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.
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.
Farschon
D M
DM
Division of Cellular Immunology, La Jolla Institute for Allergy and Immunology, San Diego, California 92121, USA.
Couture
C
C
Mustelin
T
T
Newmeyer
D D
DD
D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D053148:Caspase 3; D020169:Caspases; D002458:Cell Fractionation; D002467:Cell Nucleus / Q000737:chemistry / Q000502:physiology; D002474:Cell-Free System; D002518:Ceramides / Q000378:metabolism*; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D004792:Enzyme Precursors / Q000378:metabolism*; D005260:Female; D008565:Membrane Proteins / Q000378:metabolism; D016296:Mutagenesis / Q000502:physiology; D009865:Oocytes / Q000737:chemistry / Q000502:physiology; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000502:physiology; D013997:Time Factors; D014981:Xenopus; D018909:src Homology Domains / Q000235:genetics*
D002518:Ceramides; D004792:Enzyme Precursors; D008565:Membrane Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D011993:Recombinant Fusion Proteins; D053148:Caspase 3; D020169:Caspases; D003546:Cysteine Endopeptidases