Growth Suppression by an E2F-Binding-Defective Retinoblastoma Protein (RB): Contribution from the RB C Pocket

ABSTRACT

Growth suppression by the retinoblastoma protein (RB) is dependent on its ability to form complexes with transcription regulators. At least three distinct protein-binding activities have been identified in RB: the large A/B pocket binds E2F, the A/B pocket binds the LXCXE peptide motif, and the C pocket binds the nuclear c-Abl tyrosine kinase. Substitution of Trp for Arg 661 in the B region of RB (mutant 661) inactivates both E2F and LXCXE binding. The tumor suppression function of mutant 661 is not abolished, because this allele predisposes its carriers to retinoblastoma development with a low penetrance. In cell-based assays, 661 is shown to inhibit G₁/S progression. This low-penetrance mutant also induces terminal growth arrest with reduced but detectable activity. We have constructed mutations that disrupt C pocket activity. When overproduced, the RB C-terminal fragment did not induce terminal growth arrest but could inhibit G₁/S progression, and this activity was abolished by the C-pocket mutations. In full-length RB, the C-pocket mutations reduced but did not abolish RB function. Interestingly, combination of the C-pocket and 661 mutations completely abolished RB’s ability to cause an increase in the percentage of cells in G₁ and to induce terminal growth arrest. These results suggest that the A/B or C region can induce a prolongation of G₁ through mechanisms that are independent of each other. In contrast, long-term growth arrest requires combined activities from both regions of RB. In addition, E2F and LXCXE binding are not the only mechanisms through which RB inhibits cell growth. The C pocket also contributes to RB-mediated growth suppression.

ACKNOWLEDGMENTS

We thank Paolo Vigneri, Lauren Wood, and Pam Woodring for critically reading the manuscript. We also thank the following researchers for the generous provision of reagents: B. Vogelstein (Johns Hopkins University); E. Harlow (Massachusetts General and Harvard); H. Land (Imperial Cancer Research Fund, London, United Kingdom); W. Krek, D. Livingston, W. Sellers, and W. Kaelin, Jr. (Dana-Farber Cancer Institute); S. Hiebert (St. Jude Children’s Hospital); C. Murre (University of California, San Diego); and A. Means and P. Farnham (McArdle Laboratory for Cancer Research, University of Wisconsin Medical School).

H.S. was supported by National Institutes of Health grant CA66314. R.B. was supported by Council for Tobacco Research grant CTR3762. This research was supported by National Institutes of Health grant CA58320 to J.Y.J. Wang.