DYRK1A interaction network in development and disease

DYRKA 1（Dual-specificity tyrosine-regulated kinase 1A，双特异性酪氨酸调节激酶1A）是一个位于酪氨酸激酶关键区域的基因。 21号染色体，过量的编码蛋白质被认为有助于唐氏症的许多方面。 综合征相关的出生缺陷，包括颅面畸形。在子宫内调节DYRK 1A的功能 已经被提出作为一种方法来改善唐氏综合症儿童的结果。然而，不受限制 由于DYRK 1A是一种肿瘤抑制因子，因此这种蛋白激酶的抑制引起了主要的关注。因此，我们理想地 需要开发能够在胚胎发育过程中更精确地操纵DYRK 1A的工具， 导致有害的副作用。我们已经确定了两种蛋白质，LZTS 2和SIPA 1 L1，它们形成了一种新的调节蛋白。 与DYRK 1A复合。这个探索性项目的主要目标是测试这两个新的候选人是否 DYRK 1A调节剂确实可以调节胚胎中这种重要激酶的功能。我们建议 整合人类细胞中的生物化学测定和发育模型非洲爪蟾体内研究的使用 laevis，以更好地理解LZTS 2和SIPA 1 L1对DYRK 1A功能的分子作用。爪蟾 这三种蛋白质的同源物显示出与它们的人类对应物（包括Lzts 2）的高度序列同一性 (50.2%）、Sipa 1 l1（75.9%）和Dyrk 1a（92.4%）。我们的初步研究和已发表的文献表明， 将这些蛋白质转化为各种生物的发育过程。然而， 它们的相互作用尚未在任何模型系统中进行研究。在这里，我们建议填补这一知识空白， 这是理解和潜在地减轻颅面异常的第一步， 唐氏综合症。在Aim 1中，我们将确定Lzts 2和Sipa 1 l1是否在颅面过程中与Dyrk 1a相互作用。 在Xenopus的发展。我们将评估Lzts 2、Sipa 1 l1和Dyrk 1a是否在同一通路中， 表型修饰物测定。然后，我们将确定是否修改这些蛋白质的水平可以改变 使用Litovchick实验室开发的体外方法在胚胎中检测Dyrk 1a的活性。此外，为了 在开发非洲爪蟾作为唐氏综合症研究的工具时，我们将询问是否抑制Lzts 2和/或Sipa 1 l1 可以补偿Dyrk 1a过量。在目的2中，我们将描述LZTS 2和SIPA 1 L1介导的 DYRK 1A在哺乳动物细胞中的调节，并验证我们在胚胎中的发现。人类细胞的初步数据 表明LZTS 2促进DYRK 1A活性，可能是通过增强DYRK 1A在一个新的磷酸化位点的磷酸化， 通过未知的机制。我们将通过测试LZTS 2是否需要SIPA 1 L1来扩展这些数据 介导的活性和磷酸化。我们将探讨DYRK 1A的这种和其他修改的性质， 需要LZTS 2和SIPA 1 L1。最后，在这一目标中，LZTS 2-SIPA 1 L1调节的磷酸化的作用 还将在胚胎中探索位点。本研究的结果将为未来的R 01项目奠定基础 在那里，我们将专注于这种新的DYRK 1A调控复合物的机制研究，其整合到其他 发育信号通路和治疗测试使用非洲爪蟾和小鼠模型。

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.