Award details

Functional analysis of a novel Rapgef5 mediated nuclear transport system

Reference	BB/T003766/1
Principal Investigator / Supervisor	Professor Grant Wheeler
Co-Investigators / Co-Supervisors	Dr John Griffin
Institution	University of East Anglia
Department	Biological Sciences
Funding type	Research
Value (£)	472,387
Status	Current
Type	Research Grant
Start date	01/12/2019
End date	30/06/2023
Duration	43 months

Abstract

Canonical Wnt signaling coordinates many critical aspects of embryonic development and adult tissue homeostasis. The nuclear localization of beta-catenin is the defining step in pathway activation, yet despite decades of intense investigation, how beta-catenin gets into the nucleus remains a fundamental question in the field of Wnt signaling. Beta-catenin does not have a canonical nuclear localization signal nor does it use the well known importin-beta1/Ran mediated transport system. However, beta-catenin does enter through the nuclear pores and entry requires ATP/GTP. We recently discovered that RAPGEF5 and Rap GTPases are critical for beta-catenin nuclear transport, answering a fundamental question about the pathway. Indeed, our results suggest that these proteins constitute an independent alternative nuclear transport system. Here we propose to thoroughly investigate this system and its requirements in embryonic development. In particular, we will first use a combination of state of the art photoconvertible tags and biochemical assays to identify the key players and molecular logic underlying this system, as well as quantitate alterations to nuclear import and export when Rapgef5 is depleted. We will also use newly obtained mutant mice and Xenopus to determine developmental requirements for Rapgef5. Finally, we will investigate whether the Rapgef5 transport system is specific for beta-catenin or if it is also required for the nuclear transport of additional proteins. Together, these experiments will transform our understanding of the canonical Wnt signaling pathway and open new avenues of basic and therapeutic research.

Summary

For an embryo to develop normally, or an adult to function correctly, cells need to communicate with each other. The Wnt signaling pathway is an important cellular communication system that controls how cells behave during embryonic development and in adults. Disruption of Wnt signaling causes birth defects and diseases, including cancers. The critical step in the Wnt pathway requires the movement of a protein called beta-catenin into the nucleus. However, despite intensive research, the mechanisms that control the movement of beta-catenin remain mysterious. We discovered that a gene called Rapgef5 functions in a previously unknown nuclear transport system, which is required for the nuclear localization of beta-catenin. Loss of Rapgef5 function disrupts Wnt signaling and impairs development of the heart and face. Here, I propose to investigate exactly how this Rapgef5 transport system functions, to confirm its role in Wnt signaling, and to determine how defects in Rapgef5 function affect embryonic development. Furthermore, I will also investigate whether the new Rapgef5 nuclear transport system is required by other proteins that move in and out of the nucleus. By investigating a novel nuclear transport system for beta-catenin this research will transform our understanding of the Wnt signaling pathway and open up new avenues of basic and therapeutic research.

Impact Summary

This work investigates a novel nuclear transport system, required for canonical Wnt signaling during embryonic development. It will have a direct impact on many groups Basic Science Impact: This is a basic science project. It investigates 1) the nuclear transport of beta-catenin, a central mystery in the canonical Wnt signaling pathway, and 2) the workings of a previously unknow nuclear transport system. The existence of a Ran/Importin independent nuclear transport mechanism has long been speculated but remained unidentified until now. Due to the fundamental importance of nuclear transport and Wnt signaling, the project will have long term impact in numerous research fields, including developmental and cell biology, stem cells, regeneration, oncology and biotechnology. Impact for human (and animal) health: Increased Wnt activity often causes cancer. For example, colorectal cancer is the third most common cancer, affecting 9.4 million people in 2015. Strikingly, abnormal Wnt signaling causes over 90% of all colorectal cancer cases. In particular, different mutations (in APC for example) lead to cytoplasmic stabilization of beta-catenin, which allows it to enter the nucleus and activate a transcriptional program for uncontrolled growth. I have made the remarkable discovery that RAPGEF5 regulates beta-catenin nuclear translocation. As such, investigation of RAPGEF5 and its target RAP proteins may provide promising targets for the treatment of colorectal cancer. Benefit for congenital disease: The information gained from the phenotypic analysis will be shared with clinical geneticists. As genetic screening becomes more common, these functional insights can directly inform clinical decisions and allow development of personally tailored treatments for developmental defects. They also facilitate the provision of genetic counselling to patients and their families. This genetic counselling is immensely important, as understanding the cause of congenital anomalies can provide comfort to parents. Institutional and Workforce Impact: The award would support a newly established research group at UEA and allow for recruitment of a PDRA. The PDRA will receive three years of training, research experience, publications and career mentorship at a top UK university. The project will also strengthen ties between the Yale School of Medicine and the University of East Anglia. It is likely this work will result in high impact publications that will benefit both the researcher and institution, by increasing their profiles and facilitating the development of related projects and grant proposals. Impact for Pharma and Biotech: The results will greatly expand our understanding of nuclear transport and signal transduction, a critical step in rational drug design. Conclusion: The study will contribute to long term basic, translational and industrial science in the UK.

Committee	Research Committee C (Genes, development and STEM approaches to biology)
Research Topics	Stem Cells
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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