BRAF35 as target of MID1/TRIM18 E3 ligase activity

TRIM proteins are a family of ubiquitin E3 ligase enzymes characterized by the presence of a conserved N-terminus, the tripartite motif, which consists of a RING finger domain, two B-box motifs and an alpha-helical Coiled-coil region (RBCC). We focused our attention on TRIM18/MID1, the gene responsible for the X-linked form of Opitz G/BBB syndrome, a congenital disease characterized by defects in midline development and mental retardation. More recently, MID1 has also been found as overexpressed in some aggressive prostate cancers. The role of MID1 within the cell and the target(s) of its E3 ubiquitin activity during cellular processes are still not completely unravelled. In order to better investigate MID1 function and to find new cellular partners for this protein a two hybrid assay was performed in our laboratory. By means of this screening, BRCA2-Associated Factor 35 (BRAF35 or HMG20b) was identified as a novel MID1 interacting protein. BRAF35 is a non-canonical High-Mobility-Group (HMG) protein that has a role in both neuronal differentiation and in cell cycle progression. Moreover BRAF35 sumoylation has been shown to be fundamental for its antineurogenic activity and is inhibited by the interaction with its homologue iBraf. The aim of the project was to characterise the functional role of MID1/BRAF35 interaction and to understand if MID1, as an E3 ubiquitin ligase, regulates BRAF35 during cytokinesis. The first evidence obtained from the preliminary screening was confirmed through MBP pull-down assay and co-immunoprecipitation, identifying the coiled-coil region of both proteins as responsible for the binding. We further investigated on a possible regulation of BRAF35 by the ubiquitin proteasome system and we recognized BRAF35 as a poly-ubiquitinated protein and we found that its abundance is regulated in a proteasome-dependent manner. In addition, overexpression of MID1 or its domain-deleted mutants altered BRAF35 stability and post-translational modification suggesting a MID1-dependent BRAF35 ubiquitination that implicates also K63-polyUb dependent signalling involvement. Additionally, we found that MID1 and BRAF35 colocalize not only during interphase but also at the intercellular bridge during cytokinesis. Consistent with this observation, we observed a cell cycle-related regulation of BRAF35 protein level. Moreover, MID1 depletion rescued the cytokinetic defect caused by BRAF35 silencing, leading to a decrease of binucleation, but promoted a blebbing phenotype in dividing cells. This indicates that a fine regulation of the two proteins for the completion of cytokinesis is required and suggests an additional and new role for MID1 in cytokinesis regulation.