DYRK1A interaction network in development and disease

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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.