Disruption of retinoblastoma protein function by coexpression of its C pocket fragment.
The growth suppression function of the retinoblastoma protein (RB) is mediated by its interaction with a variety of cellular proteins. RB contains at least two protein-binding pockets: the large A/B pocket, which interacts with E2F and the D-type cyclins, and the C pocket, which interacts with the nuclear c-Abl tyrosine kinase. The large A/B pocket and the C pocket are shown here to be functionally distinct and can be occupied simultaneously. A complex containing E2F, RB, and c-Abl is detected in vivo and can be assembled in vitro. We propose that the biological activity of RB not only depends on the inhibition of its targets but also on its ability to properly assemble specific protein complexes. Consistent with this hypothesis, a fragment of RB, SE delta, containing only the C pocket is shown to act as a dominant-negative inhibitor of RB function. SE delta does not have growth inhibitory activity of its own. When coexpressed with full-length RB, SE delta does not disrupt the RB-E2F or RB-D2 complexes nor does it affect the expression, phosphorylation, or nuclear tethering of the full-length RB. SE delta does compete with RB for binding to c-Abl and is fully capable of inhibiting the c-Abl tyrosine kinase. Thus, SE delta can inactivate RB while maintaining the inhibition of E2F and c-Abl. These results suggest that the inhibition of RB-binding proteins is not sufficient to suppress cell growth and that the assembly of RB-mediated protein complexes is also important for the promotion of cell-cycle arrest.
Welch
P J
PJ
Department of Biology, University of California at San Diego, La Jolla 92093-0347.
Wang
J Y
JY
D002352:Carrier Proteins; D002453:Cell Cycle / Q000502:physiology; D018797:Cell Cycle Proteins; D002467:Cell Nucleus / Q000378:metabolism; D056742:Cyclin D2; D016213:Cyclins / Q000378:metabolism*; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D005786:Gene Expression Regulation; D006801:Humans; D008957:Models, Genetic; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D016315:Proto-Oncogene Proteins c-abl / Q000378:metabolism*; D016160:Retinoblastoma Protein / Q000235:genetics / Q000378:metabolism*; D056465:Retinoblastoma-Binding Protein 1; D013329:Structure-Activity Relationship; D050680:Transcription Factor DP1; D014157:Transcription Factors / Q000378:metabolism*; D014407:Tumor Cells, Cultured
C080336:CCND2 protein, human; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D056742:Cyclin D2; D016213:Cyclins; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D010446:Peptide Fragments; D016160:Retinoblastoma Protein; D056465:Retinoblastoma-Binding Protein 1; D050680:Transcription Factor DP1; D014157:Transcription Factors; D004247:DNA; D016315:Proto-Oncogene Proteins c-abl