Direct binding of apoptosis signal-regulating kinase 1 to retinoblastoma protein: novel links between apoptotic signaling and cell cycle machinery.

The retinoblastoma protein Rb has antiproliferative and antiapoptotic functions. Our previous studies have shown that certain apoptotic signals can inactivate Rb via the p38 pathway. Here we show that Rb associates with the apoptosis signal-regulating kinase ASK1 in response to specific apoptotic signals. An LXCXE motif on ASK1 was required for Rb binding; this correlated with increased E2F1 transcriptional activity and up-regulation of the proapoptotic protein p73. Overexpression of Rb inhibited ASK1-induced apoptosis; in addition, an ASK1 mutant incapable of binding Rb could not induce apoptosis, indicating that ASK1 has to overcome the antiapoptotic properties of Rb to kill cells. Chromatin immunoprecipitation assays show that in asynchronous cells the p73P1 promoter is occupied predominantly by E2F3; upon tumor necrosis factor (TNF)-alpha stimulation, E2F3 is dissociated from the promoter and replaced by E2F1. At the same time, TNF-alpha stimulation causes Rb to dissociate from the p73P1 promoter. These are promoter-specific events because Rb binds to the mitogenic cdc25A promoter upon TNF-alpha stimulation. These studies suggest that Rb acts as a link between apoptotic and proliferative pathways by interacting with distinct kinases and occupying different promoters.