GROWTH-REGULATORY TARGETS OF DROSOPHILA CYCLIND/CDK4

Cyclin D1/p16INK4a/pRb通路失控是人类肿瘤中最常见的缺陷之一。定义这一途径的基因被描述为G1特异性的细胞周期调节因子，它们的致癌特性归因于这一功能。果蝇细胞周期蛋白D激酶(Cycd/CDK4)不是一种特异性的G1/S调节因子。相反，它促进了细胞的大量积累(生长)，进而在某些情况下促进了细胞的增殖。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。差异凝胶电泳法(DGE)将被用来定位由过度表达的CyCD/CDK4引起的细胞蛋白质组成的变化(如蛋白质磷酸化)。将使用一种使用ATP类似物的高度特异的技术来分离体内的CyCD/CDK4底物。在体内被过度表达的CyCD/CDK4影响转录的基因将使用代表大约9000个果蝇mRNAs的DNA微阵列来鉴定。快速的F1基因筛查将识别抑制或增强由异位CyCD/CDK4引起的果蝇眼睛肥大的基因。这些研究应该确定作为CyCD/CDK4底物、激活物或抑制物的基因和蛋白质。在靶标确定后，将使用分子和遗传学方法来表征靶标与CyCD/CDK4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/CDK4如何改变细胞生理以促进生长，并提供一个解释细胞生长和细胞分裂如何耦合的分子范式。苍蝇生长调节基因的识别也应该为研究人类正常和肿瘤发育过程中的细胞生长控制提供切入点。

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.