GEF-independent Ran activation shifts a fraction of the protein to the cytoplasm and promotes cell proliferation

Jinhan Zhou,Yuping Tan,Qingxiang Sun,Da Jia,Xiaofei Shen,Yuqing Zhang,Xiaodong Sun,Aiping Tong

doi:10.1186/s43556-020-00011-2

Abstract

Ran (Ras-related nuclear protein) plays several important roles in nucleo-cytoplasmic transport, mitotic spindle formation, nuclear envelope/nuclear pore complex assembly, and other functions in the cytoplasm, as well as in cellular transformation when switched on. Unlike other members of the GTPase superfamily, Ran binds more tightly to GDP than to GTP due to the presence of an auto-inhibitory C-terminal tail. Multiple missense mutations in the C-terminus of Ran occur in cancers, but their biological significance remains unclear. Here, the quantitative GDP/GTP binding preference of four engineered mutations with unstable C-termini was analyzed using a devised mant-GDP dissociation assay. The results showed that the impact of different C-terminal mutations depends on multiple factors. Although these mutants were more GTP-loaded in human cells, they were shown to be more cytoplasmic, and to support nuclear transport with minimally or partially reduced efficiency. Further, several Ran cancer mutants were compromised in autoinhibition, slightly more GTP-bound, more cytoplasmic, and enhanced the proliferation of A549 and HeLa cells in vitro. Thus, our work reveals a new route of Ran activation independent of guanine nucleotide exchange factor (GEF), which may account for the hyper-proliferation induced by Ran cancer mutations.

Highlights

Ras-related nuclear protein (Ran) (Ras-related nuclear) protein is a member of the Ras superfamily of small GTPases
The designed C-tail disrupting (C-dis) mutants were more biased to bind Guanosine triphosphate (GTP) In order to study the function of C-terminal mutations, we designed four C-dis mutants (A133D, L182A, M189D, and Y197A) to disrupt C-tail binding to the G domain (Fig. 1a)
To quantify the change of relative affinities, we devised a mant-Guanosine diphosphate (GDP) dissociation experiment wherein different proteins were first charged with mant-GDP in the presence of Regulator of chromosome condensation (RCC1), the bound mant-GDP was dissociated by incubating with increasing concentrations of GDP or GTP (Fig. 1b)

Summary

Introduction

Ran (Ras-related nuclear) protein is a member of the Ras superfamily of small GTPases. Like other GTPases, Ran switches between GDP-bound (inactive) and GTPbound (active) states. The guanine nucleotide exchange factor (GEF) RCC1 (Regulator of Chromosome Condensation 1), which is chromatin-bound, increases the nucleotide exchange rate and charges Ran with GTP in the presence of abundant cellular GTP [1]. Ran is well-studied for its role in nucleo-cytoplasmic transport [4, 5]. RanGTP unloads a nuclear localization signal (NLS)-containing cargo from an importin (e.g. importin β1), or forms a trimeric nuclear export complex with a nuclear export signal (NES)-containing-cargo and an exportin (e.g. CRM1) [6]. The different Ran complexes are disassembled by RanGAP-mediated RanGTP hydrolysis with the help of Ran-binding protein 1 or 2 (RanBP1 or (2020) 1:18

Methods

Results

Conclusion