The Impact of Cyclin B1 on Tuberin Stabilization

Tuberous Sclerosis Complex (TSC) is an autosomal dominant disorder caused by various mutations in either TSC1 or TSC2, genes that encode the proteins Hamartin and Tuberin respectively. Hamartomas (benign tumours), skin lesions, neurological symptoms, renal dysfunctions, and retinal malformations are often present in TSC patients with varying severity. Tuberin and Hamartin regulate protein synthesis through mTORC1 inhibition. Tuberin can also delay mitotic onset at the G2/M cell cycle transition by binding to Cyclin B1. Hamartin has been shown to stabilize Tuberin by inhibiting its ubiquitination by HERC1 and subsequent degradation. Preliminary data from our lab suggests that Cyclin B1 may also contribute to the stabilization of Tuberin levels during the G2/M transition. Phosphorylation status of the cytoplasmic retention sequence (CRS) of Cyclin B1 plays an important role in the formation of the Tuberin/Cyclin B1 complex. The unphosphorylated CRS form of Cyclin B1 (Cyclin B1 5xA) binds stronger to Tuberin compared to the phosphorylated form (Cyclin B1 5xE). My thesis investigates the role of Cyclin B1 in Tuberin stabilization and how CRS phosphorylation status impacts Tuberin/Cyclin B1 complex formation. HEK293-TSC1 null cells (IC2) will be transfected with varying concentrations of Cyclin 5xA DNA and the levels of the Tuberin protein will be quantified by Western blot techniques. The importance of each residue in the CRS region for the Tuberin/Cyclin B1 complex formation will also be evaluated using Immunoprecipitation studies. Understanding the role of Cyclin B1 in Tuberin stabilization will shed light on cell proliferation and growth mechanisms that underlie tumorigenic disorders