Macromolecular Interactions of t-Darpp and Darpp-32

t-Darpp 和 Darpp-32 的大分子相互作用

• 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
• 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

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）：在研究癌症耐药的过程中，我们和其他人发现了一种名为t-Darpp （tDp）的蛋白质，它是耐药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的一个较长的异构体，其在多巴胺能神经传递中的蛋白激酶a （PKA）信号传导中的作用得到了很好的研究。Dp32通过蛋白相互作用起作用；它是一种双重功能蛋白，可以抑制蛋白磷酸酶-1 （PP1）或PKA，这取决于Dp32在T34或T75残基的磷酸化状态。它在非神经元细胞中的作用尚不清楚，但有证据表明它可能抑制细胞生长和迁移。我们发表的研究表明，短蛋白tDp通过PKA激活（而不是抑制）促进细胞增殖（而不是抑制）。这种作用的分子机制尚未确定，但它可能也通过蛋白质-蛋白质相互作用起作用。tDp缺乏与PP1相互作用所需的n端结构域，但T75位点的磷酸化（使用Dp32编号方案）似乎是其促进细胞增殖活性所必需的。因此，Dp32和tDp似乎对细胞生长和PKA信号传导具有拮抗作用，可能是通过对彼此大分子相互作用的显性-负性作用。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和生长抑制之间的平衡。为了解决这一假设，我们需要更好地了解细胞中Dp32和tDp的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。解剖这些相互作用将需要多个具有分子生物学、细胞生物学和蛋白质生物化学专业知识的研究人员的贡献；现代蛋白质组学策略和生物物理方法的应用以及基于成像的创新方法的发展。我们提出了三个具体目标：1)确定细胞裂解物中Dp32和tDp的功能相关大分子相互作用；2）