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）：在研究癌症耐药性的过程中，我们和其他人鉴定了一种称为 t-Darpp (tDp) 的蛋白质，它是耐药表型的关键组成部分。 tDp 的正常功能完全未知。 Darpp-32 (Dp32) 是 tDp 的较长亚型，因其在多巴胺能神经传递中的蛋白激酶 A (PKA) 信号转导中的作用而得到充分研究。 Dp32 通过蛋白质-蛋白质相互作用发挥作用；它是一种双功能蛋白，可抑制蛋白磷酸酶-1 (PP1) 或 PKA，具体取决于 Dp32 在残基 T34 或 T75 处的磷酸化状态。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，较短的蛋白质 tDp 通过 PKA 激活（而非抑制）促进细胞增殖（而非抑制）。这种效应的分子机制尚未确定，但它可能也通过蛋白质-蛋白质相互作用发挥作用。 tDp 缺乏与 PP1 相互作用所需的 N 端结构域，但 T75 处的磷酸化（使用 Dp32 编号方案）似乎是其促进细胞增殖的活性所必需的。因此，Dp32 和 tDp 似乎对细胞生长和 PKA 信号传导具有拮抗作用，可能是通过对彼此大分子相互作用的显性负效应而实现的。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 Dp32 和 tDp 在细胞中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。剖析这些相互作用需要具有分子生物学、细胞生物学和蛋白质生物化学专业知识的多名研究人员的贡献；当代蛋白质组学策略和生物物理方法的应用；以及基于成像的创新方法的开发。我们提出了三个具体目标：1）识别细胞裂解物中 Dp32 和 tDp 功能相关的大分子相互作用； 2） 确定活细胞中是否发生功能相关的大分子相互作用； 3) 确定介导 Dp32 和 tDp 相互作用的物理性质和分子界面。