Separation of anti-proliferation and anti-apoptotic functions of retinoblastoma protein through targeted mutations of its A/B domain.
BACKGROUND
The human retinoblastoma susceptibility gene encodes a nuclear phosphoprotein RB, which is a negative regulator of cell proliferation. The growth suppression function of RB requires an evolutionarily conserved A/B domain that contains two distinct peptide-binding pockets. At the A/B interface is a binding site for the C-terminal trans-activation domain of E2F. Within the B-domain is a binding site for proteins containing the LxCxE peptide motif.


RESULTS
Based on the crystal structure of the A/B domain, we have constructed an RB-K530A/N757F (KN) mutant to disrupt the E2F- and LxCxE-binding pockets. The RB-K530A (K) mutant is sufficient to inactivate the E2F-binding pocket, whereas the RB-N757F (N) mutant is sufficient to inactivate the LxCxE-binding pocket. Each single mutant inhibits cell proliferation, but the RB-KN double mutant is defective in growth suppression. Nevertheless, the RB-KN mutant is capable of reducing etoposide-induced apoptosis.


CONCLUSIONS
Previous studies have established that RB-dependent G1-arrest can confer resistance to DNA damage-induced apoptosis. Results from this study demonstrate that RB can also inhibit apoptosis independent of growth suppression.
Chau
B Nelson
BN
Department of Medicine, Division of Hematology/Oncology, Moores-UCSD Cancer Center, University of California San Diego, La Jolla, California, United States of America.
Pan
Chris W
CW
Wang
Jean Y J
JY
D019943:Amino Acid Substitution; D017209:Apoptosis / Q000235:genetics / Q000502:physiology*; D001665:Binding Sites / Q000235:genetics; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D050684:E2F Transcription Factors / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D016297:Mutagenesis, Site-Directed; D054730:Protein Interaction Domains and Motifs; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016160:Retinoblastoma Protein / Q000737:chemistry* / Q000235:genetics* / Q000502:physiology
D050684:E2F Transcription Factors; D011994:Recombinant Proteins; D016160:Retinoblastoma Protein