EphB4 localises to the nucleus of prostate cancer cells.
The EphB4 receptor tyrosine kinase is over-expressed in a variety of different epithelial cancers including prostate where it has been shown to be involved in survival, migration and angiogenesis. We report here that EphB4 also resides in the nucleus of prostate cancer cell lines. We used in silico methods to identify a bipartite nuclear localisation signal (NLS) in the extracellular domain and a monopartite NLS sequence in the intracellular kinase domain of EphB4. To determine whether both putative NLS sequences were functional, fragments of the EphB4 sequence containing each NLS were cloned to create EphB4NLS-GFP fusion proteins. Localisation of both NLS-GFP proteins to the nuclei of transfected cells was observed, demonstrating that EphB4 contains two functional NLS sequences. Mutation of the key amino residues in both NLS sequences resulted in diminished nuclear accumulation. As nuclear translocation is often dependent on importins we confirmed that EphB4 and importin-α can interact. To assess if nuclear EphB4 could be implicated in gene regulatory functions potential EphB4-binding genomic loci were identified using chromatin immunoprecipitation and Lef1 was confirmed as a potential target of EphB4-mediated gene regulation. These novel findings add further complexity to the biology of this important cancer-associated receptor.
Institute of Health and Biomedical Innovation, Queensland University of Technology, Woolloongabba, QLD, Australia; Australian Prostate Cancer Research Centre-Queensland, Translational Research Institute, 37 Kent Street, Woolloongabba 4102, QLD, Australia. Electronic address: inga.mertenswalker@qut.edu.au.
Inga
I
Mertens-Walker
Institute of Health and Biomedical Innovation, Queensland University of Technology, Woolloongabba, QLD, Australia; Australian Prostate Cancer Research Centre-Queensland, Translational Research Institute, 37 Kent Street, Woolloongabba 4102, QLD, Australia.
Jessica E
JE
Lisle
Institute of Health and Biomedical Innovation, Queensland University of Technology, Woolloongabba, QLD, Australia.
William A
WA
Nyberg
Institute of Health and Biomedical Innovation, Queensland University of Technology, Woolloongabba, QLD, Australia; Australian Prostate Cancer Research Centre-Queensland, Translational Research Institute, 37 Kent Street, Woolloongabba 4102, QLD, Australia.
Carson R
CR
Stephens
Institute of Health and Biomedical Innovation, Queensland University of Technology, Woolloongabba, QLD, Australia.
Leslie
L
Burke
Institute of Health and Biomedical Innovation, Queensland University of Technology, Woolloongabba, QLD, Australia; Australian Prostate Cancer Research Centre-Queensland, Translational Research Institute, 37 Kent Street, Woolloongabba 4102, QLD, Australia.
Raphael
R
Rutkowski
Institute of Health and Biomedical Innovation, Queensland University of Technology, Woolloongabba, QLD, Australia; Australian Prostate Cancer Research Centre-Queensland, Translational Research Institute, 37 Kent Street, Woolloongabba 4102, QLD, Australia.
Adrian C
AC
Herington
Institute of Health and Biomedical Innovation, Queensland University of Technology, Woolloongabba, QLD, Australia; Australian Prostate Cancer Research Centre-Queensland, Translational Research Institute, 37 Kent Street, Woolloongabba 4102, QLD, Australia.
Sally-Anne
SA
Stephenson
D021581:Active Transport, Cell Nucleus; D000595:Amino Acid Sequence; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism*; D004247:DNA / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D051821:Lymphoid Enhancer-Binding Factor 1 / Q000235:genetics / Q000378:metabolism; D008297:Male; D008969:Molecular Sequence Data; D019913:Nuclear Localization Signals; D011471:Prostatic Neoplasms; D011485:Protein Binding; D036224:Receptor, EphB4 / Q000737:chemistry / Q000378:metabolism*; D028901:alpha Karyopherins / Q000378:metabolism
C497573:LEF1 protein, human; D051821:Lymphoid Enhancer-Binding Factor 1; D019913:Nuclear Localization Signals; D028901:alpha Karyopherins; D004247:DNA; D036224:Receptor, EphB4
EphB4; Importin; Lef1; Nuclear localisation signal; Prostate cancer; Receptor tyrosine kinase