TEMPORAL PHASES IN APOPTOSIS DEFINED BY THE ACTIONS OF SRC HOMOLOGY-2DOMAINS, CERAMIDE, BCL-2, INTERLEUKIN-1-BETA CONVERTING-ENZYME FAMILYPROTEASES, AND A DENSE MEMBRANE-FRACTION

We have begun to explore the mechanisms of apoptosis using a cell-free system based on extracts from Xenopus eggs. Nuclei assembled or place d in these extracts undergo the morphological changes typical pf apopt osis and eventually disintegrate. We used this system to investigate t he potential involvement in apoptosis of proteins containing Src homol ogy 2 (SH2) domains, which are known to interact with specific tyrosin e-phosphorylated ligands. SH2 domains from a number of signaling prote ins, 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 synthet ic 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. SH 2 domains and ceramide act throughout the first 60-90 min of the proce ss (the ''initiation'' phase). Next, Bcl-2, interleukin-1 beta convert ing enzyme family(CPP32-like) proteases, and the heavy membrane fracti on act in a period occurring similar to 90-120 min after the start of incubation (the ''sentencing'' phase). In the final phase (''execution ''), the process of active nuclear destruction ensues.