Disruption of BRCA1 LXCXE motif alters BRCA1 functional activity and regulation of RB family but not RB protein binding.
The tumor suppressor activity of the BRCA1 gene product is due, in part, to functional interactions with other tumor suppressors, including p53 and the retinoblastoma (RB) protein. RB binding sites on BRCA1 were identified in the C-terminal BRCT domain (Yarden and Brody, 1999) and in the N-terminus (aa 304-394) (Aprelikova et al., 1999). The N-terminal site contains a consensus RB binding motif, LXCXE (aa 358-362), but the role of this motif in RB binding and BRCA1 functional activity is unclear. In both in vitro and in vivo assays, we found that the BRCA1:RB interaction does not require the BRCA1 LXCXE motif, nor does it require an intact A/B binding pocket of RB. In addition, nuclear co-localization of the endogenous BRCA1 and RB proteins was observed. Over-expression of wild-type BRCA1 (wtBRCA1) did not cause cell cycle arrest but did cause down-regulation of expression of RB, p107, p130, and other proteins (e.g., p300), associated with increased sensitivity to DNA-damaging agents. In contrast, expression of a full-length BRCA1 with an LXCXE inactivating mutation (LXCXE-->RXRXH) failed to down-regulate RB, blocked the down-regulation of RB by wtBRCA1, induced chemoresistance, and abrogated the ability of BRCA1 to mediate tumor growth suppression of DU-145 prostate cancer cells. wtBRCA1-induced chemosensitivity was partially reversed by expression of either Rb or p300 and fully reversed by co-expression of Rb plus p300. Our findings suggest that: (1) disruption of the LXCXE motif within the N-terminal RB binding region alters the biologic function of BRCA1; and (2) over-expression of BRCA1 inhibits the expression of RB and RB family (p107 and p130) proteins.
Fan
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S
Department of Radiation Oncology, Long Island Jewish Medical Center, The Long Island Campus for the Albert Einstein College of Medicine, 270-05 76th Avenue, New Hyde Park, New York, NY 11040, USA. fan@lij.edu
Yuan
R
R
Ma
Y X
YX
Xiong
J
J
Meng
Q
Q
Erdos
M
M
Zhao
J N
JN
Goldberg
I D
ID
Pestell
R G
RG
Rosen
E M
EM
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019313:BRCA1 Protein / Q000737:chemistry / Q000502:physiology*; D001665:Binding Sites; D015536:Down-Regulation; D016161:Genes, Retinoblastoma; D006801:Humans; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics; D016160:Retinoblastoma Protein / Q000378:metabolism*; D015534:Trans-Activators / Q000235:genetics; D014407:Tumor Cells, Cultured
D019313:BRCA1 Protein; D009687:Nuclear Proteins; D016160:Retinoblastoma Protein; D015534:Trans-Activators