A New Paradigm for Cell Size Control by the RB Tumor Suppressor Pathway

描述（由申请人提供）：大小控制是细胞的基本性质，涉及生长和分裂的协调调节，这些过程在人类疾病如癌症中经常功能失调。这项研究的长期目标是建立管理尺寸控制的机械原理。我们已经利用了一个独特的信息模型的细胞大小调节，单细胞衣藻莱茵衣藻。使用衣原体，我们已经确定了一个大小检查点途径的关键组成部分，其行为表明耦合细胞大小的细胞周期激活的机制。该检查点的基因鉴定组分各自具有人类对应物，并且包括视网膜母细胞瘤（RB）肿瘤抑制途径的蛋白质、使RB磷酸化的细胞周期蛋白依赖性激酶CDKG 1和RNA结合蛋白TNY 1。我们推测，激酶CDKG 1的活性是由TNY 1控制的，TNY 1是将细胞大小与细胞周期进入偶联的一种手段，这两种活性的平衡通过打开和关闭RB通路来控制细胞周期。该提案的发现将提供有关细胞生长和分裂控制机制的基础知识，这些机制与理解人类类似过程有关。本提案的具体目标如下：目标1。研究CDKG 1作为sizer蛋白的作用：我们将i）确定CDKG 1的丰度和活性在细胞周期中是如何控制的; ii）鉴定和表征CDKG 1在其底物RB相关蛋白MAT 3上的磷酸化位点; iii）使用条件等位基因确定CDKG 1在细胞周期中的时间需求。目标2.确定TNY 1抑制细胞周期的机制：TNY 1在每个细胞周期产生一次，然后随着细胞在G1期的生长而稀释。我们将测试TNY 1作为细胞大小的阈值调节剂的作用，其对抗CDKG 1的活性。我们将i）确定TNY 1是否通过结合其mRNA来调节CDKG 1; ii）测试TNY 1的剂量效应; iii）鉴定内源性TNY 1 RNA靶标;以及iv）确定将TNY 1靶向mRNA是否使编码蛋白的表达受到TNY 1和/或细胞周期的控制。目标3：通过抑制筛选鉴定新的大小途径基因：tny 1无效突变体和CDKG 1过表达（CDKG 1-OE）系都具有小细胞表型，提供了一个敏感的遗传背景，在其中鉴定大小控制的相互作用组分。i）我们将在正向筛选中使用这些细胞系来寻找其小细胞表型的抑制因子。预测这些抑制子编码靶蛋白或相互作用蛋白。ii）我们将使用二次筛选来对大小途径中的突变体进行排序，并鉴定候选者以供进一步研究和克隆。

DESCRIPTION (provided by applicant): Size control is a fundamental property of cells that involves coordinated regulation of growth and division, processes that are frequently dysfunctional in human diseases such as cancer. The long-term goal of this research is to establish the mechanistic principles that govern size control. We have exploited a uniquely informative model for cell-size regulation, the unicellular alga Chlamydomonas reinhardtii. Using Chlamydomonas we have identified key components of a size checkpoint pathway whose behavior suggests a mechanism for coupling cell size to cell cycle activation. The genetically identified components of this checkpoint each have human counterparts and include proteins of the retinoblastoma (RB) tumor suppressor pathway, a cyclin dependent kinase, CDKG1, that phosphorylates RB, and an RNA binding protein, TNY1. We hypothesize that the activity of the kinase, CDKG1, is held in check by TNY1 as a means of coupling cell size to cell cycle entry, and that the balance of these two activities controls the cell cycle by switching on and off the RB pathway. The findings from this Proposal will provide fundamental knowledge regarding mechanisms of cell growth and division control that are relevant for understanding similar processes in humans. The Specific Aims of this Proposal are as follows: Aim 1. Investigate the role of CDKG1 as sizer protein: We will i) determine how CDKG1 abundance and activity are controlled during the cell cycle; ii) identify and characterize CDKG1 phosphorylation sites on its substrate, the RB related protein MAT3; iii) determine the temporal requirement for CDKG1 in the cell cycle using conditional alleles. Aim 2. Determine the mechanism by which TNY1 represses the cell cycle: TNY1 is produced once per cell cycle and then becomes diluted as cells grow during G1. We will test the role of TNY1 as a threshold regulator of cell size that opposes the activity of CDKG1. We will i) establish whether TNY1 regulates CDKG1 through binding its mRNA; ii) test dosage effects of TNY1; iii) identify endogenous TNY1 RNA targets; and iv) establish whether targeting TNY1 to a mRNA brings expression of the encoded protein under control of TNY1 and/or the cell cycle. Aim 3. Identify new size pathway genes through suppressor screens: tny1 null mutants and CDKG1 over- expressing (CDKG1-OE) lines both have small-cell phenotypes that provide a sensitized genetic background in which to identify interacting components of size control. i) We will use these lines in forward screens to find suppressors of their small-cell phenotypes. These suppressors are predicted to encode either targets or interacting proteins. ii) We will use secondary screens to order the mutants in the size pathway and to identify candidates for further investigation and cloning.