Regulation of ADAM13 function during cranial neural crest migration

描述（申请人提供）：颅神经嵴（CNC）细胞的迁移是胚胎发育过程中面部和颌骨正确形成的关键过程。这些细胞迁移到正确位置的失败可能导致出生时严重的颅面畸形，然而，在我们如何预防或治疗这些缺陷的知识方面存在重大差距。该建议旨在增加我们目前对CNC细胞迁移及其调节机制的理解。在非洲爪蟾中，CNC的迁移需要细胞表面金属蛋白酶ADAM13，而ADAM13在CNC中具有双重功能。细胞外蛋白酶裂解Cadherin-11使其粘附结构域从表面脱落，促进迁移，而ADAM13胞质结构域被裂解，调控细胞核内基因表达。本研究的长期目标是阐明ADAM13控制CNC细胞迁移的机制。这一建议的目的是确定如何开展活动

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.