GROWTH-REGULATORY TARGETS OF DROSOPHILA CYCLIND/CDK4

Cyclin D1/p16ink4a/pRB通路的失调是人类癌症中最常见的缺陷之一。定义这一途径的基因被描述为g1特异性细胞周期调节因子，它们的致癌特性归因于这一功能。果蝇周期蛋白D激酶（CycD/Cdk4）不是特异性G1/S调节因子。相反，它促进细胞质量的积累（生长），而这反过来又在某些情况下促进细胞增殖。我们提出了几种平行的方法来确定果蝇中CycD/Cdk4的关键生长调控靶点。差异凝胶电泳（DIGE）将用于绘制由CycD/Cdk4过表达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被用于分离体内的CycD/Cdk4底物。体内转录受过表达CycD/Cdk4影响的基因将使用代表大约9000个果蝇mrna的DNA微阵列进行鉴定。快速F1基因筛选将确定抑制或增强由异位CycD/Cdk4引起的果蝇眼睛肥大的基因。这些研究应该确定哪些基因和蛋白质是CycD/Cdk4的底物、激活因子或抑制因子。在确定靶点后，将利用分子和遗传学方法来表征这些靶点与CycD/Cdk4的相互作用，并在果蝇体内测试它们在细胞生长中的功能。这些研究将揭示CycD/Cdk4如何改变细胞生理以促进生长，并提供解释细胞生长和细胞分裂如何耦合的分子范式。果蝇生长调控基因的鉴定也将为人类正常和肿瘤发育过程中细胞生长控制的研究提供切入点。

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.