Figure 2
HECTD3 ubiquitinates caspase-8 with K63-linked polyubiquitin chains at K215. (a) HECTD3 ubiquitinates caspase-8. HEK293T cells were co-transfected with expressing plasmids for HA-Ub, HECTD3, and Flag-caspase-8 as indicated for 2 days. The ubiquitinated Flag-caspase-8 proteins were detected by an anti-HA antibody after immunoprecipitation caspase-8 by anti-Flag M2 beads under denaturing conditions. (b) The ubiquitination of caspase-8 by HECTD3 was assessed with linkage-specific antibodies that recognize K48- or K63-linked polyubiquitin chains. (c) Different HA-Ub mutants were used to assess the HECTD3-mediated caspase-8 polyubiquitin chain linkage in HEK293T. (d) Alignment of the HECT domains of 10 HECT type E3 ligases using the Glustal W multiple alignment program.47 HECTD3 does not have the conserved ‘HTCFN’ catalytic loop. (e) HECTD3-C823A loses the E3 ubiquitin ligase activity, as determined by the in vitro ATP-dependent ubiquitination assay. UbcH5b was used as the E2, which can mediate the K63-linked polyubiquitination.43 (f) Both C823 and C744 contribute to the E3 ligase activity of HECTD3 toward caspase-8. In contrast to WT HECTD3, the HECTD3-C744A, -C823A, and -C744, 823A mutants cannot efficiently ubiquitinate caspase-8 in HEK293T. (g) In contrast to WT caspase-8, the caspase-8-K215R mutant cannot be efficiently ubiquitinated by HECTD3 in HEK293T
