A highly conserved pocket on PP2A-B56 is required for hSgo1 binding and cohesion protection during mitosis

The shugoshin proteins are universal protectors of centromeric cohesin during mitosis and meiosis. The binding of human hSgo1 to the PP2A‐B56 phosphatase through a coiled‐coil (CC) region mediates cohesion protection during mitosis. Here we undertook a structure function analysis of the PP2A‐B56‐hSgo1 complex, revealing unanticipated aspects of complex formation and function. We establish that a highly conserved pocket on the B56 regulatory subunit is required for hSgo1 binding and cohesion protection during mitosis in human somatic cells. Consistent with this, we show that hSgo1 blocks the binding of PP2A‐B56 substrates containing a canonical B56 binding motif. We find that PP2A‐B56 bound to hSgo1 dephosphorylates Cdk1 sites on hSgo1 itself to modulate cohesin interactions. Collectively our work provides important insight into cohesion protection during mitosis

Structural basis for PP2A-mediated regulation of accurate chromosome segregation

The dynamic crosstalk between kinases and phosphatases is essential to ensure faithful mitosis. Protein phosphatases PP2A(B55) and PP2A(B56), and the Chromosomal Passenger Complex -CPC, containing Aurora B kinase-, establish a signalling network that controls sister chromatid cohesion, kinetochore-microtubule attachments, and timely chromosome segregation. PP2A(B56), together with Shugoshin1 (Sgo1), opposes Aurora B during early mitosis to protect cohesin from being disassembled at the inner centromere. PP2A(B56) and Aurora B also act antagonistically in controlling the processes of error correction and activation of the Spindle Assembly Checkpoint, both of which are essential to achieve chromosome biorientation. PP2A(B55) is the master regulator of mitotic exit, as it triggers late mitotic events by counteracting Cdk1 activity. Combining biochemistry, structural biology and biophysics, my work sheds light into the mechanistic basis of how PP2A regulates these key mitotic events through its interaction with Shugoshin1, the Chromosomal Passenger Complex and the Ska complex. Recent studies have shown that individual PP2A(B56) isoforms might have differential roles during mitosis. Nevertheless, the structural and functional basis of this isoform specificity and how it affects Sgo1 function remain unclear. In my thesis, I have explored the intricacies of PP2A(B56)-Sgo1 interactions and have discovered that Sgo1 binds PP2A(B56) in a B56- isoform-dependent manner. Our ongoing work hints that this specificity is mediated by dynamic interactions between Sgo1 and the B56 subunits, and aims to decipher the functional contribution of these interactions in achieving faithful chromosome segregation My work also unveils a novel interaction between PP2A(B56) and the CPC. Structural analysis of this complex shows that Borealin – one of the components of the CPC – contains a short linear motif that binds PP2A(B56) via the LxxIxE-binding pocket of B56. This highly conserved pocket is key for the recognition of PP2A(B56) interactors, and it is heavily involved in the regulation of PP2A activity and localisation during mitosis. The importance of this interaction in PP2A(B56) and/or CPC localisation and function in vivo is an outstanding question that our ongoing research aims to resolve. Recent studies suggested that PP2A(B55) might interact with the Spindle and Kinetochoreassociated (Ska) complex. The Ska complex is a key protein assembly -consisting of Ska1, Ska2 and Ska3- essential for stabilising load-bearing end-on kinetochore-microtubule attachments, and it is also found in the central spindle and midbody at the end of mitosis. Our research shows that the interaction between PP2A(B55) and the Ska complex is mediated by a short unstructured region of Ska3. This region binds the B55 subunit of PP2A and, according to our predicted models, it might involve its substrate-binding groove. Ongoing efforts in human cell lines aim to explain how this interaction controls the localisation and function of the Ska complex during late mitotic events

Chemogenetic profiling reveals PP2A-independent cytotoxicity of proposed PP2A activators iHAP1 and DT-061

Protein phosphatase 2A (PP2A) is an abundant phosphoprotein phosphatase that acts as a tumor suppressor. For this reason, compounds able to activate PP2A are attractive anticancer agents. The compounds iHAP1 and DT‐061 have recently been reported to selectively stabilize specific PP2A‐B56 complexes to mediate cell killing. We were unable to detect direct effects of iHAP1 and DT‐061 on PP2A‐B56 activity in biochemical assays and composition of holoenzymes. Therefore, we undertook genome‐wide CRISPR‐Cas9 synthetic lethality screens to uncover biological pathways affected by these compounds. We found that knockout of mitotic regulators is synthetic lethal with iHAP1 while knockout of endoplasmic reticulum (ER) and Golgi components is synthetic lethal with DT‐061. Indeed we showed that iHAP1 directly blocks microtubule assembly both in vitro and in vivo and thus acts as a microtubule poison. In contrast, DT‐061 disrupts both the Golgi apparatus and the ER and lipid synthesis associated with these structures. Our work provides insight into the biological pathways perturbed by iHAP1 and DT‐061 causing cellular toxicity and argues that these compounds cannot be used for dissecting PP2A‐B56 biology