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Deciphering the code of RB phosphorylation

破译RB磷酸化密码

基本信息

批准号：
10187530
负责人：
NICHOLAS J DYSON
金额：
$ 50.32万
依托单位：
MASSACHUSETTS GENERAL HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-07-01 至 2023-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10187530
关键词：
Affect Alleles Binding Cell Culture Techniques Cell Proliferation Cells Cellular Stress Classification Code Communities Complex Cyclin-Dependent Kinases Data Elements Epitopes Event Future Gene Expression Profile Gene Targeting Genetic Predisposition to Disease Genetic Transcription Genomics Human Individual Knowledge Laboratories Lesion Literature Location Malignant Neoplasms Maps Mass Spectrum Analysis Mediating Methods Modeling Molecular Mutation Normal Cell Phosphorylation Phosphorylation Site Play Property Protein Isoforms Proteins Proteomics RB1 gene Reagent Reporting Research Retinoblastoma Retinoblastoma Protein Role Series Set protein Site Somatic Mutation Testing Time Transcriptional Regulation Tumor Suppressor Genes Tumor Suppressor Proteins United States Viral Oncogene Virus Inactivation Work base cancer cell experimental study genomic locus information framework molecular modeling mortality mutant prevent programs protein complex protein purification prototype recruit response retinoblastoma tumor suppressor tool tumor tumor progression tumorigenesis

项目摘要

Project summary For simplicity we tend to imagine that most proteins have a single mechanism of action. The retinoblastoma tumor suppressor is an example of a protein that defies this simple classification. RB1 is functionally inactivated in most human cancers and the molecular properties of its protein product (RB) have been studied intensively. Despite this research, RB's mechanism of action has remained an enigma. RB has been reported to physically associate with hundreds of proteins and many different interactions have been proposed to contribute to its tumor suppressive properties. The RB research community is faced with a conundrum: which of these interactions are real, which are not, and how could one protein co-ordinate its effects on so many potential targets? Recent studies from several laboratories have suggested that the answers to these questions lie in a code of RB phosphorylation. The concept is that normal cells do not contain a single form of RB, but that differential phosphorylation generates multiple isoforms of RB that have different binding properties and, presumably, perform different roles. In essence, the action of RB is tailored by phosphorylation. RB is known to have 14 sites of CDK phosphorylation. We have recently developed methods that allow us to use mass spectrometry-based proteomics to profile RB complexes. We have also generated panels of isogenic cell cultures in which we can replace the endogenous RB protein with mutant RB proteins that contain just a single cdk phosphorylation site, or a single phospho-mimicking mutation. In this application we propose to use these tools to decipher this code of RB phosphorylation. In Aim 1 we will use state-of-the-art proteomics to define the binding properties of each of the mono-phosphorylated isoforms of RB. In Aim 2 we will identify the functional consequences of these interactions by identifying the transcriptional programs that they control and by genomic loci that they target. Using this binding information and transcription profiles we will identify the molecular interactions that allow specific mono-phosphorylated isoforms of RB to control distinct programs of transcription. Together these experiments will generate a framework of molecular information that is critical to be able to understand RB's mechanism of action.

项目总结