Restraint of apoptosis during mitosis through interdomain phosphorylation of caspase-2

Abstract

The apoptotic initiator caspase-2 has been implicated in oocyte death, in DNA damage- and heat shock-induced death, and in mitotic catastrophe. We show here that the mitosis-promoting kinase, cdk1-cyclin B1, suppresses apoptosis upstream of mitochondrial cytochrome c release by phosphorylating caspase-2 within an evolutionarily conserved sequence at Ser 340. Phosphorylation of this residue, situated in the caspase-2 interdomain, prevents caspase-2 activation. S340 was susceptible to phosphatase 1 dephosphorylation, and an interaction between phosphatase 1 and caspase-2 detected during interphase was lost in mitosis. Expression of S340A non-phosphorylatable caspase-2 abrogated mitotic suppression of caspase-2 and apoptosis in various settings, including oocytes induced to undergo cdk1-dependent maturation. Moreover, U2OS cells treated with nocodazole were found to undergo mitotic catastrophe more readily when endogenous caspase-2 was replaced with the S340A mutant to lift mitotic inhibition. These data demonstrate that for apoptotic stimuli transduced by caspase-2, cell death is prevented during mitosis through the inhibitory phosphorylation of caspase-2 and suggest that under conditions of mitotic arrest, cdk1-cyclin B1 activity must be overcome for apoptosis to occur.

Figure 1

Mitotic suppression of apoptosis occurs upstream of cytochrome c release. (A) Interphase and mitotic extracts were incubated at RT alone or in the presence of roscovitine (0.28 mM) or U0126 (200 μM). Caspase activity was determined by a colorimetric measurement of DEVD-pNA cleavage. (B) The activity of the ERK inhibitor U0126 was verified by immunoblotting for phospho-ERK. (C) Interphase and mitotic extracts were incubated at RT. Samples were collected at indicated times, fractionated to make mitochondria-free cytosolic supernatants, and immunoblotted for cytochrome c, actin and VDAC (P=cell pellet containing mitochondria). (D) Mitotic and interphase extracts were incubated with caspase-8-cleaved Bid (tBid) at a concentration of 1 nM. Release of cytochrome c was measured as in panel (C).

Figure 2

Caspase-2 is suppressed in mitosis. (A) ³⁵S-labelled caspase-2 was incubated in interphase and mitotic extract. Samples collected at the indicated times were resolved by SDS–PAGE/phosphorimager. (B) ³⁵S-labelled Bid was incubated in interphase and mitotic extract. Samples collected at the indicated times were resolved as in panel (A). (C) NADPH levels from samples of interphase and mitotic extract, collected as in panel (A), were measured by spectrophotometric colour change (kit supplied by BioVision). (D) ³⁵S-labelled caspase-2 (WT or S135A) was incubated in mitotic extract and resolved by SDS–PAGE/film. (E) ³⁵S-labelled caspase-2 was incubated in interphase and mitotic extracts in the presence of GST–RAIDD (10 μg). At the indicated times, GST–RAIDD was retrieved, washed once in PBS, and resolved as in panel (D).

Figure 3

Caspase-2 is phosphorylated in mitosis at S340 (S308 in Xenopus numbering). (A) GST–caspase-2, incubated in interphase and mitotic extract, was retrieved, washed, and resolved by SDS–PAGE. Excised caspase-2 bands were analysed by tandem mass spectrometry. Red arrows point to the relevant shifted peaks. (B) GST–caspase-2 (WT and S308A) was incubated in interphase or mitotic extract in the presence of [γ-³²P]ATP. Samples were resolved by SDS–PAGE/phosphorimager. (C) GST–caspase-2 (WT and S308A) was incubated in the presence of purified cdk1–cyclin B1. Samples were resolved by SDS–PAGE/film. (D) Sequence alignment of caspase-2 from different species. Letters highlighted in red indicate the conserved cdk1 phospho site. Asterisks denote the aspartate cleavage sites used to form processed active caspae-2. (E) Mitotic and cycling lysates from U20S cells were subjected to 2D gel electrophoresis followed by immunoblotting for endogenous caspase-2. (F) Mitotic and cycling lysates from U20S cells were immunoblotted with anti-phospho S340 caspase-2, anti-phospho histone H3, and actin antibodies.

Figure 4

PP1 activity is required for mitotic suppression of caspase-2. (A) Okadaic acid, at indicated concentrations, was incubated in interphase extract with ³⁵S-labelled caspase-2. Processing of capase-2 was measured by SDS–PAGE/film. (B) ³⁵S-labelled caspase-2 was incubated with buffer or with the PP1-specific inhibitor I2. Processing of capase-2 at various time points was measured as in panel (A). (C) GST–caspase-2 was incubated in vitro with the indicated combinations of purified cdk1–cyclin B1 (0.5 μM), PP1 (2 μM) and I2 (0.5 μM), in the presence of γ³²P. Samples were resolved by SDS–PAGE/phosphorimager. (D) Glutathione sepharose-bound caspase-2, or GST alone, were incubated in interphase or mitotic extract for 30 min, retrieved, washed, and subjected to immunoblotting for endogenous PP1.

Figure 5

Phosphorylation of caspase-2 at S308 is required for mitotic suppression of apoptosis. (A) ³⁵S-labelled caspase-2 (WT and S308A) was incubated in interphase or mitotic extract. Processing of caspase-2 was resolved by SDS–PAGE/film. (B) ³⁵S-labelled caspase-2 (WT and S308E) were incubated with GST–RAIDD. Processing of caspase-2 was measured by SDS–PAGE/phosphorimager (C) GST–caspase-2 (WT and S308A) were incubated in mitotic extract. Samples were collected at indicated times, fractionated, and immunoblotted for cytochrome c. (D) Results of oocyte injection, in the presence of progesterone, with flag-tagged WT and S308A caspase-2. Apoptotic oocytes were scored as described previously by Nutt et al (2005). (E) Representative images of oocytes injected with the indicated RNA. (F) Oocytes injected with RNA were lysed, and expression of caspase-2 protein was determined by immunoblotting with anti-flag antibodies. (G) Oocytes with and without progesterone treatment were lysed and subjected to immunoblotting with anti-phospho histone H3 antibodies.

Figure 6

Metabolic and mitotic factors combine to suppress caspase-2 in Xenopus extract. (A) ³⁵S-labelled caspase-2 both WT and, S308 and S308A/S135A double mutant were incubated in mitotic extract. Processing of caspase-2 was measured SDS–PAGE/phosphorimager. (B) ³⁵S-labelled caspase-2 (S308A) was incubated in mitotic extract±glucose-6-phosphate (5 mM). Processing of caspase-2 was measured by SDS–PAGE/film.

Figure 7

Loss of phosphorylation of human caspase-2 at S340 sensitizes cells to mitotic cell death. (A) U20S cells were transfected with the indicated siRNA. At 24 h post-transfection, cells were infected with lentivirus expressing human caspase-2 cDNA (WT or S340A). At 36 h after viral infection, cells were collected and immunoblotted for caspase-2. (B) Cells treated as in panel (A) were placed in medium supplemented with nocodazole (starting 24 h after lentiviral infection). After 30 h of nocodazole treatment, cells were collected, incubated in PBS + propidium iodide and analysed by flow cytometry. (C) Representative images of cells from panel (B).

Figure 8

Comparison of caspases with cdk1 phospho sites within or adjacent to the interdomain. Human caspase sequences were scanned for S/T-P sites, denoted by red arrowheads.