Active site-adjacent phosphorylation at Tyr-397 by c-Abl kinase inactivates caspase-9

Abstract

Caspase-9 (casp-9) is an initiator caspase and plays a central role in activating apoptotic cell death. Control of all caspases is tightly regulated by a series of phosphorylation events enacted by several different kinases. Caspase-9 is the most heavily phosphorylated of all caspases, with phosphorylation of at least 11 distinct residues in all three caspase-9 domains by nine kinases. Caspase-9 phosphorylation by the non-receptor tyrosine kinase c-Abl at Tyr-153 reportedly leads to caspase-9 activation. All other phosphorylation events on caspases have been shown to block proteolytic function by a number of mechanisms, so we sought to unravel the molecular mechanism of the putative caspase-9 activation by phosphorylation. Surprisingly, we observed no evidence for Tyr-153 phosphorylation of caspase-9 in vitro or in cells, suggesting that Tyr-153 is not phosphorylated by c-Abl. Instead, we identified a new site for c-Abl-mediated phosphorylation, Tyr-397. This residue is adjacent to the caspase-9 active site but, as a member of the second shell, not a residue that directly contacts substrate. Our results further indicate that Tyr-397 is the dominant site of c-Abl phosphorylation both in vitro and upon c-Abl activation in cells. Of note, phosphorylation at this site inhibits caspase-9 activity, and the bulk of the added phosphate moiety appeared to directly block substrate binding. c-Abl plays both proapoptotic and prosurvival roles, and our findings suggest that c-Abl's effects on caspase-9 activity promote the prosurvival mode.

Keywords: ABL tyrosine kinase; apoptosis; caspase; phosphocapture; phosphoenrichment; protease; protein phosphorylation; substrate-binding groove.

Figure 1.

Phosphomimetic Y153E is inactive. A, domain architecture of casp-9 showing the N-terminal CARD and the core, which is composed of a large (Lg) and a small (Sm) subunit connected by an intersubunit linker. Three cleavage sites in the intersubunit linker are indicated by arrows: Glu-306 (minor, self-cleavage), Asp-315 (major, self-cleavage), and Asp-330 (by casp-3). Reported phosphorylation sites are indicated by a circled P in white; the c-Abl phosphorylation site Tyr-153 (Y153) in the large subunit is highlighted in yellow. Cognate kinases are indicated with arrows pointing at the phosphorylation site. B, structure of full-length, dimeric casp-9 showing the phosphorylation site, Tyr-153, as spheres. The structure shown here was modeled from individual structures of the CARD-deleted casp-9 (amino acids 138–416; PDB entry 1JXQ) and the casp-9 CARD (amino acids 1–95; PDB entry 3YGS) from a dimeric complex with Apaf-1 CARD. The region between the CARD and core is not present in either structure and is most likely highly disordered; hence, it was modeled as gray coil. The loops forming the substrate binding groove (L2, L3, and L4 from one monomer and L2′ from the opposite monomer) are labeled. C, Tyr-153 forms a hydrogen bond with Asp-350 in loop L2′, supporting the “up” position of L2′ to properly interact with L2 and L4 and achieve a catalytically competent active site. D, Casp-9 Tyr-153 variants (phosphomimetics Y153E and Y153D and non-phosphorylatable Y153F) remained in the uncleaved zymogen (full-length) state upon overexpression, whereas WT casp-9 forms the mature, cleaved state upon overexpression. The CT versions of casp-9 are generated by independent translation of the CARD+Large (Lg) and Small (Sm) subunits. Due to the separate expression of the subunits, all casp-9 CT variants (CT Y153E, CT Y153F, and CT Y153Q) are in the “cleaved” state following overexpression. E, the WT casp-9 zymogen readily undergoes self-processing into the CARD+Large and small subunits. Full-length Y153E is unable to self-process and remains in the zymogen (uncleaved) form, suggesting that it is inherently inactive. F, Casp-3 natively cleaves casp-9 at Asp-330. Casp-9 WT, catalytic site–inactivated variant C287A, phosphomimetic Y153E, and variants Y153D and Y153F were incubated alone or with casp-3. Cleavage at Asp-330 to generate mature proteins only occurred in the presence of casp-3, except for WT, which immediately self-processes, and Y153F, which can weakly self-process. G, Casp-9 full-length variants cleaved by casp-3 (D) were tested in an LEHDase assay. Y153E, Y153D, and Y153F did not gain activity following casp-3 cleavage. Error bars represent S.E. of three independent trials done on three separate days.

Figure 2.

c-Abl phosphorylates casp-9 in vitro at the small subunit. A and C, recombinant c-Abl constructs used to phosphorylate casp-9 in vitro. The construct c-Abl KD comprises only the kinase domain, whereas the c-Abl 3D construct contains the SH3-SH2 regulatory/binding domains as well as the kinase domain. B and D, casp-9 catalytic site–inactivated variant C287A (full-length) and WT (cleaved) were subjected to in vitro phosphorylation by c-Abl KD or 3D in the presence of ATP + [γ-³²P]ATP for 2 h. c-Abl undergoes autophosphorylation/autoactivation upon treatment with ATP. Both forms of c-Abl phosphorylated casp-9 in the zymogen (C287A) and cleaved (WT) forms. No phosphorylation in the CARD+Large region (Tyr-153 site) was detected, but phosphorylation in the small subunit was clearly visible, as shown in the autoradiograph labeled here and in the succeeding figures as ³²P.

Figure 3.

Tyr-397 is the predominant site for c-Abl phosphorylation in vitro. A, structure of casp-9 core showing all the tyrosine residues (yellow spheres). Tyr-397 (Y397) resides in loop L4 and is noticeably solvent-exposed, due to crystal contacts in this structure (PDB entry 1JXQ). B, substrate sequence logo for the consensus recognition sequence of c-Abl (downloaded from PhosphoSitePlus (33)). C, sequence of residues surrounding each tyrosine present in casp-9. Residues in favorable positions are underlined. The sequence surrounding Tyr-397 conforms well to the consensus sequence for c-Abl phosphorylation. D, unphosphorylatable casp-9 variants (phenylalanine substitutions at putative phosphorylated tyrosines) in both FL and CT versions were subjected to in vitro phosphorylation by c-Abl for 2 h. The asterisk denotes that FL Y397F was constructed in the background of C287A (catalytic cysteine–inactivated variant) to prevent self-processing because Y397F is active. FL Y153F casp-9 was still visibly phosphorylated; the CT version of Y153F revealed that the phosphorylation is in the small subunit. An absence of phosphorylation was observed for Y397F (both FL and CT) and the double mutant Y153F/Y397F. E, removal of the CARD in casp-9 (ΔCARD) did not promote phosphorylation of the Tyr-153 site in the large subunit. Only the small subunit was robustly phosphorylated. An unidentified nonspecific 12 kDa band from ΔCARD C287A was also observed be phosphorylated.

Figure 4.

Phosphorylation of Tyr-397 leads to casp-9 inactivation. A, WT Casp-9 and the unphosphorylatable variant Y397F were subjected to in vitro phosphorylation by Abl in the presence or absence of ATP for 2 h. Phosphorylation of the small subunit was clearly observed in WTcasp-9 but was essentially absent for casp-9 Y397F. Only background levels of phosphorylation were visible in the CARD+Large region. Gels and corresponding autoradiographs shown are representative of four independent trials performed on 4 separate days. B, inhibition of WT and Y397F casp-9 by phosphorylation. The activities of samples in A after incubation with c-Abl for 2 h were measured using the casp-9 preferred substrate N-acetyl-Leu-Glu-His-Asp-7-aminotrifluoromethylcoumarin. Casp-9 WT was moderately inhibited, whereas casp-9 Y397F was insensitive to c-Abl–mediated inhibition. The reduced LEHDase activity for phosphorylated WT casp-9 (+ATP) was statistically different from that of unphosphorylated WT (−ATP) (**, p < 0.05) as determined by Student's t test. Data shown are means ± S.E. (error bars) from four independent experiments performed on 4 separate days. C, the level of casp-9 phosphorylation correlates with the extent of inhibition. Phosphorylation levels of casp-9 were determined from [γ-³²P]ATP standards exposed on the same autoradiograph as the Coomassie-stained SDS-polyacrylamide gel (supplemental Fig. S2). The percentage inhibition for phosphorylated casp-9 (both c-Abl and ATP present) was normalized against activity in the non-phosphorylated form (with c-Abl but no ATP). Data shown are means ± S.E. from four independent experiments performed on 4 separate days. D, correlation plot between casp-9 inhibition and casp-9 phosphorylation. WT casp-9 was initially phosphorylated in vitro by c-Abl and was subjected to phosphoprotein enrichment to capture a greater fraction of phosphorylated casp-9. Data shown are means ± S.D. from three independent experiments performed on 3 separate days.

Figure 5.

Phosphorylated casp-9 exhibits slower protein cleavage kinetics. WT (A) or Y397F casp-9 (B) was incubated with c-Abl in the presence or absence of ATP. 1 μm casp-9 from the phosphorylation reaction was allowed to cleave 3 μm full-length casp-3 C163A (catalytic site–inactivated variant) (C3 FL) for 30 min. Cleavage kinetics for each reaction are plotted as a function of the disappearance of the casp-3 FL band. C, representative Coomassie-stained gels and corresponding autoradiographs of phosphorylation reactions used in A and B. Casp-9 WT is visibly phosphorylated at Tyr-397. D, correlation between casp-9 phosphorylation as detected by autoradiography in C and inhibition of casp-3 (C3) cleavage after 30 min as shown in A and B. E and F, casp-9 WT or Y397F was incubated with c-Abl in the presence or absence of ATP. 1 μm casp-9 from the phosphorylation reaction was allowed to cleave 3 μm full-length casp-7 C186S (catalytic site–inactivated variant) (C7 FL) for 30 min. Cleavage kinetics for each reaction are plotted as a function of the disappearance of the casp-7 FL band. G, representative Coomassie-stained gel and corresponding autoradiograph of phosphorylation reactions used in E and F. Casp-9 WT is phosphorylated at Tyr-397. H, correlation between casp-9 phosphorylation as detected by autoradiography in G and inhibition of casp-7 (C7) cleavage after 30 min as shown in E and F. Data shown for all of the above experiments are means ± S.E. (error bars) from three independent experiments done on 3 separate days.

Figure 6.

Activation of c-Abl leads to casp-9 phosphorylation at Tyr-397 intracellularly. A, recombinant casp-9 was phosphorylated in HEK 293T lysates. Lysates were supplemented with 20 nm c-Abl, 200 μm orthovanadate, and 1 mm ATP + [γ-³²P]ATP to ensure in trans activation of c-Abl. Where indicated, lysates were also treated with the c-Abl inhibitor imatinib (200 μm) 30 min before the addition of c-Abl. WT or Y397F casp-9 (30 μg) were added to lysates to allow casp-9 phosphorylation. WT but not Y397F casp-9 showed phosphorylation in the small (Sm) subunit, which was not visible with imatinib-treated lysates. In some trials, the small subunit of Y397F also appears to be labeled, albeit at a significantly lower level than that of WT (supplemental Fig. S5, B and C). The phosphorylation observed for CARD+Large appeared to be c-Abl-independent, because imatinib did not eliminate its phosphorylation. B, c-Abl is activated by DPH in synergy with orthovanadate treatment. HEK 293T cells were treated with DMSO, the known c-Abl–activating compound DPH + orthovandate, or imatinib for 2 h. Lysates were probed for active c-Abl as assessed by immunoblotting (IB). Active c-Abl is phosphorylated at Tyr-412. DPH/vanadate treatment clearly resulted in c-Abl activation, as manifested by phosphorylation at Tyr-412 and downstream phosphorylation of a well-known c-Abl substrate, CrkII, at Tyr-221. C, Casp-9 is phosphorylated at Tyr-397 by active c-Abl intracellularly. HEK 293T cells were transfected with vector alone (p3xFLAG-CMV^TM-14), catalytic site–inactivated casp-9 (C9 C287A-3xFLAG), or the unphosphorylatable variant (C9 C287A/Y397F-3xFLAG). 24 h post-transfection, cells were treated with DPH/vanadate for 2 h, harvested, and lysed. Immunoblot of total proteins confirmed c-Abl activation and casp-9 expression. Casp-9 was immunoprecipitated from lysates with an anti-FLAG antibody and probed with anti-phosphotyrosine (p-Tyr) and anti-casp-9 by immunoblotting. Cells transfected with C287A/Y397F showed significantly lower levels of phosphotyrosine in uncleaved caspase-9 compared with those transfected with C287A, although the levels of immunoprecipitated casp-9 in both C287A and C287A/Y397F were similar.