Regulation of ADAM13 function during cranial neural crest migration

描述（由申请方提供）：颅神经嵴（CNC）细胞的迁移是胚胎发育期间面部和颌骨正确形成的关键过程。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重大差距。该提案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迁移需要细胞表面金属蛋白酶ADAM 13，其在CNC中具有双重功能。细胞外蛋白酶切割钙粘蛋白-11以使其粘附结构域从表面脱落以促进迁移，而ADAM 13的细胞质结构域被切割掉并调节细胞核中的基因表达。本研究的长期目标是阐明ADAM 13控制CNC细胞迁移的机制。本提案的目的是确定如何开展 胞质结构域受到调节。基于初步数据，假设是激酶GSK 3引发ADAM 13的胞质结构域，用于随后被Polo样激酶（Plk）磷酸化，并且这些事件对于ADAM 13的下游核活性是必需的。将通过确定Plk（目的1）和GSK 3（目的2）对CNC中ADAM 13功能的贡献来检验该假设。将采用体内迁移试验来确定迁移是否需要Plk和GSK 3，以及在缺乏Plk和GSK 3激酶活性的情况下，ADAM 13的组成型磷酸化形式是否可以促进CNC迁移。免疫沉淀和蛋白质印迹分析将用于检查阻断GSK 3或Plk磷酸化对胞质结构域的切割和核转位的影响，以及GSK 3磷酸化对Plk结合的要求。此外，将使用真实的时间PCR来分析ADAM 13的非磷酸化形式调节CNC中的基因表达的能力。这项研究的结果预计将确定GSK 3和Plk是否调节ADAM 13的活性，以及这如何有助于CNC迁移。这将更好地定义ADAM蛋白在发育过程中如何在特定组织中发挥作用，并最终提供对CNC细胞体内迁移机制的更清晰理解。 公共卫生相关性：这项拟议的研究与公共卫生有关，因为它解决了阐明促进颅面发育的分子过程的关键需求。提高我们对颅神经嵴迁移是如何控制的理解，将为颅面异常如何发生以及如何预防或治疗提供有价值的见解。此外，增加我们对细胞迁移中ADAM功能的了解也是相关的，因为它们通常在侵袭性癌症中上调。

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. PUBLIC HEALTH RELEVANCE: The proposed study is relevant to public health because it addresses a critical need to elucidate the molecular processes that contribute to craniofacial development. Improving our understanding of how cranial neural crest migration is controlled will provide valuable insight into how craniofacial abnormalities can arise and potentially be prevented or treated. In addition, increasing our knowledge of ADAM function in cell migration is also relevant as they are often upregulated in invasive cancers.