Macromolecular Interactions of t-Darpp and Darpp-32

描述(申请人提供)：在研究癌症耐药性的过程中，我们和其他人发现了一种称为t-DARPP(TDP)的蛋白质，它是耐药表型的关键成分。TDP的正常功能完全未知。DARPP-32(Dp32)是TDP的一种较长的异构体，因其在蛋白激酶A(PKA)信号转导中的作用而被广泛研究。Dp32通过蛋白质间的相互作用发挥作用；它是一种双功能蛋白，可以抑制蛋白磷酸酶-1(PP1)或蛋白激酶A，这取决于Dp32‘S在T34或T75残基的磷酸化状态。它在非神经细胞中的作用尚不清楚，但有证据表明，它可能会抑制细胞的生长和迁移。我们发表的研究表明，TDP是一种较短的蛋白质，通过激活PKA(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-蛋白质相互作用发挥作用。TDP缺乏与PP1相互作用所需的N-末端结构域，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信号具有拮抗作用，可能是通过对彼此的大分子相互作用产生显性-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。剖析这些相互作用将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)确定细胞裂解产物中Dp32和TDP的功能相关的大分子相互作用；2) 确定活细胞中是否存在功能相关的大分子相互作用；以及3)确定介导Dp32和TDP相互作用的物理性质和分子界面。

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.