A40
RAN GTPase-controlled tumour suppression: RASSF1A tumour suppressor gene which is frequently inactivated in human cancers interacts with the RAN GTPase network
Ashraf Dallol1, Luke Hesson1, Wendy Cooper1, Eamonn Maher1, Walter Kolch2, Farida Latif1
1University of Birmingham, Birmingham, UK, 2The Beatson Institute for Cancer Research, Glasgow, UK
We identified RAN as a novel RASSF1-interacting protein using FLAG-immunoprecipitation and mass spectrometry. The interaction of RAN with RASSF1A was confirmed by the ability of GST-RASSF1A to pull-down endogenous RAN or GST-RAN associating with HA-RASSF1A. Furthermore, the RASSF1A-RAN interaction is mediated by residues 120-285aa in RASSF1A. In addition, endogenous RASSF1A and RAN colocalised at the centrosomes. We and others have reported the RASSF1A-induced microtubule hyperstabilisation (or RIMH). To test whether RIMH is RAN-dependent, we used the tsBN2 cell line that harbours a temperature-sensitive mutation in the RCC1 gene. Culturing the RASSF1A-expressing tsBN2 cells at the restrictive temperature depleted RCC1 and abrogated RIMH. This suggested that RASSF1A over-expression maybe altering the RAN-GTP: RAN-GDP ratio and RIMH is RAN-dependent. In support of this, RASSF1A enhanced the association of importin beta with RAN (Importin beta specifically binds RAN-GTP but not RAN-GDP). Expression of gfp-importin beta also abrogated RIMH presumably by sequestering the excess RAN-GTP generated by RASSF1A expression. Co-expression of RASSF1A and its associated S/T kinase, MST2, enhanced the RAN-GTP levels which implicated the MST2 kinase activity in the RASSF1A-RAN interaction. To this end, we have identified RCC1 to be an MST2 substrate in vitro. Furthermore, modulating the RAN-GTP: RAN-GDP ratio is associated with increased MST2 cleavage and kinase activity. Our findings suggest that RASSF1A/MST2 could be under the influence of exquisite regulation measures mediated by the RAN network that would limit the MST kinase activity to the right cellular compartment or mediate apoptotic signals sensed by the RASSF1A/MST2 complex.
