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 G 1 /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 G 1 /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 G 1 and to induce terminal growth arrest. These results suggest that the A/B or C region can induce a prolongation of G 1 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.

The retinoblastoma tumor suppressor inhibits cellular proliferation through two distinct mechanisms: inhibition of cell cycle progression and induction of cell death

“…Importantly, C33-A cells are HPV-negative and thus the short-circuiting of RB-mediated cell cycle arrest is not due to the expression of the HPV E7 protein. Furthermore, a large number of biochemical studies on RB have been carried out in C33-A cells and it is known that the function of RB in protein binding and repressing transcription are intact (Knudsen and Wang, 1997;Whitaker et al, 1998). However, it is possible that cellular proteins which can inactivate RB may be present in abundance in C33-A cells.…”

“…However, our ®ndings suggest instead that the pathways through which RB functions to impinge on the cell cycle are short-circuited in these cells. Since C33-A cells have been used previously to study RB biochemical activity (Knudsen and Wang, 1997;Whitaker et al, 1998), we focused on elucidating how these cells bypass the requirement of RB inactivation for cell cycle progression.…”

“…To determine how PSM-RB inhibits C33-A proliferation we followed the behavior of C33-A cells transfected with PSM-RB or a small C-terminal fragment of RB (7S-SE) which has no activity (Whitaker et al, 1998). To monitor those cells transfected with RB we used an antibody directed against the C-terminal of RB for direct immunofluorescence (851) (Welch and Wang, 1993).…”

The Aryl Hydrocarbon Receptor Binds to E2F1 and Inhibits E2F1-induced Apoptosis

“…Expression of the peptides encoded by each AHR truncation mutant presented in Supplemental Figure S2 was confirmed by Western blot (data not shown). The reporter plasmid p3XE2FLuc, containing three E2F-responsive elements, has also been described previously (Whitaker et al, 1998). The kinase-dead (KD) and constitutively active (CA) CHK2 expression vectors were a gift of P. Stambrook (University of Cincinnati).…”

Detection of allelic imbalance in the gene expression of hMSH2 or RB1 in lymphocytes from pedigrees of hereditary, nonpolyposis, colorectal cancer and retinoblastoma by an RNA difference plot

“…4a). Low penetrance could be attributed to this specific amino acid substitution because this mutant RB protein (W661) was shown to have lost binding activity with E2F or adenovirus E1A proteins but retained the wild-type properties of nuclear localization, the ability to undergo hyperphosphorylation in vitro, and the capacity to suppress the growth of RB-deficient cells when overexpressed (Kratzke et al 1994;Whitaker et al 1998). RDP analysis revealed that the expression ratios of a mutant to a wild-type RB1 in the affected children (II-1 and II-2) were 0.45±0.03 and 0.40±0.03, respectively although the difference between the children (II-1 and II-2) was not statistically significant (Fig.…”