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.
Fenton
Sarah L
SL
Section of Medical and Molecular Genetics, Department of Reproductive and Child Health, University of Birmingham, The Medical School, Edgbaston, Birmingham, United Kingdom.
Dallol
Ashraf
A
Agathanggelou
Angelo
A
Hesson
Luke
L
Ahmed-Choudhury
Jalal
J
Baksh
Shairaz
S
Sardet
Claude
C
Dammann
Reinhard
R
Minna
John D
JD
Downward
Julian
J
Maher
Eamonn R
ER
Latif
Farida
F
D017500:Adenovirus E1A Proteins / Q000235:genetics; D017504:Adenovirus E4 Proteins / Q000235:genetics* / Q000378:metabolism*; D003001:Cloning, Molecular; D006801:Humans; D019656:Loss of Heterozygosity; D010957:Plasmids; D034741:RNA, Small Interfering / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics* / Q000378:metabolism*; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics; D017384:Sequence Deletion; D025521:Tumor Suppressor Proteins / Q000235:genetics*
D017500:Adenovirus E1A Proteins; D017504:Adenovirus E4 Proteins; C411762:RASSF1 protein, human; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D025521:Tumor Suppressor Proteins; C411663:transcription factor p120(E4F)