Characterization of RanBPM Molecular Determinants that Control Its Subcellular Localization

Abstract

RanBPM/RanBP9 is a ubiquitous, nucleocytoplasmic protein that is part of an evolutionary conserved E3 ubiquitin ligase complex whose function and targets in mammals are still unknown. RanBPM itself has been implicated in various cellular processes that involve both nuclear and cytoplasmic functions. However, to date, little is known about how RanBPM subcellular localization is regulated. We have conducted a systematic analysis of RanBPM regions that control its subcellular localization using RanBPM shRNA cells to examine ectopic RanBPM mutant subcellular localization without interference from the endogenously expressed protein. We show that several domains and motifs regulate RanBPM nuclear and cytoplasmic localization. In particular, RanBPM comprises two motifs that can confer nuclear localization, one proline/glutamine-rich motif in the extreme N-terminus which has a dominant effect on RanBPM localization, and a second motif in the C-terminus which minimally contributes to RanBPM nuclear targeting. We also identified a nuclear export signal (NES) which mutation prevented RanBPM accumulation in the cytoplasm. Likewise, deletion of the central RanBPM conserved domains (SPRY and LisH/CTLH) resulted in the relocalization of RanBPM to the nucleus, suggesting that RanBPM cytoplasmic localization is also conferred by protein-protein interactions that promote its cytoplasmic retention. Indeed we found that in the cytoplasm, RanBPM partially colocalizes with microtubules and associates with α-tubulin. Finally, in the nucleus, a significant fraction of RanBPM is associated with chromatin. Altogether, these analyses reveal that RanBPM subcellular localization results from the combined effects of several elements that either confer direct transport through the nucleocytoplasmic transport machinery or regulate it indirectly, likely through interactions with other proteins and by intramolecular folding.

Ran Binding Protein 9 (RanBP9) is a novel mediator of cellular DNA damage response in lung cancer cells

“…We investigated the role in the DDR of Ran Binding Protein 9 (RanBP9, also known as RanBPM), a protein whose biological functions are still unknown [ 30 – 34 ]. RanBP9 interacts with several factors involved in various cellular processes such as signal transduction, gene expression, cell adhesion and migration [ 35 – 37 ].…”

“…Relevant to the present work, RanBP9 becomes phosphorylated in cells exposed to different types of genotoxic stress including cisplatin, IR, UV exposure and osmotic shocks [ 43 , 44 ]. Finally, a recent study dissected out the role of different regions of RanBP9 involved in its nuclear-cytoplasmic localization [ 34 ]. However, the stimuli and the mechanisms of RanBP9 nucleus-cytoplasmic shuttling remain largely unknown.…”

Integrative meta-analysis of protein interaction data identified multiple GID/MRCTLH protein complexes in plants

“…This is not surprising because most of the components contain LisH and CTLH protein domains that are known to mediate protein-protein interactions (Salemi et al, 2015). For example, to date, more than 50 protein interactions have been demonstrated for RanBPM (Francis et al, 2013;Suresh et al, 2012;Kobayashi et al, 2007).…”

Death domain of p75 neurotrophin receptor: a structural perspective on an intracellular signalling hub

“…RanBPM is a 90‐kDa protein with a long stretch of Pro and Gln residues in its highly disordered N‐terminal region followed by conserved Sp1a kinase and ryanodine receptor (SPRY), lissencephaly type‐1‐like homology (LisH), C‐terminal to LisH (CTLH) and the C terminal CT‐11‐RanBPM (CRA) domains (Bai, Chen & Huang, ; Suresh, Ramakrishna & Baek, ; Salemi, Loureiro & Schild‐Poulter, ; Salemi et al, ). The interaction domain of p75 NTR with RanBPM was mapped to the p75 NTR ‐DD (Bai et al, ).…”