GROWTH-REGULATORY TARGETS OF DROSOPHILA CYCLIND/CDK4

细胞周期蛋白D1/p16 ink 4a/pRB通路的失调是人类癌症中最常见的缺陷之一。 定义该途径的基因已被表征为G1特异性细胞周期调节因子，并且其致癌特性归因于该功能。 果蝇细胞周期蛋白D激酶（CycD/Cdk 4）不是一个特异的G1/S期调节因子。 相反，它促进细胞质量积累（生长），这反过来又在某些情况下促进细胞增殖。 我们提出了几个平行的方法来确定关键的生长调节CycD/Cdk 4在果蝇的目标。差异凝胶电泳（DIGE）将用于绘制由过表达的CycD/Cdk 4引起的细胞蛋白质组成（如蛋白质磷酸化）的变化。 使用ATP类似物的高度特异性技术将用于分离CycD/Cdk 4的体内底物。 将使用代表约9000个果蝇mRNA的DNA微阵列鉴定其转录受体内过表达CycD/Cdk 4影响的基因。 快速F1基因筛选将识别抑制或增强由异位CycD/Cdk 4引起的果蝇眼睛肥大的基因。这些研究应确定基因和蛋白质的底物，激活剂，或抑制剂的CycD/Cdk 4。在靶点鉴定之后，将使用分子和遗传学方法来表征靶点与CycD/Cdk 4的相互作用，并测试它们在果蝇体内细胞生长中的功能。 这些研究将揭示CycD/Cdk 4如何改变细胞生理学以促进生长，并提供一个分子范式来解释细胞生长和细胞分裂是如何耦合的。 果蝇生长调节基因的鉴定也为人类正常和肿瘤发育过程中细胞生长控制的研究提供了切入点。

Deregulation of the Cyclin D1/p16ink4a/pRB pathway is one of the most common defects in human cancers. The genes defining this pathway have been characterized as G1-specific cell cycle regulators, and their oncogenic properties attributed to this function. Drosophila Cyclin D kinase (CycD/Cdk4) is not a specific G1/S regulator. Rather, it promotes cell mass accumulation (growth), and this in turn promotes cell proliferation in some contexts. We propose several parallel approaches for identifying critical growth regulatory targets of CycD/Cdk4 in Drosophila. Difference gel electrophoresis (DIGE) will be used to map alterations in cellular protein composition (such as protein phosphorylation) caused by overexpressed CycD/Cdk4. A highly specific technique using ATP analogs will be employed to isolate in vivo substrates of CycD/Cdk4. Genes whose transcription is affected in vivo by overexpressed CycD/Cdk4 will be identified using DNA microarrays representing approximately 9000 Drosophila mRNAs. Rapid F1 genetic screens will identify genes that suppress or enhance hypertrophy of the Drosophila eye caused by ectopic CycD/Cdk4. These studies should identify genes and proteins that are substrates, activators, or suppressors of CycD/Cdk4. Following target identification, molecular and genetic methods will be used to characterize interactions of the targets with CycD/Cdk4, and test their functions in cell growth in vivo in Drosophila. These studies should reveal how CycD/Cdk4 alters cell physiology to promote growth, and provide a molecular paradigm explaining how cell growth and cell division are coupled. The identification of growth regulatory genes in flies should also provide entry points for studies of cell growth control in humans during both normal and neoplastic development.