terminal domain of cav-1) in the Fas-ligand attenuated extrinsic cell death pathway-associated cytotoxicity, demonstrating that the relationship between the Fas-ligand and cav-1 provides a molecular basis for the location of the ligand to lipid rafts and Fas-ligand-dependent apoptosis [151]. As for the intrinsic apoptosis pathway, mitochondrial outer membrane permeabilization is the central event of the intrinsic pathway: in response to apoptotic stimuli, mitochondria release intermembrane space proteins, such as cytochrome C and apoptosis-inducing factor, which exert a cryptic cytotoxic activity following their mitochondrial release [152].…”