Wee1-regulated apoptosis mediated by the crk adaptor protein in Xenopus egg extracts.
Many of the biochemical reactions of apoptotic cell death, including mitochondrial cytochrome c release and caspase activation, can be reconstituted in cell-free extracts derived from Xenopus eggs. In addition, because caspase activation does not occur until the egg extract has been incubated for several hours on the bench, upstream signaling processes occurring before full apoptosis are rendered accessible to biochemical manipulation. We reported previously that the adaptor protein Crk is required for apoptotic signaling in egg extracts (Evans, E.K., W. Lu, S.L. Strum, B.J. Mayer, and S. Kornbluth. 1997. EMBO (Eur. Mol. Biol. Organ.) J. 16:230-241). Moreover, we demonstrated that removal of Crk Src homology (SH)2 or SH3 interactors from the extracts prevented apoptosis. We now report the finding that the relevant Crk SH2-interacting protein, important for apoptotic signaling in the extract, is the well-known cell cycle regulator, Wee1. We have demonstrated a specific interaction between tyrosine-phosphorylated Wee1 and the Crk SH2 domain and have shown that recombinant Wee1 can restore apoptosis to an extract depleted of SH2 interactors. Moreover, exogenous Wee1 accelerated apoptosis in egg extracts, and this acceleration was largely dependent on the presence of endogenous Crk protein. As other Cdk inhibitors, such as roscovitine and Myt1, did not act like Wee1 to accelerate apoptosis, we propose that Wee1-Crk complexes signal in a novel apoptotic pathway, which may be unrelated to Wee1's role as a cell cycle regulator.
Smith
J J
JJ
Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Evans
E K
EK
Murakami
M
M
Moyer
M B
MB
Moseley
M A
MA
Vande Woude
G
G
Kornbluth
S
S
D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D018797:Cell Cycle Proteins; D002457:Cell Extracts; D008969:Molecular Sequence Data; D009687:Nuclear Proteins; D010063:Ovum / Q000166:cytology* / Q000378:metabolism*; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D011518:Proto-Oncogene Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D050737:Proto-Oncogene Proteins c-crk; D011687:Purines / Q000494:pharmacology; D000077546:Roscovitine; D015398:Signal Transduction / Q000187:drug effects; D029867:Xenopus Proteins; D014982:Xenopus laevis; D018909:src Homology Domains
D018797:Cell Cycle Proteins; D002457:Cell Extracts; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; D050737:Proto-Oncogene Proteins c-crk; D011687:Purines; D029867:Xenopus Proteins; C493873:crk2 protein, Xenopus; D000077546:Roscovitine; D019000:Phosphotyrosine; C095970:MYT1 kinase, Xenopus; C530005:WEE1 protein, Xenopus; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases