Characterizing early neural crest phosphoregulation using antiphosphatase targets

描述（由申请人提供）：神经嵴细胞是可迁移的、侵袭性多能细胞，形成多种脊椎动物结构，包括颅面骨骼。迄今为止，大多数关于早期神经嵴发育的研究都集中在外胚层中指定神经嵴祖细胞的转录因子网络上。然而，这些转录因子的表达并不能保证最终迁移为神经嵴细胞。此外，目前还不清楚迁移机制是如何部署和协调，以实现神经嵴细胞的复杂迁移行为。一种可能性是，翻译后修饰调节和多样化的神经嵴调节因子的活性，细胞分层和迁移。为了研究这种可能性，以前的工作将新蛋白Paladin定义为神经嵴发育所需的抗磷酸酶。作为揭示这种需求机制的第一步，双杂交筛选确定肌球蛋白重链9（MYH9）作为Paladin的候选磷酸化依赖性靶点。MYH9被磷酸化，调节细胞运动（包括转移），并与非综合征性唇腭裂相关。然而，MYH9尚未在神经嵴中表征。该应用测试了Paladin通过保护在神经嵴细胞中至关重要的靶蛋白如MYH9的磷酸化状态来调节神经嵴发育的假设。目的1通过基因和蛋白表达分析以及免疫共沉淀研究，证明MYH9及其与Paladin的相互作用与神经嵴细胞相关。目的2将确定神经嵴发育中需要MYH9作为前奏的事件，以确定MYH9磷酸化对这些功能的重要性。最后，目标3将可视化MYH9酪氨酸磷酸化，以评估Paladin对这种磷酸化的影响。总之，这些研究将确定MYH9在早期神经嵴发育中的作用，并将MYH9表征为神经嵴中由Paladin调控的磷酸化蛋白。这些结果将创建一个更清晰的了解早期神经嵴发育的分子机制，并将验证使用Paladin来识别由磷酸化调节的神经嵴蛋白。了解神经嵴在其正常的胚胎环境中的发育将使我们能够破译疾病中神经嵴通路的异常功能和激活。

DESCRIPTION (provided by applicant): Neural crest cells are migratory, invasive multipotent cells that form diverse vertebrate structures, including the craniofacial skeleton. To date, most research on early neural crest development has focused on the network of transcription factors that specifies neural crest progenitors in the ectoderm. However, expression of these transcription factors does not guarantee eventual migration as a neural crest cell. Furthermore, it is not understood how the migratory machinery is deployed and coordinated to achieve neural crest cells' complex migratory behaviors. One possibility is that post-translational modifications modulate and diversify the activity of neural crest regulatory factors as cells delaminate and migrate. To investigate this possibility, previous work defined the novel protein Paladin as an antiphosphatase that is required for neural crest development. As a first step in revealing the mechanism of this requirement, two-hybrid screening identified myosin heavy chain 9 (MYH9) as a candidate phosphorylation-dependent target of Paladin. MYH9 is phosphorylated, regulates cell motility (including metastasis), and associates with non-syndromic cleft lip and palate. However, MYH9 has not been characterized in the neural crest. This application tests the hypothesis that Paladin regulates neural crest development by protecting the phosphorylation status of target proteins like MYH9 that are crucial in neural crest cells. Aim 1 will demonstrate that MYH9 and its interaction with Paladin are relevant to neural crest cells through gene and protein expression analyses and co-immunoprecipitation studies. Aim 2 will determine the events in neural crest development that require MYH9 as a prelude to defining the importance of MYH9 phosphorylation for these functions. Finally, Aim 3 will visualize MYH9 tyrosine phosphorylation in order to evaluate the impact of Paladin on this phosphorylation. Altogether, these studies will determine the role of MYH9 in early neural crest development and characterize MYH9 as a phosphorylated protein regulated by Paladin in the neural crest. These outcomes will create a clearer understanding of the molecular mechanisms of early neural crest development and will validate the use of Paladin to identify neural crest proteins regulated by phosphorylation. Understanding neural crest development in its normal, embryonic context will allow us to decipher the abnormal function and activation of neural crest pathways in disease.