Role of Tuberin in Mitotic Progression

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A great deal of ground breaking work has determined that the Tuberin and Hamartin complex function as a negative regulator of protein synthesis and cell cycle progression through G1/S. This is largely attributed to the GTPase activity of Tuberin functioning to indirectly inhibit the mammalian target of rapamycin (mTOR). While current work on this pathway is focused on the regulation of G1/S, there is significant evidence which points to a potential role for the Tuberin-Hamartin complex in G2/M regulation. Data from our NSERC program has characterized a direct interaction between Tuberin and the mitotic cyclin, Cyclin B1. We have mapped the Cyclin B1 binding sites in Tuberin and have shown that functionally this interaction delays the shuttling of Cyclin B1 to the nucleus, and subsequent mitotic onset. Interestingly, we have demonstrated that in the absence of nutrients and/or Akt phosphorylation of Tuberin the Tuberin-Cyclin B1 interaction is reduced and cells enter mitosis. This raises the exciting possibility that Tuberin regulates a novel cell cycle checkpoint linking mitotic onset with the nutritional status of the cell. Resolving the molecular mechanism by which Tuberin and Cyclin B1 interact, and the consequences of this on cell cycle regulation and overall cell physiology are the focus of this research program. Hypothesis: A direct interaction between Tuberin and Cyclin B1 constitutes a key component of a novel cell cycle checkpoint whereby initiation of mitosis is linked to the external growth conditions of the cell. This checkpoint functions in part to regulate cell size and differentiation decisions in the presence of specific growth/differentiation factors and nutrients. Short-term objectives: Using a number of key technologies that we have established in our lab over the past grant period we will: 1. dissect the dynamics of the Cyclin B1-Ck1 interaction with Tuberin-Hamartin. 2. determine whether the Cyclin B1-Tuberin interaction constitutes a cell size checkpoint and whether this is involved in differentiation decisions. 3. address the importance of nuclear accumulation of Tuberin during mitosis. Long term objectives: To study specific Tuberin mutations in animal model systems to address the physiological consequence of this checkpoint in system development, maintenance and response to a number of different stressors. Our data has revealed a novel and exciting regulatory mechanism controlling the onset of mitosis. This research program seeks to resolve the molecular complexities and the cellular consequences of this interaction; data which is essential for understanding normal cell growth mechanisms.