RaDAR guides nuclear membrane transport

Physiology 2014 (London, UK) (2014) Proc Physiol Soc 31, PCA118

Poster Communications: RaDAR guides nuclear membrane transport

M. Lu1, J. Zak1, X. Lu1

1. NDM department, University of Oxford, Ludwig Institute for Cancer Research, Oxford, United Kingdom.

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MOTIVATION Regulation of nuclear import is fundamental to eukaryotic biology. The only general nuclear import pathway identified in the past 30 years is the Nuclear Localization Signal (NLS) pathway, yet it cannot account for the entire nuclear proteome. The ankyrin repeat (AR) is a common protein structural motif. ARs containing proteins (ARPs) play pivotal roles in the development and maintenance of tissue homeostasis. Mutation and mislocalization of ARPs are associated with various human diseases. It is mysterious how ARPs without a NLS enter the nucleus and play their various physiological and pathological roles.METHODS 1) Nuclear iASPP is a key ARP maintaining tumor growth. We employed co-immunoprecipitation to search the potential nuclear transport factors involved in iASPP nuclear entry. Mutagenesis was applied to identify the essential residues (barcode), in iASPP, that bind nuclear transport factors and trigger nuclear import. 2) The barcode in human proteomic ARPs was determined by protein sequence alignment, aiming to predict their nuclear localization. 3) The barcode explained the mechanism behind p16 (a key ARP suppressing tumor) function in melanoma. Additionally, we screened for compounds that mask the iASPP barcode to block iASPP nuclear entry and kill cancer cells.RESULTS 1) Co-immunoprecipitation identified NTF2/RanGDP as an iASPP binding partner in the cytoplasm. In the nucleus, RanGDP is converted to RanGTP, disassembling NTF2/RanGDP/iASPP complex and releasing iASPP. Mutagenesis identified the hydrophobic 13th residues from two consecutive ARs as contributing to RanGDP binding and nuclear import. 2) 17 ARPs were experimentally tested. The barcode predicts the nuclear localization of >150 human proteomic ARPs with high accuracy. Interestingly, > 70% of barcode containing ARPs participate in NF-kB signaling, implying a role of the RaDAR (RanGDP-AR) guided nuclear import pathway in human cancers, autoimmune and inflammatory diseases. 3) The most commonly occurring p16 mutation, M13I, confers the barcode to p16, resulting in aberrant p16 nuclear accumulation and uncontrolled melanoma growth. In the oncoprotein iASPP, masking the barcode by compounds sequestered iASPP in cytoplasm, inhibiting tumor growth in mice.SIGNIFICANCE The finding shows a precise molecular barcode that flags proteins for nuclear import. This discovery will help illuminate the molecular dysfunction that underpins a broad array of ailments. The RaDAR is being exploited to develop entirely novel diagnostics and therapies (Lu et al, Cell, 2014). We experimentally proved, in melanoma treatment (Lu et al, Cancer Cell, 2013), it is a promising strategy (Nat. Rev. Cancer, 2013). The barcode masking compounds is scheduled for further pre-clinical study (Lu et al, WIPO, 2013).



Where applicable, experiments conform with Society ethical requirements.

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