Mitosis ensures equal segregation of the genome and is controlled by a variety of ubiquitylation signals on substrate proteins. However, it remains unexplored how the versatile ubiquitin code is read out during mitotic progression. Here, we identify the ubiquitin receptor protein UBASH3B as an important regulator of mitosis. UBASH3B interacts with ubiquitylated Aurora B, one of the main kinases regulating chromosome segregation, and controls its subcellular localization but not protein levels. UBASH3B is a limiting factor in this pathway and is sufficient to localize Aurora B to microtubules prior to anaphase. Importantly, targeting Aurora B to microtubules by UBASH3B is necessary for the timing and fidelity of chromosome segregation in human cells. Our findings uncover an important mechanism defining how ubiquitin attachment to a substrate protein is decoded during mitosis.

PDZ domains recognise short sequence motifs at the extreme C-termini of proteins. A model based on microarray data has been recently published for predicting the binding preferences of PDZ domains to five residue long C-terminal sequences. Here we investigated the potential of this predictor for discovering novel protein interactions that involve PDZ domains. When tested on real negative data assembled from published literature, the predictor displayed a high false positive rate (FPR). We predicted and experimentally validated interactions between four PDZ domains derived from the human proteins MAGI1 and SCRIB and 19 peptides derived from human and viral C-termini of proteins. Measured binding intensities did not correlate with prediction scores, and the high FPR of the predictor was confirmed. Results indicate that limitations of the predictor may arise from an incomplete model definition and improper training of the model. Taking into account these limitations, we identified several novel putative interactions between PDZ domains of MAGI1 and SCRIB and the C-termini of the proteins FZD4, ARHGAP6, NET1, TANC1, GLUT7, MARCH3, MAS, ABC1, DLL1, TMEM215 and CYSLTR2. These proteins are localised to the membrane or suggested to act close to it and are often involved in G protein signalling. Furthermore, we showed that, while extension of minimal interacting domains or peptides toward tandem constructs or longer peptides never suppressed their ability to interact, the measured affinities and inferred specificity patterns often changed significantly. This suggests that if protein fragments interact, the full length proteins are also likely to interact, albeit possibly with altered affinities and specificities. Therefore, predictors dealing with protein fragments are promising tools for discovering protein interaction networks but their application to predict binding preferences within networks may be limited.

The E6 oncoproteins from high-risk mucosal human papillomavirus (HPV) induce cervical cancer via two major activities, the binding and the degradation of the p53 protein and PDZ domain-containing proteins. Human MAGI-1 is a multi-PDZ domain protein implicated into protein complex assembly at cell-cell contacts. High-risk mucosal HPV E6 proteins interact with the PDZ1 domain of MAGI-1 via a C-terminal consensus binding motif. Here, we developed a medium throughput protocol to accurately measure by surface plasmon resonance affinity constants of protein domains binding to peptidic sequences produced as recombinant fusions to the glutathione-S-transferase (GST). This approach was applied to measure the binding of MAGI-1 PDZ1 to the C-termini of viral or cellular proteins. Both high-risk mucosal HPV E6 C-terminal peptides and cellular partners of MAGI-1 PDZ1 bind to MAGI-1 PDZ1 with comparable dissociation constants in the micromolar range. MAGI-1 PDZ1 shows a preference for C-termini with a valine at position 0 and a negative charge at position -3, confirming previous studies performed with HPV18 E6. A detailed combined analysis via site-directed mutagenesis of the HPV16 C-terminal peptide and PDZ1 indicated that interactions mediated by charged residues upstream the PDZ-binding motif strongly contribute to binding selectivity of this interaction. In addition, our work highlighted the K(499) residue of MAGI-1 as a novel determinant of binding specificity. Finally, we showed that MAGI-1 PDZ1 also binds to the C-termini of LPP and Tax proteins, which were already known to bind to PDZ proteins but not to MAGI-1.