Identification of the E1A-Regulated Transcription Factor p120 ^E4F as an Interacting Partner of the RASSF1A Candidate Tumor Suppressor Gene

Epigenetic inactivation of the candidate tumor suppressor gene RASSF1A is a frequent and critical event in the pathogenesis of many human cancers. The RASSF1A protein contains a Ras association domain, suggesting a role in Ras-like signaling pathways, and has also been implicated in cell cycle progression. However, the preliminary data suggests that the RASSF1A gene product is likely to have multiple functions. To identify novel RASSF1A functions, we have sought to identify interacting proteins by yeast two-hybrid analysis in a human brain cDNA library. We identified the E1A-regulated transcription factor p120^E4F as a RASSF1A. interacting partner in yeast and mammalian cells, and demonstrated that RASSF1A protein and p120 ^E4F form a complex in vivo. The interaction between RASSF1A and p120^E4F was confirmed by both in vitro and in vivo pull downs and coimmunoprecipitation assays. In addition, specific inactivation of RASSF1A by short interfering RNA disrupts binding of RASSF1A to p120^E4F in coimmunoprecipitation assays. In addition, we demonstrated enhanced G ₁ cell cycle arrest and S phase inhibition by propidium iodide staining of p120^E4F in the presence of RASSF1A. As p120 ^E4F has been reported previously to interact with p14ARF, retinoblastoma, and p53, these findings provide an important link between the function of RASSF1A and other major human tumor suppressor genes.