Role of LXCXE motif-dependent interactions in the activity of the retinoblastoma protein

Cell cycle control by pRb requires the integrity of the pocket domain, whic h is a region necessary for interactions with a variety of proteins, includ ing E2F and LXCXE-motif containing proteins. Through knowledge of the cryst al structure of pRb we have prepared a panel of pRb mutant derivatives in w hich a cluster of lysine residues that demark the LXCXE peptide binding dom ain were systematically mutated. One of the mutant derivatives, Rb6A, exhib its significantly reduced LXCXE-dependent interactions with HPV E7, cyclinD 1 and HDAC2, but retained LXCXE-independent binding to E2F. Consistent with these results, Rb6A could down-regulate E2F-1-dependent activation of diff erent E2F responsive promoters, but was compromised in Rb-dependent repress ion. Most importantly, Rb6A retained wild-type growth arrest activity, and colony forming activity similar to wild-type pRb. It is compatible with the se results that directly targeting HDAC2 to E2F responsive promoters as an E2F/HDAC hybrid protein failed to effect cell cycle arrest. These results s uggest that LXCXE-dependent interactions are not essential for pRb to exert growth arrest.