FA1: 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.

Farschon DM, Couture C, Mustelin T, Newmeyer DD - J. Cell Biol. (1997)

Bottom Line: 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.

Affiliation: Division of Cellular Immunology, La Jolla Institute for Allergy and Immunology, San Diego, California 92121, USA.

Abstract: 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.