Doctor of Philosophy
Dr. Caroline Schild-Poulter
RanBPM has been shown to interact with numerous proteins implicating it in a variety of cellular processes including apoptosis, transcription regulation, cell migration, adhesion and morphology and has been shown to have tumour suppressive functions. RanPBM has been hypothesized to be a scaffolding protein and is part of a large protein complex termed the CTLH complex. Although the homologous complex in yeast has been demonstrated to have E3 ubiquitin ligase activity, whether the evolutionarily conserved human complex retains this activity remains to be determined. In this work we aim to characterize the regions of RanBPM that regulate its subcellular localization and identify structures that retain RanBPM in the nuclear and cytoplasmic compartments. In addition, we aim to characterize the role of RanBPM in aggresome formation and characterize its association with HDAC6.
In this study we have identified 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 and a second motif in the C-terminus. We also identify a nuclear export signal and demonstrate that the SPRY, LisH and CTLH domains function to retain RanBPM in the cytoplasm. We demonstrate that RanBPM associates with chromatin in the nucleus and microtubules in the cytoplasm. We reveal that RanBPM is an essential component of aggresomes induced by both ionizing radiation and proteasomal inhibition. Aggresomes are aggregates of misfolded ubiquitinated proteins that form in response to proteasomal impairment. RanBPM was found to interact with HDAC6, a central regulator of aggresome formation, and inhibits deacetylase activity. We found that the RanBPM-mediated inhibition of HDAC6 α-tubulin deacetylase activity is dependent on its association with HDAC6. We show that HDAC6 does not require RanBPM to associate with microtubules, but that RanBPM requires HDAC6 to associate with microtubules. Furthermore, we demonstrate that components of the CTLH complex associate with both microtubules and HDAC6. Lastly, RanBPM was found to inhibit HDAC6-mediated cell migration.
This suggests that the tumour suppressor functions of RanBPM stem, in part, from an inhibition of the oncogenic activities of HDAC6.
Salemi, Louisa M., "Characterization of RanBPM Subcellular Localization and Function in HDAC6 Regulation" (2016). Electronic Thesis and Dissertation Repository. 4301.