Wee1-regulated apoptosis mediated by the Crk adaptor protein in Xenopus egg extracts

Many of the biochemical reactions of apoptotic cell death, including mitoch ondrial cytochrome c release and caspase activation, can be reconstituted i n cell-free extracts derived from Xenopus eggs. In addition, because caspas e activation does not occur until the egg extract has been incubated for se veral hours on the bench, upstream signaling processes occurring before ful l apoptosis are rendered accessible to biochemical manipulation. We reporte d previously that the adaptor protein Crk is required for apoptotic signali ng in egg extracts (Evans, E.K., W. Lu, S.L. Strum, B.J. Mayer, and S. Korn bluth. 1997. EMBO (Eur. Mel. Biol. Organ.) J. 16:230-241). Moreover, we dem onstrated that removal of Crk Src homology (SH)2 or SH3 interactors from th e extracts prevented apoptosis. We now report the finding that the relevant Crk SH2-interacting protein, important for apoptotic signaling in the extr act, is the well-known cell cycle regulator, Wee1. We have demonstrated a s pecific interaction between tyrosine-phosphorylated Wee1 and the Crk SH2 do main and have shown that recombinant Wee1 can restore apoptosis to an extra ct depleted of SH2 interactors. Moreover, exogenous Wee1 accelerated apopto sis in egg extracts, and this acceleration was largely dependent on the pre sence of endogenous Crk protein. As other Cdk inhibitors, such as roscoviti ne 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 unrel ated to Wee1's role as a cell cycle regulator.