Regulation of ADAM13 function during cranial neural crest migration

描述(由申请人提供)：在胚胎发育过程中，颅神经脊细胞的迁移是面部和颌骨正常形成的关键过程。这些细胞未能迁移到正确的位置可能会导致出生时严重的颅面畸形，但我们对如何预防或治疗这些缺陷的知识存在着显著的差距。这项建议旨在增加我们目前对数控细胞迁移及其调控机制的理解。在非洲爪哇中，细胞迁移需要细胞表面金属蛋白酶ADAM13，它在细胞表面具有双重功能。胞外的蛋白水解酶将钙粘素-11从细胞表面剥离，从而促进其迁移，而ADAM13的胞浆结构域则被切割并调节细胞核内的基因表达。本研究的长期目标是阐明ADAM13控制数控细胞迁移的机制。这项提案的目标是确定 胞质结构域受调控。根据初步数据，假设GSK3激活了ADAM13的胞浆结构域，随后被Polo样激酶(PLK)磷酸化，这些事件对ADAM13下游的核活动是必不可少的。这一假说将通过确定PLK(目标1)和GSK3(目标2)对ADAM13在计算机数控系统中的功能的贡献来检验。体内迁移分析将被用来确定PLK和GSK3是否需要迁移，以及在缺乏PLK和GSK3激酶活性的情况下，结构性磷酸化形式的ADAM13是否可以促进CNC迁移。免疫沉淀和Western印迹分析将检测GSK3或PLK阻断磷酸化对细胞质结构域的切割和核转位的影响，以及GSK3磷酸化对PLK结合的要求。此外，实时定量聚合酶链式反应将用于分析非磷酸化形式的ADAM13在计算机控制系统中调节基因表达的能力。这项研究的结果有望确定GSK3和PLK是否调节ADAM13的活性，以及这如何促进数控系统的迁移。这将更好地定义ADAM蛋白在发育过程中特定组织中的功能，并最终提供对体内CNC细胞迁移机制的更清晰的理解。

DESCRIPTION (provided by applicant): The migration of cranial neural crest (CNC) cells is a critical process for proper formation of the face and jaw during embryo development. Failure of these cells to migrate to the correct locations can result in severe craniofacial abnormalities at birth, yet there is a significant gap in our knowledge of how to prevent or treat these defects. This proposal aims to increase our current understanding of CNC cell migration and the mechanisms by which it is regulated. In Xenopus laevis, CNC migration requires the cell surface metalloprotease ADAM13, which has a dual function in the CNC. The extracellular protease cleaves Cadherin-11 to shed its adhesive domain from the surface to promote migration, while the cytoplasmic domain of ADAM13 is cleaved off and regulates gene expression in the nucleus. The long-term goal of this research is to elucidate the mechanisms by which ADAM13 controls CNC cell migration. The objective of this proposal is to determine how the activity of the cytoplasmic domain is regulated. Based on preliminary data, the hypothesis is that the kinase GSK3 primes the cytoplasmic domain of ADAM13 for subsequent phosphorylation by Polo-like kinase (Plk), and that these events are essential for the downstream nuclear activity of ADAM13. This hypothesis will be tested by determining the contribution of Plk (Aim 1) and GSK3 (Aim 2) to the function of ADAM13 in the CNC. In vivo migration assays will be employed to determine if Plk and GSK3 are required for migration and if constitutively phosphorylated forms of ADAM13 can promote CNC migration in the absence of Plk and GSK3 kinase activity. Immunoprecipitation and western blot analysis will be used to examine the effect of blocking phosphorylation by GSK3 or Plk on both the cleavage and nuclear translocation of the cytoplasmic domain, as well as the requirement of GSK3 phosphorylation on Plk binding. Additionally, real time PCR will used to analyze the ability of non-phosphorylatable forms of ADAM13 to regulate gene expression in the CNC. The results of this study are expected to determine if GSK3 and Plk regulate the activity of the ADAM13 and how this contributes to CNC migration. This will better define how ADAM proteins function in specific tissues during development and ultimately provide a clearer understanding of the mechanisms of CNC cell migration in vivo.