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Molecular and Cellular Biology, May 2000, p. 3715-3727, Vol. 20, No. 10
MGH Cancer Center, Charlestown, Massachusetts
02129
Received 29 September 1999/Returned for modification 10 November
1999/Accepted 15 February 2000
The pocket domain of pRB is required for pRB to arrest the cell
cycle. This domain was originally defined as the region of the protein
that is necessary and sufficient for pRB's interaction with adenovirus
E1A and simian virus s40 large T antigen. These oncoproteins, and other
pRB-binding proteins that are encoded by a variety of plant and animal
viruses, use a conserved LXCXE motif to interact with pRB. Similar
sequences have been identified in multiple cellular pRB-binding
proteins, suggesting that the viruses have evolved to target a highly
conserved binding site of pRB that is critical for its function. Here
we have constructed a panel of pRB mutants in which conserved amino
acids that are predicted to make close contacts with an LXCXE peptide
were altered. Despite the conservation of the LXCXE binding site
throughout evolution, pRB mutants that lack this site are able to
induce a cell cycle arrest in a pRB-deficient tumor cell line. This
G1 arrest is overcome by cyclin D-cdk4 complexes but is
resistant to inactivation by E7. Consequently, mutants lacking the
LXCXE binding site were able to induce a G1 arrest in HeLa
cells despite the expression of HPV-18 E7. pRB mutants lacking the
LXCXE binding site are defective in binding to adenovirus E1A and human
papillomavirus type 16 E7 protein but exhibit wild-type binding to E2F
or DP, and they retain the ability to interact with CtIP and HDAC1, two transcriptional corepressors that contain LXCXE-like sequences. Consistent with these observations, the pRB mutants are able to actively repress transcription. These observations suggest that viral
oncoproteins depend on the LXCXE-binding site of pRB for interaction to
a far greater extent than cellular proteins that are critical for cell
cycle arrest or transcriptional repression. Mutation of this binding
site allows pRB to function as a cell cycle regulator while being
resistant to inactivation by viral oncoproteins.
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Mutagenesis of the pRB Pocket Reveals that Cell
Cycle Arrest Functions Are Separable from Binding to Viral
Oncoproteins
*
Corresponding author. Mailing address: MGH Cancer
Center, Building 149, 13th St., Charlestown, MA 02129. Phone: (617)
726-7800. Fax: (617) 726-7808. E-mail: dyson{at}helix.mgh.harvard.edu.
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