DYRK1A interaction network in development and disease

摘要：DYRKA1(双特异性酪氨酸调节激酶1A)是一种位于 21号染色体，过量的编码蛋白被认为与唐氏综合症的许多方面有关 综合征相关的出生缺陷，包括头面部畸形。DYRK1A在子宫内的功能调控 已经被提出作为一种改善唐氏综合症儿童预后的方法。然而，不受限制 由于DYRK1A是一种肿瘤抑制因子，因此对该蛋白激酶的抑制引起了人们的极大关注。因此，理想情况下，我们 需要开发能够在胚胎发育期间更精确地操作DYRK1A的工具，而不需要 造成有害的副作用。我们已经鉴定出两种蛋白，LZTS2和SIPA1L1，它们形成了一种新的调控蛋白 DYRK1A型复合体。这个探索性项目主要目标是测试这两个新候选人 DYRK1A型调节剂确实可以调节胚胎中这一重要激酶的功能。我们建议 结合在人类细胞中的生化分析和在发育模型非洲爪哇的体内研究 为了更好地了解LZTS2和SIPA1L1在DYRK1A功能中的分子作用。非洲爪哇 这三种蛋白的同源物显示出与其人类同源蛋白高度序列的同源性，包括Lzts2 (50.2%)、Sipa1l1(75.9%)和Dyrk1a(92.4%)。我们的初步研究和出版的文献都牵涉到 这些蛋白质在不同生物体中的发育过程。然而，生理学意义在于 它们的相互作用在任何模型系统中都没有被研究过。在这里，我们建议填补这一知识空白，因为 了解和潜在地缓解与以下疾病相关的颅面畸形的第一步 唐氏综合症。在Aim1中，我们将确定Lzts2和Sipa1l1是否在颅面部与Dyrk1a相互作用 非洲爪哇的发展。我们将评估Lzts2、Sipa1l1和Dyrk1a是否处于相同的途径中 表型修饰物分析。然后，我们将确定改变这些蛋白质的水平是否会改变 利用Litovchick实验室开发的体外方法研究Dyrk1a在胚胎中的活性。此外，目的是 将非洲爪哇发展为唐氏综合症研究的工具，我们将询问是否抑制Lzts2和/或Sipa1l1 可以补偿Dyrk1a的过量。在目标2中，我们将描述LZTS2和SIPA1L1介导的机制 在哺乳动物细胞中调节DYRK1A，并在胚胎中验证我们的发现。人类细胞的初步数据 表明LZTS2促进了DYRK1A的活性，可能是通过促进DYRK1A在一个新的 网站被未知的机制。我们将通过测试LZTS2是否需要SIPA1L1来扩展这些数据 介导的活性和磷酸化。我们将探讨DYRK1A的这一修改和其他修改的性质 需要LZTS2和SIPA1L1。最后，在这一目标中，LZTS2-SIPA1L1调节的磷酸化的作用 还将在胚胎中探索S的位置。这项研究的结果将为未来的R01项目奠定基础 在那里我们将专注于对这个新的DYRK1A监管复合体的机械研究，它与其他 利用非洲爪哇和小鼠模型进行发育信号通路和治疗测试。

Summary: DYRKA1 (Dual-specificity tyrosine-regulated kinase 1A) is a gene within the critical region of chromosome 21, and excess of the encoded protein is thought to contribute to many facets of the Down Syndrome associated birth defects, including craniofacial anomalies. Modulating the function of DYRK1A in utero has been proposed as a method to improve outcomes for children with Down Syndrome. However, unrestricted inhibition of this protein kinase poses major concerns since DYRK1A is a tumor suppressor. Therefore, we ideally need to develop the tools that can more precisely manipulate DYRK1A during embryonic development without causing harmful side effects. We have identified two proteins, LZTS2 and SIPA1L1, that form a novel regulatory complex with DYRK1A. The broad goal of this exploratory project is to test whether these two NEW candidate DYRK1A regulators can indeed modulate the function of this important kinase in the embryo. We propose to integrate the use of biochemical assays in human cells and in vivo studies in the developmental model, Xenopus laevis, to better understand the molecular effects of LZTS2 and SIPA1L1 on DYRK1A function. Xenopus homologues of these three proteins display high sequence identity to their human counterparts, including Lzts2 (50.2%), Sipa1l1 (75.9%) and Dyrk1a (92.4%). Our preliminary studies and published literature implicate each of these proteins into developmental processes in various organisms. However, the physiological significance of their interaction has not been investigated in any model system. Here, we propose to fill this knowledge gap as a first step towards the understanding and potentially alleviating the craniofacial abnormalities associated with Down syndrome. In Aim1 we will determine if Lzts2 and Sipa1l1 interact with Dyrk1a during craniofacial development in Xenopus. We will assess whether Lzts2, Sipa1l1 and Dyrk1a are in the same pathway using a phenotypic modifier assay. Then, we will determine whether modifying the levels of these proteins can alter the activity of Dyrk1a in the embryo using an in vitro approach developed in the Litovchick lab. Also, with the aim of developing Xenopus as a tool for Down syndrome research we will ask whether inhibition of Lzts2 and/or Sipa1l1 can compensate for Dyrk1a excess. In Aim 2 we will characterize mechanisms of LZTS2 and SIPA1L1 mediated regulation of DYRK1A in mammalian cells and validate our findings in embryos. Preliminary data in human cells indicates that LZTS2 promotes DYRK1A activity, possibly by enhancing phosphorylation of DYRK1A at a novel site by unknown mechanism. We will extend these data by testing whether SIPA1L1 is required for LZTS2 mediated activity and phosphorylation. We will explore the nature of this and other modifications of DYRK1A that require LZTS2 and SIPA1L1. Finally, in this aim, the role of the LZTS2-SIPA1L1-regulated phosphorylation site(s) will also be explored in embryos. The outcomes of this study will set the stage for a future R01 project where we will focus on mechanistic studies of this new DYRK1A regulatory complex, its integration into other developmental signaling pathways and therapeutic testing using both Xenopus and mouse models.