Inactivation of effector caspases through nondegradative polyubiquitylation

Abstract

Ubiquitin-mediated inactivation of caspases has long been postulated to contribute to the regulation of apoptosis. However, detailed mechanisms and functional consequences of caspase ubiquitylation have not been demonstrated. Here we show that the Drosophila Inhibitor of Apoptosis 1, DIAP1, blocks effector caspases by targeting them for polyubiquitylation and nonproteasomal inactivation. We demonstrate that the conjugation of ubiquitin to drICE suppresses its catalytic potential in cleaving caspase substrates. Our data suggest that ubiquitin conjugation sterically interferes with substrate entry and reduces the caspase's proteolytic velocity. Disruption of drICE ubiquitylation, either by mutation of DIAP1's E3 activity or drICE's ubiquitin-acceptor lysines, abrogates DIAP1's ability to neutralize drICE and suppress apoptosis in vivo. We also show that DIAP1 rests in an "inactive" conformation that requires caspase-mediated cleavage to subsequently ubiquitylate caspases. Taken together, our findings demonstrate that effector caspases regulate their own inhibition through a negative feedback mechanism involving DIAP1 "activation" and nondegradative polyubiquitylation.

Figure 1. DIAP1 Cleavage Enhances Caspase Binding

(A) Model depicting DIAP1’s hypothetical “closed” and “open” configuration. A vertical arrow marks the site of caspase cleavage. (B–E) Coimmunoprecipitation (IP) assays with the indicated constructs. Lysates and V5 immunoprecipitates were analyzed by immunoblotting with the indicated antibodies. The two arrows on the DIAP1 panels indicate the positions of full-length and cleaved DIAP1.

Figure 2. Cleaved DIAP1 Binds to UBR Domains and Promotes Ubiquitylation of drICE and DCP-1

(A) Schematic representation of caspase-mediated DIAP1 cleavage, maturation, and UBR binding. See text for details. (B) Co-IP assays with DIAP1^21–438 and various UBR domains. S2 cells were cotransfected with GTC-tagged DIAP1^21–438 and HA-tagged UBR domains of the indicated UBR proteins. Copurified proteins were analyzed by immunoblotting with the indicated antibodies. (C) Co-IPs were performed as in (B) with N-, RD-, and M-DIAP1^21–438 and the UBR domain of UBR1. (D and E) Requirement of DIAP1’s RING domain and N-exposure in mediating effector caspase ubiquitylation. 293T (D) or S2 (E) cells were cotransfected with the indicated DIAP1 constructs together with Ub-AKG-DCP-1^C>A (D) or Ub-ALG-drICE^C>A (E) and His-Ub. Cells were lysed under denaturing conditions and ubiquitylated proteins isolated with Ni²⁺ columns. The presence of ubiquitylated caspases was identified by immunoblotting with the indicated antibodies. (F) DIAP1 and drICE bind through a bimodular interaction. Mutation of the IBM weakens but does not abrogate DIAP1 binding. Ubiquitylation assays with the indicated constructs were performed as in (D). Asterisks mark a nonspecific band ([D] and [F]) and unmodified drICE (E) that is due to a nonspecific drICE:matrix association. (G) Endogenous drICE is ubiquitylated in a DIAP1-dependent manner. S2 cells were either left untreated or exposed to UV, and α-drICE or preimmune serum was used to immunoprecipitate drICE from cellular extracts. The presence of ubiquitylated endogenous drICE was determined by immunoblotting with an α-Ub antibody (top panel). Note, treatment with UV causes depletion of DIAP1 (bottom panel, lane 3).

Figure 3. DIAP1 Requires Cleavage and a Functional RING to Suppress Apoptosis In Vivo

(A) Overview of the Ub fusion technique. Tubulin-driven DIAP1 variants were expressed as V5-DHFR/Ub-DIAP1 fusion in which the reference protein DHFR/Ub is cotranslationally cleaved off by DUBs. Expression of the reference protein indirectly indicates the expression level of the protein of interest. DIAP1’s NH₂-terminal sequence and the caspase cleavage site (DQVD, arrowhead) are indicated. (B) Selection of transgenic lines with “near” equivalent expression levels of the reference protein (upper panel). Quantitative, infrared fluorescence-based measurement of V5-DHFR-Ub expression levels (lower panel). (C–J) The ability of DIAP1 to suppress the eye phenotypes of Rpr expression. Rpr expression results in a small-eye phenotype (C) that is rescued by tubulin-driven coexpression of DIAP1^wt (D) and N-DIAP1^21–438 (F), but not DIAP1^D20A (E), M-DIAP1^21–438 (G), or RING mutants (H–J). Representative phenotypes are shown.

Figure 4. DIAP1-Mediated Ubiquitylation of drICE Correlates with Its Inactivation

(A) The E3 ligase activity of DIAP1 is required to suppress effector caspase activity induced by Rpr. S2 cells were cotransfected with the indicated constructs, and cell lysates were assayed for DEVDase activity. Values of Rpr-expressing controls were set to 100% (lanes 2 and 8). Immunoblot analysis of the lysates indicates near-equivalent expression levels of the DHFR reference protein (upper panels). (B) RING-dependent ubiquitylation of drICE. Ubiquitylation assays were performed as in Figure 2E. Asterisks represent nonspecific bands. (C) DIAP1’s E3 activity is required to suppress appearance of processed drICE (p10) and DEVDase activity. Schematic representation of cleavage-mediated activation of drICE (upper panel). Arrows indicate cleavage sites. DIAP1 coexpression, but not the indicated range of ubiquitylation-deficient DIAP1 mutants, efficiently prevents detection of drICE’s small subunit. S2 cells were cotransfected with the indicated constructs. Following a 6 hr induction of drICE, cell lysates were simultaneously analyzed for DEVDase activity and by immunoblotting with the indicated antibodies. Values were normalized to drICE in vector controls (lane 2). DEVDase values and immunoblot analysis of a representative experiment are shown. (D) M-DIAP1^21–438 is less efficient than N-DIAP1^21–438 in suppressing drICE activation and activity, as indicated by the presence of the p10 subunit of drICE and significant amounts of DEVDase activity. Experiments were performed as in (C). DEVDase values and immunoblot analysis of a representative experiment are shown. Error bars denote SD from three independent experiments.

Figure 5. Ubiquitylation of drICE Suppresses Its Catalytic Potential

(A) Inhibition of the proteasome does not alter the ubiquitylation pattern of drICE. 293T cells were cotransfected with the indicated constructs and treated with Lactacystin or DMSO (control). The presence of ubiquitylated drICE was assayed as in Figure 2E. (B) Proteasome inhibition failed to restore the appearance of the small subunit of drICE. S2 cells were cotransfected with the indicated constructs. Following a 2 hr drICE induction, cells were treated for a further 4 hr with MG132 or DMSO. (C) Clonal expression of DIAP1 in the developing eye does not reduce the levels of drICE. Eye discs overexpressing DIAP1 in clones (marked by the absence of GFP, first and last panel) were stained using an α-drICE antibody (red, middle panel). (D) S2 cells were cotransfected with the indicated constructs. Immunoblot analysis (left panel) revealed that zymogenic ALG-drICE accumulated at the expense of processed drICE (p10) in the presence of DIAP1. LI-COR Odyssey quantification (right panel) of the drICE signal.

Figure 6. Conjugation of Ub to drICE Acts as a “Mixed” Inhibitor

(A) In vitro ubiquitylation of recombinant drICE suppresses its catalytic ability to cleave PARP. Schematic representation of the assay procedure (left panels). In vitro ubiquitylation assay of active drICE with DIAP1 or DIAP1^F437A. Arrows on the DIAP1 panel indicate DIAP1 cleavage. (Bottom panel) Step 2 depicting in vitro PARP cleavage assay: the ubiquitylation reactions from step 1 were incubated with recombinant PARP. Shown is immunoblot analysis with α-PARP antibodies. Graphs indicate LI-COR Odyssey quantification of the signal expressed as % of processed PARP in relation to total PARP (lower panel). Values and immunoblot analysis of a representative experiment are shown. (B and C) Ubiquitylation changes kinetic parameters of active drICE. drICE was incubated in an in vitro ubiquitylation reaction with DIAP1 or DIAP^F437A as in Figure 5D. (B) 10.3 nmole of PARP (left panel, a representative experiment is shown) or increasing concentrations of PARP (2.1–34.5 nmole, 15 min, right panel) was incubated with drICE or ubiquitylated drICE. PARP cleavage was monitored by immunoblot analysis. Western blot data were analyzed by LI-COR Odyssey quantification as in Figure 5D. Nonlinear regression using the Michaelis-Menten equation was applied to obtain a curve fit (right panel). (C) drICE was incubated in an in vitro ubiquitylation reaction with DIAP1 or DIAP^F437A as in Figure 5D. drICE and ubiquitylated drICE were incubated with 20 μM DEVD-AMC, and DEVDase activity was analyzed at the indicated time points (left panel, shown is a representative experiment). (Right panel) Increasing substrate concentrations were used, and DEVDase activity was measured at 20 min. Curve fit was performed as in (B). RFU, relative fluorescence units. (D) K_M and V_max (±SE) were determined from the curves shown in (B) and (C) using Prism software.

Figure 7. Nonubiquitylatable drICE Is Refractory to DIAP1-Mediated Inhibition

(A) Schematic representation showing the position of surface-exposed K residues in the large subunit. (B–E) 3D model of drICE dimers. drICE monomers are colored in blue and gray, and surface-exposed K residues are highlighted in red. Residues involved in catalysis are highlighted in yellow. Four different viewpoints of drICE are shown. (F) K>R mutation schedule. Additive rounds of K>R mutations were carried out, and their effects on DIAP1-mediated regulation of drICE were assessed in cell-based DEVDase assays (as in Figure 4C). “++++” reflects no change and “−” reflects loss of DIAP1-mediated inhibition of drICE. (G) drICE^9K>R is refractory to DIAP1-mediated ubiquitylation. S2 cells were cotransfected with DIAP1^21–438, His-Ub, and the indicated drICE-V5/FLAG constructs, and cell lysates were analyzed for the presence of ubiquitylated caspases. (H) drICE^9K>R readily binds to DIAP1. Experiment performed as in Figure 1D. (I) drICE^9K>R is refractory to DIAP1-mediated inhibition. drICE^9K>R was examined as in Figure S6C. The mean values of triplicate experiments and their SD are shown. Immunoblot analysis of a representative experiment is shown. (J and K) Model of caspase regulation in living (J) and dying cells (K). See Discussion for details.