RETINOBLASTOMA PROTEIN IN LUNG AND OTHER TISSUES

Rb是一种肿瘤抑制因子，是细胞周期从G1期向S期运动所必需的。现在认为，Rb要么是突变，要么是肿瘤中调节Rb活性的途径被破坏，导致几乎所有肿瘤细胞中Rb功能的丧失。Rb在正常的生物过程中也起作用，这从Rb基因敲除小鼠的胚胎致死表型中可以看出。最近的研究表明Rb在骨骼肌和中枢神经系统的形成以及肺分支的形态发生中起作用。在上一期拨款期内，我们详细研究了Rb的结构/功能。这些研究产生了两个主要发现。第一个和Rb的抑制域结构有关。我们发现Rb的中心口袋区（肿瘤中Rb基因大多数失活突变的目标）是抑制基序。在口袋里有两个高度保守的区域，分别是A和B。我们发现这两个区域相互作用形成阻遏子基序。更重要的是，这种A-B相互作用被G1 cdks催化的超磷酸化破坏，这阻断了Rb的功能。最近，Rb口袋已经结晶，我们将利用这种晶体结构以及Rb结构/功能的结果来指导Rb口袋结构的靶向突变分析。在上一个资助期间的第二个发现是Rb可以通过两种不同的机制抑制转录。当靶向细胞周期启动子时，它可以结合周围的转录因子并阻断其活性。此外，Rb通过招募组蛋白去乙酰化酶到启动子来抑制转录。基因启动子可以被组蛋白八聚体缠绕到核小体中，从而抑制转录因子的结合。核心组蛋白的乙酰化抑制核小体的组装，组蛋白去乙酰化酶去除乙酰基，促进核小体组装并抑制转录。我们建议进一步研究Rb在培养细胞和转基因小鼠中的作用机制和作用。

Rb is a tumor suppressor that is required for movement of cells from G1 to S phase of the cell cycle. It is now thought that Rb is either mutated or that the pathway that regulates Rb activity is disrupted in tumors, resulting in the loss of Rb function in virtually all tumor cells. Rb also functions in normal biologic processes, which is evident from the embryonic lethal phenotype, observed in Rb knockout mice. Recent studies have suggested roles for Rb in formation of skeletal muscle and CNS, and for lung branching morphogenesis. In the previous grant period we have examined the structure/function of Rb in some detail. Two major findings arose from these studies. The first has to do with the structure of the repressor domain of Rb. We found that the central so called pocket region of Rb (the target of most inactivating mutations of the Rb gene in tumors) is the repressor motif. There are two highly conserved regions, designated A and B, within the pocket. We found that these two regions interact with one another to form the repressor motif. And more importantly, this A-B interaction is disrupted by the hyper-phosphorylation catalyzed by G1 cdks, which blocks Rb function. Recently, the Rb pocket has been crystallized, and we will use this crystal structure along with our results on Rb structure/function to direct a targeted mutational analysis of structures in the Rb pocket. A second finding during the previous grant period was that Rb can repress transcription through two distinct mechanisms. When targeted to cell cycle promoters, it an bind surrounding transcription factors and block their activity. Additionally, Rb represses transcription by recruiting histone deacetylase to the promoter. Gene promoters can be wound into nucleosomes by histone octamers-this inhibits binding of transcription factors. Nucleosome assembly is inhibited by acetylation of the core histones, and histone deacetylase removes the acetyl groups, promoting nucleosome assembly and repressing transcription. We propose additional studies to further examine the mechanism of action and the role of Rb in cultured cells and transgenic mice.