ARTICLE ABSTRACTThe retinoblastoma protein pRB and its two homologs p130 and p107 form the family of pocket proteins and play a major role in cell-cycle regulation and suppression of human and mouse tumorigenesis. Pocket proteins regulate the activity of E2F transcription factors during G1–S transition. Two mechanisms have been described: (i) pocket protein binding blocks the transactivation domain of activator E2Fs, inhibiting E2F-dependent transcription and (ii) E2F-bound pocket proteins can recruit chromatin remodeling proteins containing an LxCxE motif (x encoding any amino acid), resulting in active repression of E2F target genes. To investigate the importance of pRB's LxCxE-interacting motif in cell-cycle control and tumor suppression, we generated mouse embryonic fibroblasts and mice expressing a mutant pRB protein carrying an asparagine for phenylalanine substitution at position 750, abrogating LxCxE binding. Because p130 may compensate for loss of pRB, we studied pRBN750F activity in the presence and absence of p130. The pRB–LxCxE interaction was not required for cell-cycle arrest upon mitogen deprivation and cell-cell contact, but did contribute to RASV12- and radiation-induced cell-cycle arrest. Remarkably, the pRB–LxCxE interaction was not required for suppression of in vitro and in vivo transformation, even in the absence of p130. These results indicate that pRB's tumor suppressor activity is not effectuated by active silencing of E2F target genes, but rather by regulation of activator E2Fs or another unidentified mechanism. Furthermore, the in vitro response of pocket protein–perturbed cells to mitogen deprivation and cell–cell contact seems a better predictor of tumor development than the response to ectopic RASV12 expression. Cancer Res; 74(18); 5266–76. ©2014 AACR.