LSF AND MAMMALIAN CELL CYCLE PROGRESSION

哺乳动物细胞的生长是由信号转导途径驱动的，信号转导途径的靶标包括调节细胞周期进程关键基因表达的转录因子。在G1期晚期的一个关键点，即限制点，细胞信号被整合，以决定是复制细胞DNA(进展到S期)还是恢复到静止状态。从G1向S转运所需的信号分子包括D和E周期蛋白与细胞周期蛋白依赖性蛋白激酶(CDK)的复合体。细胞周期蛋白D/CDKs使Rb磷酸化，激活E2F转录因子家族的成员，从而激活对细胞DNA合成至关重要的基因。然而，人们对其他转录因子的调控知之甚少，这些转录因子激活了DNA合成机制的其他组成部分，包括在细胞周期的这个关键点被Cyclin E/CDK靶向的潜在转录因子。我们最近的研究表明，哺乳动物转录因子Lsf在细胞周期中起关键作用，它通过调节胸苷合成酶(TS)在G1/S转换期的表达而发挥作用。由于TS对细胞生长至关重要，二十多年来它一直是癌症化疗的靶点。同样，作为TS表达的重要调节因子，LSF最终可能被证明是癌症治疗的有价值的靶点。通过对已存在的核LSF池的修饰，LSF的DNA结合活性在G1末期得到增强。在体外，LSF是细胞周期蛋白E/CDK2的有效底物。因此，了解调控LSF的信号通路，以及LSF调控TS表达的分子机制，可能有助于阐明哺乳动物细胞周期进程中的新信号事件。具体来说，这项建议将解决以下问题：1.LSF在G1期晚期是否被细胞周期蛋白E/CDK2磷酸化？2.被Cyclin E/CDK2(或其他相关修饰)磷酸化是否改变LSF的DNA结合和转录活性？3.LSF是否是CDK2(或其他途径)在细胞向S进展过程中的主要靶点？4.LSF在细胞周期进程中是否对TS以外的基因进行关键调控？

The growth of mammalian cells is driven by signal transduction pathways, whose targets include transcription factors that regulate expression of genes critical to cell cycle progression. At one pivotal point in late G1 phase, the restriction point, cellular signals are integrated to determine whether to replicate cellular DNA (progress to S phase) or revert to a resting state. The signalling molecules required for transit from G1 to S include both D and E cyclins complexed with cyclin-dependent kinases (CDKs). Cyclin D/CDKs phosphorylate Rb and activate members of the E2F family of transcription factors, resulting in activation of genes important for cellular DNA synthesis. Little is known, however, about the regulation of other transcription factors that activate additional components of the DNA synthesis machinery, including potential transcription factors that are targeted by cyclin E/CDK at this key point in the cell cycle. Our recent studies have shown that the mammalian transcription factor LSF plays a key role in cell cycle progression by regulating thymidylate synthase (TS) expression at the G1/S transition. Because TS is essential for cell growth, it has been a cancer chemotherapeutic target for over two decades. Likewise, as an essential regulator of TS expression, LSF may ultimately prove a valuable target for cancer therapies. The DNA-binding activity of LSF is enhanced in late G1, via the modification of pre-existing pools of nuclear LSF. In vitro, LSF is an effective substrate for cyclin E/cdk2. An understanding of the signaling pathway that regulates LSF, and the molecular mechanism by which LSF regulates TS expression may therefore illuminate novel signaling events in cell cycle progression in mammalian cells. Specifically, this proposal will address the following: 1. Is LSF phosphorylated by cyclin E/cdk2 during the late G1 phase? 2. Does phosphorylation of LSF by cyclin E/cdk2 (or other relevant modifications) alter the DNA-binding and transcriptional activities of LSF? 3. Is LSF a major target of cdk2 (or another pathway) in progression of cells into S? 4. Does LSF critically regulate genes other than TS during cell cycle progression?