A novel overlapping NLS/NES region within the PH domain of Rho Guanine Nucleotide Exchange Factor (RGNEF) regulates its nuclear-cytoplasmic localization

•RGNEF, an ALS-related RNA binding protein, localizes in the cytoplasm and the nucleus.•The PH domain of RGNEF contain a functional bipartite NLS.•The PH domain of RGNEF has a leptomycin-B sensitive NES.•This is first description of an overlapping NLS/NES pair embedded within a PH domain. Rho Guanine Nucleotide Exchange Factor (RGNEF) is a 190 kDa protein implicated in both amyotrophic lateral sclerosis (ALS) and cancer. Under normal physiological conditions, RGNEF is predominantly cytoplasmic with moderate levels of nuclear localization. We have identified a 23-amino acid region containing a bipartite nuclear localization signal (NLS) within the Pleckstrin Homology (PH) domain of RGNEF, which when deleted or mutated abolishes the nuclear localization of this protein. Fusion proteins containing only the PH domain demonstrated that this region by itself is able to translocate a 160 kDa protein to the nucleus. Interestingly, we also detected a nuclear export signal (NES) within the linker region of this bipartite NLS which is able to export from the nucleus a fusion protein containing two NLSs. Experiments using Leptomycin-B -an inhibitor of nuclear export- confirmed that this region promotes nuclear export in an exportin-1 dependent manner. This study is the first report demonstrating either of these signals embedded within a PH domain. Notably, this is also the first description of a functional overlapped NLS/NES signal.