Abstract 1311: Notch1 inhibits retinoblastoma (RB) via direct interaction with RB pocket domain
Authors: Filomena T. Papa, Ingrid Espinoza, Nicole Darack-Gutyon, Antonio Giordano, Antonio D. Luca, Christian R. Gomez, Lucio Miele
Journal: Cancer Research (2014)

Abstract Notch signaling is involved in many biological processes, ranging from cellular proliferation to differentiation and apoptosis. Mammals have four Notch genes (Notch1-4) that encode heterodimeric membrane receptors. The transmembrane subunit is cleaved to generate an intracellular active subunit (NotchIC). Gain-of-function Notch1 mutations are found in a majority of T-ALL, some adult chronic lymphocytic leukemia and some breast cancers. The Retinoblastoma (RB) protein is a tumor suppressor and negative regulator of the cell cycle. Cellular and viral proteins that interact with RB possess a LxCxE and/or ExxxDLF motifs or closely related motifs. Notch1IC affects the phosphorylation and total amount of RB in T-ALL and mouse erythroleukemia cells. Oncoproteins such as E7 and SV40 large T-antigen bind and inactivate RB but also upregulate Notch1 in some systems. Notch1 contains an AxCxD motif, a conserved negatively charged cluster similar to viral oncoproteins in ankyrin repeat 1 and an ExxLEDL motif. We tested whether Notch1 interacts with RB and determinated the binding motif responsible of this interaction. We quantified Notch transcriptional activity using CBF-1 luciferase. We performed co-immunoprecipitation experiments in T-ALL and other cell lines with elevated Notch1IC expression. To map the Notch1-RB interaction, several GST fusion proteins were constructed and used in interaction assays. Total cellular lysate of breast cancer cells transiently expressing Notch1IC with different point mutations in AxCxD and/or ExxLEDL motifs, were used for co-immunoprecipitation. Luciferase assay showed that exogenous expression of RB significantly inhibits ICN-induced stimulation of CBF1 as well as basal CBF1 activity. We found that Notch1IC containing putative LxCxE interacts with the pocket region of RB-GST and does not appear to bind or interact with other domains of RB. Co-immunoprecipitation experiments demonstrated binding of unphosphorylated RB and Notch1IC in T-ALL and other cell lines. Mutations in Notch1IC in the two putative motifs do not completely abolish interaction with RB. These mutants appear to bind preferentially the N-terminal region of RB, suggesting the presence of two binding sites or a larger complex including other nuclear proteins. We showed direct interaction of RB and Notch1IC by GST-fusion proteins and co-immunoprecipitation in T-ALL and cells. We also showed interaction of AxCxD and ExxLEDL Notch1 motifs with RB. We conclude that inactivation of RB may contribute to the oncogenic activity of Notch1IC. Citation Format: Filomena T. Papa, Ingrid Espinoza, Nicole Darack-Gutyon, Antonio Giordano, Antonio De Luca, Christian R. Gomez, Lucio Miele. Notch1 inhibits retinoblastoma (RB) via direct interaction with RB pocket domain. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1311. doi:10.1158/1538-7445.AM2014-1311