Farschon DM et al. (1997), Temporal phases in apoptosis defined by the act...

XB-ART-16384

J Cell Biol 1997 Jun 02;1375:1117-25. doi: 10.1083/jcb.137.5.1117.

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Temporal phases in apoptosis defined by the actions of Src homology 2 domains, ceramide, Bcl-2, interleukin-1beta converting enzyme family proteases, and a dense membrane fraction.

Farschon DM , Couture C , Mustelin T , Newmeyer DD .

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We have begun to explore the mechanisms of apoptosis using a cell-free system based on extracts from Xenopus eggs. Nuclei assembled or placed in these extracts undergo the morphological changes typical of apoptosis and eventually disintegrate. We used this system to investigate the potential involvement in apoptosis of proteins containing Src homology 2 (SH2) domains, which are known to interact with specific tyrosine-phosphorylated ligands. SH2 domains from a number of signaling proteins, including Lck, Src, and Abl, inhibited apoptosis when present at concentrations of 10-100 nM. The inhibition was dependent on specific interaction with endogenous tyrosine-phosphorylated ligands. A synthetic peptide ligand for Src family SH2 domains also inhibited apoptosis in a phosphotyrosine-dependent manner. Kinetic analysis defined three phases in the apoptotic process occurring in this cell-free system. SH2 domains and ceramide act throughout the first 60-90 min of the process (the "initiation" phase). Next, Bcl-2, interleukin-1beta converting enzyme family(CPP32-like) proteases, and the heavy membrane fraction act in a period occurring approximately 90-120 min after the start of incubation (the "sentencing" phase). In the final phase ("execution"), the process of active nuclear destruction ensues.

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Species referenced: Xenopus
Genes referenced: abl1 bcl2 lck

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	Figure 2. Effect of wild-type and mutant SH2 domains from Lck. The experiment was performed as in Fig. 1. GST–SH2 domain fusions were added at 100 nM. Note that the wild-type (Y192) Lck SH2 domain is unable to inhibit apoptosis, as is the E192 mutant, which mimics the phosphorylated form of Y192. However, the F192 mutant, which mimics the unphosphorylated form of Y192, inhibits apoptosis in the extract.
	Figure 3. The wild-type Lck SH2 domain is inactive in the extract because it becomes phosphorylated. (A) Genistein, an inhibitor of tyrosine kinases, restored the ability of the wild-type Lck SH2 domain to inhibit apoptosis. The experiment was done as in Fig. 2, except that genistein (10 μM) was added to the indicated samples either separately or along with the wild-type (WT) Lck SH2 domain–GST fusion (100 nM). (B) A kinase in the Xenopus egg extract can phosphorylate a synthetic peptide corresponding to the site surrounding Y192 in Lck. The peptide (RNLDNGGFYIAPR) was incubated in a buffer containing γ-[32P]ATP and Xenopus egg extract (diluted as described in Materials and Methods) for the times indicated, and the incorporated radioactivity was assayed (see Materials and Methods). Two separate experiments are shown.
	Figure 4. A synthetic peptide ligand for Src family SH2 domains, EPQYEEIPIYLK (YEEI), inhibits apoptosis in the Xenopus cell-free system in a PTyr-dependent manner. (A) The indicated concentrations of phosphopeptide were added at the start of incubation. (B) (Top) The phosphopeptide (denoted by YEEI) was added to the extract at a final concentration of 5 nM, in the presence or absence of genistein. Genistein has no effect on its activity. (Bottom) The unphosphorylated form of this peptide (YEEI) and an unphosphorylatable mutant peptide (FEEI) were added at 5 nM. Note that the FEEI peptide has no inhibitory activity. The unphosphorylated peptide (YEEI) inhibits apoptosis nearly as well as the YEEI, but this inhibition is reversed in the presence of genistein. Thus, the peptide is apparently phosphorylated by a kinase present in the extract.
	Figure 5. The Src SH2 domain inhibits an activity important in the early portion of the apoptotic process. The experiment was performed as above, except that the GST–Src SH2 fusion was added (final concentration 100 nM) at the indicated times after the start of incubation.
	Figure 6. The Bcl-2 protein acts later in the apoptotic process, after ∼1.5 h of incubation. Lysates from Sf9 cells (0.01 vol, as described; 29) infected with Bcl-2 baculovirus or, as a control, β-galactosidase baculovirus were added at the indicated times after the start of incubation. Note that Bcl-2 is much less effective when its addition is delayed at least ∼2 h after the start of incubation.
	Figure 7. The heavy membrane (HM) fraction is required relatively late in the apoptotic process. Percoll-enriched HMs (29) were added at the indicated times. Note that only when the HMs were added at least 2 h after the start of incubation was there any significant delay in the kinetics of nuclear destruction.
	Figure 8. Ceramide acts early in the cell-free apoptotic process. A progressive loss of activity occurs when the addition of ceramide (150 μM) is delayed 0.5–1 h.
	Figure 9. CPP32-like protease activity is required for apoptosis in the Xenopus system. The crucial proteolytic event occurs ∼1.5–2 h after the start of incubation. (A) The tetrapeptide inhibitors, AcDEVD-CHO (DEVD) and Ac-YVAD-CHO (YVAD), were added to the extract from the start of incubation at the concentrations indicated. Nuclear survival was quantitated at various times. (B) The experiment was performed as above, except that DEVD (100 μM) was added at the indicated times. Note that the inhibitor lost its effect only when added after ∼2 h of incubation. (C and D) Fluorescence photomicrographs of rat liver nuclei placed in apoptotic extract, incubated for 3 h in the presence (C) or absence (D) of 10 μM DEVD. Bar, 10 μM.
	Figure 10. Summary of the approximate time intervals during which various events occur in the cell-free system.
	Figure A1. Comparison of SH2 ligand peptides with regard to their effects on the Xenopus cell-free apoptosis system. The experiment was done as in Fig. 4, except that either the YEEI peptide, or a synthetic peptide corresponding to the SH2 ligand domain of phospholipase C (PLC)-γ1 surrounding Y1021 (PLC peptide, DNDY*IIPLPDPK; 35) was added to the system at the indicated concentrations before incubation. Note that the PLC-γ1 peptide had no significant inhibitory activity in this system.

References [+] :

Chittenden, Induction of apoptosis by the Bcl-2 homologue Bak. 1995, Pubmed