Role of Tuberin in Mitotic Progression

大量开创性的工作已经确定，Tuberin和Hamartin复合体通过G1/S作为蛋白质合成和细胞周期进展的负调控因子发挥作用，这在很大程度上归因于Tuberin的GTPase活性间接抑制哺乳动物的雷帕霉素靶标(MTOR)。虽然目前对该途径的研究主要集中在对G1/S的调控上，但有重要证据表明，Tuberin-Hamartin复合体在G2/M调控中具有潜在的作用。来自我们的NSERC计划的数据已经表征了Tuberin和有丝分裂周期蛋白Cyclin B1之间的直接相互作用。我们已经定位了Tuberin中的Cyclin B1结合位点，并表明在功能上，这种相互作用推迟了Cyclin B1到细胞核的穿梭，以及随后的有丝分裂开始。有趣的是，我们已经证明，在缺乏营养和/或Akt磷酸化Tuberin的情况下，Tuberin-Cyclin B1的相互作用减少，细胞进入有丝分裂。这增加了一种令人兴奋的可能性，即Tuberin调节一个新的细胞周期检查点，将有丝分裂的开始与细胞的营养状态联系起来。解决Tuberin和Cyclin B1相互作用的分子机制，以及这对细胞周期调节和整体细胞生理学的影响是本研究计划的重点。假设：Tuberin和Cyclin B1之间的直接相互作用构成了一个新的细胞周期检查点的关键组成部分，通过该检查点，有丝分裂的启动与细胞的外部生长条件有关。该检查点在一定程度上调节细胞大小和在特定生长/分化因子和营养物质存在的情况下的分化决定。短期目标：利用我们实验室在过去的资助期间建立的一些关键技术，我们将：1.剖析Cyclin B1-CK1与Tuberin-Hamartin相互作用的动力学。2.确定Cyclin B1-Tuberin相互作用是否构成细胞大小检查点，以及这是否涉及分化决策。3.解决有丝分裂期间Tuberin核积累的重要性。长期目标：研究动物模型系统中特定的Tuberin突变，以解决这一检查点在系统发展、维护和对许多不同应激源的反应中的生理后果。我们的数据揭示了一种新的和令人兴奋的调控机制来控制有丝分裂的开始。这项研究计划试图解决这种相互作用的分子复杂性和细胞后果；这些数据对于理解正常的细胞生长机制是必不可少的。

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.