This is a competing renewal from an established investigator who has had a long-standing interest in understanding the role of the retinoblastoma (RB) gene in the regulation of cell growth. RB is a tumor suppressor whose expression promotes growth arrest and maintains the long-term survival of terminally differentiated cells. RB, in its underphosphorylated form, in growth-arrested cells, and in early G1, forms complexes with transcription factors that are critical regulators of genes required for G1 progression and S phase entry. These transcription factors are released as cells progress through G1 and RB becomes hyperphosphorylated by cyclin-dependent kinases. The PI proposes to address RB function by studying the complexes that form between RB and specific transcription factors and by examining how these complexes affect the transcriptional regulation of genes critical for G1 to S progression. The three major aims are: 1. To identify and characterize genes regulated by RB-tethered protein complexes, a) by generating mutations in RB that affect the binding domain of specific proteins (such as E2F, LXCXE-containing proteins, and c-abl), and testing their growth suppressive properties in RB-/- cells and mice; b) by isolating genes differentially expressed in RB-/- and +/+ cells and testing their biologic activity in cells; and c) by isolating genes whose transcription regulation depends on the proper binding of proteins to the RB binding sites. 2.To investigate the biological relevance of RB phosphorylation: a) by studying the mechanism by which phosphorylation of Thr821 in the LXCXE-binding domain of RB affects RB interactions with proteins that carry the LXCXE motif and how these interactions, in turn, affect RB's suppressive function; b) by testing the effect of a non-phosphorylatable RB mutant on cell cycle progression. 3.To determine the role of RB in protecting cells from death by using RB mutants.
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