Fig. 10
From: Apoptotic signalling targets the post-endocytic sorting machinery of the death receptor Fas/CD95
ENTR1 is cleaved during Fas induced apoptosis. a Overexpressed ENTR1-myc (55 kDa) was cleaved upon activation of Fas-mediated apoptotic signalling to a produce a 35–40 kDa band after 3 h of treatment (indicated by an arrow) with anti-Fas (CH-11) and cycloheximide. Cleavage of Caspase 8 (p18) was also observed only upon activation with anti-Fas (CH-11). Myc-ENTR1 was not cleaved upon activation by cycloheximide only or b in the presence of pan-caspase inhibitor (z-vad-FMK). b ENTR1 is cleaved by caspase at position aa236 (isoform1) into two fragments (indicated by arrows). Myc- and flag-tagged ENTR1 mutants D179A, D181A, D234A and D236A were created by site-directed mutagenesis using D251A mutant as a template. c Immunoblot showing equal protein expression of the ENTR1 rescue constructs (includes a cartoon of the ENTR1 caspase resistant construct). Cleaved ENTR1 product indicated with an arrow. beta-tubulin was used as a loading control. d A caspase resistant ENTR1 has increased anti-apoptotic activity compared to wild-type ENTR1. Quantification of the immunofluorescence analysis of activated caspase 3 in CH11 treated wild-type HeLa cells or ENTR1 knock-out cells transfected as indicated in the graph. Relative percentage of apoptosis is shown and HeLa wild-type GFP-transfected cells is considered as 100%. Only GFP positive cells were included in the quantification. Data was collected from three independent experiments (n = 3), One-way ANOVA was performed, **p < 0.01, ***p < 0.001, error bars represent ± SEM. e Working model for endolysosomal sorting of Fas via the ENTR1/PTPN13 complex and Dysbindin. Impaired sorting of Fas into intraluminal vesicles leads to increased recycling and elevated Fas cell surface expression. Caspase dependent cleavage of ENTR1 contributes to a positive feedback apoptotic signalling loop (PM = plasma membrane)
