Defining Cell Cycle‐Specific Interactions of the pRB Tumor Suppressor

The retinoblastoma protein, pRB, functions as a tumor suppressor that is inactivated in most types of cancer cells. This tumor suppressive function is achieved through binding to E2F and regulating transcription. Other important cellular functions of pRB include DNA damage response and repair and mitotic chromosome segregation. Recently, Mass Spec approaches have identified over 300 distinct proteins that interact with pRB. Many of these proteins have known functions outside of transcriptional regulation, suggesting that they may represent the functional link between pRB and its non‐transcriptional roles in the cell. However, this approach was completed with an asynchronous population of cells, where cells undergoing mitosis are likely to be underrepresented. Nevertheless, some key interactors that were identified through the mass spec including several proteins with known roles in mitotic chromosome organization and mitotic fidelity. We hypothesize that pRB interactions with and regulation of a subset of proteins are important for pRB’s role in mitosis, and that loss of these interactions when pRB function is compromised in cancer may explain the mitotic defects seen in these cancers. To test this hypothesis, we have confirmed that pRB interacts with specific proteins during mitosis by performing reciprocal immunoprecipitation experiments in nocodazole‐synchronized cells. Using silver stain analysis of pRB immunoprecipitation samples, we find that many distinct proteins appear to interact with pRB during mitosis. We will use western blot approaches to attempt to identify specific proteins that are potential pRB mitotic interactors. We will then use Mass Spec to identify these additional interactors. By defining cell cycle specific complexes formed by pRB we hope to elucidate the non‐canonical role of pRB in regulating mitotic chromosome segregation and to understand how this function of pRB may be altered in cancer contexts.