Macromolecular Interactions of t-Darpp and Darpp-32

t-Darpp 和 Darpp-32 的大分子相互作用

基本信息

批准号：
9121583
负责人：
SUSAN E KANE
金额：
$ 30.71万
依托单位：
BECKMAN RESEARCH INSTITUTE/CITY OF HOPE
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-09-01 至 2017-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9121583
关键词：
Address Affect Affinity Affinity Chromatography Binding Binding Proteins Biological Assay Biosensor Cancer cell line Cell Proliferation Cells Cellular biology Complex Confocal Microscopy Cultured Cells Cyclic AMP-Dependent Protein Kinases Cytostatics Cytotoxic agent Development Dissociation Dominant-Negative Mutation Drug resistance Epitopes Equilibrium Fluorescence Resonance Energy Transfer Future Growth Health Image Immunoprecipitation Investigation Knowledge Life Malignant Neoplasms Maps Mass Spectrum Analysis Mediating Molecular Molecular Biology Mutate N-terminal Neuroglia Neurons Normal Cell Normal tissue morphology Pathway interactions Phase Phenotype Phosphoric Monoester Hydrolases Phosphorylation Phosphotransferases Protein Biochemistry Protein Isoforms Protein phosphatase Proteins Proteomics Publishing Recombinants Regulation Research Research Personnel Resistance Role Scheme Signal Transduction Structure Surface Plasmon Resonance Work base biophysical techniques cell growth cell motility cell type inhibitor/antagonist innovation insight interest kinase inhibitor mutant neurotransmission overexpression phosphatase inhibitor phosphoproteomics physical property protein function protein kinase A kinase protein protein interaction

项目摘要

DESCRIPTION (provided by applicant): In the course of studying drug resistance in cancer, we and others identified a protein called t-Darpp (tDp) as a key component of the resistance phenotype. The normal function of tDp is completely unknown. Darpp-32 (Dp32) is a longer isoform of tDp that is well studied for its role in protein kinase A (PKA) signaling in dopaminergi neurotransmission. Dp32 works by protein-protein interaction; it is a dual-function protein that inhibits either protein phosphatase-1 (PP1) or PKA, depending on Dp32's phosphorylation status at residues T34 or T75. Its role in non-neuronal cells is unclear, but there is evidence to suggest that it might inhibit cell growth and migration. Our published studies suggest that tDp, the shorter protein, promotes cell proliferation (not inhibition) via PKA activation (not inhibitio). The molecular mechanism of this effect has not been determined, but it presumably also functions via protein-protein interaction. tDp lacks the N-terminal domain that is required for interaction with PP1, but phosphorylation at T75 (using the Dp32 numbering scheme) appears to be required for its activity in promoting cell proliferation. Thus, Dp32 and tDp seem to have antagonistic effects on cell growth and on PKA signaling, possibly via dominant-negative effects on each other's macromolecular interactions. We hypothesize that the physical and functional interaction of tDp, Dp32, and the PKA signaling network helps determine the balance between cell growth and growth inhibition. To address this hypothesis we need a greater understanding of Dp32 and tDp physical interactions in cells and the relationship between those interactions and protein function. Dissecting these interactions will require the contribution of multiple investigators with specialized expertise in molecular biology, cell biology, and protein biochemistry; the application of contemporary proteomic strategies and biophysical methods; and the development of innovative imaging-based approaches. We propose three specific aims: 1) Identify functionally- relevant macromolecular interactions for Dp32 and tDp in cell lysates; 2) Determine if functionally relevant macromolecular interactions occur in live cells; and 3) Determine the physical properties and molecular interfaces mediating Dp32 and tDp interactions.

描述（由申请人提供）：在研究癌症中耐药性的过程中，我们和其他人将一种称为T-DARPP（TDP）的蛋白质确定为抗性表型的关键组成部分。 TDP的正常功能是完全未知的。 DARPP-32（DP32）是TDP的更长的同工型，它在多巴胺神经传递中的蛋白激酶A（PKA）信号中的作用进行了很好的研究。 dp32通过蛋白质 - 蛋白质相互作用起作用；这是一种双功能蛋白，可抑制蛋白质磷酸酶-1（PP1）或PKA，具体取决于DP32在残基T34或T75时的磷酸化状态。它在非神经元细胞中的作用尚不清楚，但是有证据表明它可能抑制细胞的生长和迁移。我们发表的研究表明，TDP（较短的蛋白质）通过PKA激活（不是抑制）促进细胞增殖（不是抑制）。该作用的分子机制尚未确定，但大概也通过蛋白质 - 蛋白质相互作用起作用。 TDP缺乏与PP1相互作用所需的N末端结构域，但是在T75处的磷酸化（使用DP32编号方案）似乎是其在促进细胞增殖中的活性所必需的。因此，DP32和TDP似乎对细胞生长和PKA信号传导具有拮抗作用，这可能是通过对彼此的大分子相互作用的显性阴性作用。我们假设TDP，DP32和PKA信号网络的物理和功能相互作用有助于确定细胞生长与生长抑制之间的平衡。为了解决这一假设，我们需要对细胞中的DP32和TDP物理相互作用以及这些相互作用与蛋白质功能之间的关系有更深入的了解。解剖这些相互作用将需要多个研究人员在分子生物学，细胞生物学和蛋白质生物化学方面具有专业专业知识的贡献。当代蛋白质组学策略和生物物理方法的应用；以及基于创新成像的方法的发展。我们提出了三个具体目的：1）识别细胞裂解物中DP32和TDP的功能相关的大分子相互作用； 2）确定活细胞中是否发生了与功能相关的大分子相互作用； 3）确定介导DP32和TDP相互作用的物理特性和分子界面。