HIP1/ΔE–mediated apoptosis is dependent on caspase-9 and functions as a dominant negative mutant. (a) Schematic of HIP1 mutants. HIP1 is a multidomain protein with an N-terminal ENTH domain; LMDMD (LMD) and DPF motifs that bind to clathrin and AP2, respectively; a central coiled coil (CC) domain with multiple interaction motifs; and a C-terminal TALIN homology domain. Each of these domains was deleted to yield the respective deletion mutants, HIP1/ΔE, HIP1/ΔLD, HIP1/ΔC, and HIP1/ΔT. (b) Correction of HIP1/ΔE–mediated apoptosis by dominant negative caspase-9 (DN casp-9) but not dominant negative caspase-8 (DN casp-8). Cells were cotransfected with HIP1/ΔE and either of the dominant negative caspases as indicated under the graph. Controls included vector-transfected cells (bar 1) and dominant negative caspases alone (bars 4 and 6). Apoptosis was assayed as in Figure 6b. (c) HIP1/ΔE–mediated apoptosis was rescued by full-length HIP1 and all HIP1 mutants except HIP1/ΔC. As in b, cells were cotransfected with various combinations of plasmids as indicated under the graph. (d) Interaction of all HIP1 mutants except HIP1/ΔC with full-length HIP1. Myc-tagged full-length HIP1 was cotransfected with various mutants as indicated. Cell extracts were immunoprecipitated with myc antibody, and pellets (P) and supernatants (S) were collected. These were then immunoblotted with HIP1 polyclonal antibody (top panel) and myc antibody (bottom panel). Note that endogenous HIP1 is co-immunoprecipitated with myc-tagged HIP1 in all lanes.