Role of Tuberin in Cell Size and 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。总体目标：本研究项目旨在揭示台糖干细胞如何调控细胞增殖和核心细胞周期机制的机制(S)。短期目标：利用我们开发的大量工具，我们将：1.剖析Tuberin与CycB1相互作用对环境应激源的反应动力学。2.在G2期早期与有丝分裂开始时相比，解析Tuberin-CycB1复合体的成分。3.研究Tuberin-CycB1在细胞命运决定过程中的体内外相互作用。长期目标：我们将把研究Tuberin与Cyclin B1相互作用的工具转移到小鼠和斑马鱼模型中，以确定这一检查点在体内对发育、维持和应激反应的影响。我们的数据揭示了一种控制有丝分裂开始的新颖而令人兴奋的调控机制。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. Overall Objective:  This research program aims to reveal the mechanism(s) of how the TSC regulates cell proliferation and the core cell cycle machinery. Short-Term Objective: Using the plethora of tools we have developed we will: 1.Dissect the dynamics of Tuberin interactions with CycB1 in response to environmental stressors. 2.Resolve the components of the Tuberin-CycB1 complex in early G2 vs at the onset of mitosis. 3.Study Tuberin-CycB1 interactions in vivo and in vitro during cell fate decisions. Long-Term Objective: We will move our tools to study Tuberin interactions with Cyclin B1 into mouse and zebrafish models to determine the in vivo consequence of this checkpoint on development, maintenance and response to stress.   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.