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

Michael V Tavolieri; Cristian A Droppelmann; Danae Campos-Melo; Kathryn Volkening; Michael J Strong

doi:10.1016/j.ejcb.2018.11.001

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

Eur J Cell Biol. 2019 Jan;98(1):27-35. doi: 10.1016/j.ejcb.2018.11.001. Epub 2018 Nov 19.

Authors

Michael V Tavolieri¹, Cristian A Droppelmann², Danae Campos-Melo³, Kathryn Volkening⁴, Michael J Strong⁵

Affiliations

¹ Department of Clinical Neurological Sciences, Schulich School of Medicine & Dentistry, Western University, London, Ontario, Canada. Electronic address: mtavolie@uwo.ca.
² Molecular Medicine Group, Robarts Research Institute, Western University, London, Ontario, Canada. Electronic address: cdroppel@uwo.ca.
³ Molecular Medicine Group, Robarts Research Institute, Western University, London, Ontario, Canada. Electronic address: dmaribel@uwo.ca.
⁴ Department of Clinical Neurological Sciences, Schulich School of Medicine & Dentistry, Western University, London, Ontario, Canada; Molecular Medicine Group, Robarts Research Institute, Western University, London, Ontario, Canada. Electronic address: kvolkening@robarts.ca.
⁵ Department of Clinical Neurological Sciences, Schulich School of Medicine & Dentistry, Western University, London, Ontario, Canada; Molecular Medicine Group, Robarts Research Institute, Western University, London, Ontario, Canada. Electronic address: Michael.Strong@schulich.uwo.ca.

PMID: 30482479
DOI: 10.1016/j.ejcb.2018.11.001

Abstract

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.

Keywords: NES; NLS; Nuclear localization; Pleckstrin homology (PH) domain; Rho Guanine Nucleotide Exchange Factor (RGNEF).

MeSH terms

Amino Acid Sequence
Cell Nucleus / metabolism*
Exportin 1 Protein
Guanine Nucleotide Exchange Factors / chemistry*
Guanine Nucleotide Exchange Factors / metabolism*
HEK293 Cells
Humans
Karyopherins / metabolism
Mutant Proteins / chemistry
Mutant Proteins / metabolism
Nuclear Export Signals*
Nuclear Localization Signals / metabolism*
Pleckstrin Homology Domains*
Protein Transport
Receptors, Cytoplasmic and Nuclear / metabolism
Structure-Activity Relationship

Substances

ARHGEF28 protein, human
Guanine Nucleotide Exchange Factors
Karyopherins
Mutant Proteins
Nuclear Export Signals
Nuclear Localization Signals
Receptors, Cytoplasmic and Nuclear