Retinal Patterning: A role for protein kinase CK2

视网膜图案：蛋白激酶 CK2 的作用

• 批准号: 6945138
• 负责人: ASHOK BIDWAI
• 金额: $ 21.9万
• 依托单位: WEST VIRGINIA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-01 至 2007-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6945138
• 关键词: Drosophilidae; RNA interference; arthropod genetics; biological models; biological signal transduction; cell differentiation; developmental genetics; enzyme activity; enzyme mechanism; gene induction /repression; phosphorylation; protein kinase; protein protein interaction; retina; retinal ganglion

DESCRIPTION (provided by applicant): During retinal patterning a diverse array of cell fates are specified with remarkable precision through a complex interplay of signaling pathways, molecular factors, and mechanisms that are highly conserved. An example is the process by which the 'founding' neural cell type, the R8-precursor, is specified in Drosophila. Notch signaling mediates this specification in a wave of differentiation, the morphogenetic furrow that sweeps across the eye disc. In an early phase, Notch elicits a broad expression of the basic-helix-loop-helix (bHLH) transcription factor Atonal. Later, Notch elicits expression of bHLH repressors encoded by the Enhancer of Split, E(spl) complex that antagonize Atonal in all but the 'founding' R8 cells. This restriction of Atonal is critical for refining the number of R8 cells, and its perturbation interferes with proper eye development. E(spl)m8 mediates this 'refinement', because its over expression and mutation in the E(spl)D allele (encoding M8*) severely antagonizes Atonal and results in reduced eyes. We have shown that the conserved protein kinase CK2 phosphorylates E(spl)M8 at Ser159. We now demonstrate that replacement of Ser159 with Asp (M8SD) elicits a reduced eye phenotype that mimics the eye phenotype and mechanism of E(spl)D. We hypothesize that phosphorylation by CK2 permits E(spl)M8 to antagonize Atonal and is, therefore, critical for proper retinal patterning. We propose three specific aims to test this hypothesis. (1) Analyze the residual ommatidia of flies expressing M8SD, define the mechanism of its dominant behavior via over expression of Atonal, and define the role of CK2 via analysis of CK2 mutants, RNA-interference/mosaics, and over expression of CK2 in R8-precursors. (2) Test the hypothesis that phosphorylation stabilizes M8 and/or mediates hierarchical phosphorylation. (3) Test the hypothesis that CK2 phosphorylation switches M8 into an active repressor of Atonal, and identify phosphatases that counteract CK2. Our studies are likely to be relevant to humans, because specification of fly R8 photoreceptors and mammalian retinal ganglion cells involve similar mechanisms, and, as in the E(spl)M8-Atonal interaction, CK2 activity mediates interaction of Hes6-Hesl, two vertebrate bHLH proteins involved with rod-photoreceptor specification. Our studies will provide a better understanding of the roles of CK2 in this process, and may provide insights into how misregulated cell fate specification contributes to eye defects.

描述（由申请人提供）：在视网膜模式期间，通过信号通路，分子因子和高度保守的机制的复杂相互作用，指定了各种细胞命运的多种细胞命运。一个例子是果蝇中指定的“创建”神经细胞类型，R8-驱虫器的过程。 Notch信号传导在分化波中介导了这种规范，即横穿眼盘的形态发生沟。在早期阶段，Notch引起了基本螺旋 - 环螺旋（BHLH）转录因子的广泛表达。后来，Notch引发了由拆分，E（SPL）复合物的增强子编码的BHLH阻遏物的表达，除了“基础” R8细胞以外，所有均具有拮抗utonal的表达。对亚顿的这种限制对于完善R8细胞的数量至关重要，并且其扰动会干扰适当的眼睛发育。 E（SPL）M8介导了这种“改进”，因为它在E（SPL）D等位基因（编码M8*）中的表达和突变严重拮抗了atonal并导致眼睛减少。我们已经表明，保守的蛋白激酶CK2在Ser159处磷酸化E（SPL）M8。现在，我们证明，用ASP（M8SD）替换Ser159引起了模仿E（SPL）d的眼表型和机制的减少眼表型。我们假设CK2磷酸化允许E（SPL）M8对抗atonal，因此对于正确的视网膜模式至关重要。我们提出了三个特定的目的来检验这一假设。 （1）分析表达M8SD的苍蝇的残留 - 瘤，通过过度表达来定义其主导行为的机制，并通过分析CK2突变体，RNA介入/马赛克，以及CK2在R8-代表中的表达来定义CK2的作用。 （2）检验磷酸化稳定M8和/或介导层次磷酸化的假设。 （3）检验CK2磷酸化将M8切成Atonal的活性阻遏物，并鉴定抵抗CK2的磷酸酶的假设。我们的研究可能与人类有关，因为蝇R8光感受器和哺乳动物视网膜神经节细胞的规范涉及相似的机制，并且就像在E（SPL）M8-芳族相互作用中一样，CK2的CK2活性介导了Hes6-Hesl的相互作用，两种脊椎动物BHLH蛋白涉及Rod-Phod-Phodphotorepeptififie。我们的研究将更好地理解CK2在此过程中的作用，并可以提供有关细胞命运规格如何导致眼睛缺陷的见解。