Biology subjects

Kettenbach, A.

Publications and source records attributed to Kettenbach, A..

4 recordsLinked to original sources

bioRxiv2021

Cellular toxicity of iHAP1 and DT-061 does not occur through PP2A-B56 targeting

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 small molecule compounds iHAP1 and DT-061 have recently been reported by Leonard et al. (2020) and Morita et al. (2020) in Cell to selectively stabilize specific PP2A-B56 complexes which mediate cell killing. Here, we show that this is not the case and question key findings in these papers. Through genome wide CRISPR-Cas9 screens, we uncover the biological pathways targeted by these compounds. We find 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 endoplasmic reticulum and we directly visualize DT-061 in cytoplasmic granules that co-localize with Golgi markers. Our work demonstrates that iHAP1 and DT-061 cannot be used for dissecting PP2A-B56 biology.

cell biology↗

bioRxiv2021

A complex of BRCA2 and PP2A-B56 is required for DNA repair by homologous recombination

Mutations in the tumour suppressor gene BRCA2 are associated with predisposition to breast and ovarian cancers. BRCA2 has a central role in maintaining genome integrity by facilitating the repair of toxic DNA double-strand breaks (DSBs) by homologous recombination (HR). BRCA2 acts by promoting RAD51 nucleoprotein filament formation on resected single-stranded DNA, but how BRCA2 activity is regulated during HR is not fully understood. Here, we delineate a pathway where ATM and ATR kinases phosphorylate a highly conserved region in BRCA2 in response to DSBs. These phosphorylations stimulate the binding of the protein phosphatase PP2A-B56 to BRCA2 through a conserved binding motif. We show that the phosphorylation-dependent formation of the BRCA2-PP2A-B56 complex is required for efficient RAD51 loading to sites of DNA damage and HR-mediated DNA repair. Moreover, we find that several cancer-associated mutations in BRCA2 deregulate the BRCA2-PP2A-B56 interaction and sensitize cells to PARP inhibition. Collectively, our work uncovers PP2A-B56 as a positive regulator of BRCA2 function in HR with clinical implications for BRCA2 and PP2A-B56 mutated cancers.

cell biology↗

bioRxiv2020

A highly conserved binding pocket on PP2A-B56 is required for shugoshin binding and cohesion protection

The shugoshin proteins are universal protectors of centromeric cohesin during mitosis and meiosis. The binding of human Sgo1 to the PP2A-B56 phosphatase through a coiled coil (CC) region is believed to mediate cohesion protection during mitosis. Here we undertook a structure function analysis of the PP2A-B56-Sgo1 complex, revealing unanticipated aspects of complex formation and function. We establish that a highly conserved pocket of the B56 regulatory subunit is required for Sgo1 binding and cohesion protection. Consistent with this, we show that Sgo1 blocks the binding of PP2A-B56 substrates containing a canonical B56 binding motif. Surprisingly, we identify B56 and Sgo1 mutants that prevent complex formation yet support cohesion protection and normal mitotic progression. This suggests that Sgo1 and PP2A-B56 have cohesion protection activity independently of complex formation. Collectively our work provides important insight into cohesion protection during mitosis.

cell biology↗

bioRxiv2020

Coupling of Cdc20 inhibition and activation by BubR1

Tight regulation of the APC/C-Cdc20 ubiquitin ligase that targets Cyclin B1 for degradation is important for mitotic fidelity. The spindle assembly checkpoint (SAC) inhibits Cdc20 through the mitotic checkpoint complex (MCC). In addition, phosphorylation of Cdc20 by Cyclin B1-Cdk1 independently inhibits APC/C-Cdc20 activation. This creates a conundrum for how Cdc20 gets activated prior to Cyclin B1 degradation. Here we show that the MCC component BubR1 harbours both Cdc20 inhibition and activation activities, allowing for cross-talk between the two Cdc20 inhibition pathways. Specifically BubR1 acts as a substrate specifier for PP2A-B56 to enable efficient Cdc20 dephosphorylation in the MCC. A mutant Cdc20 mimicking the dephosphorylated state escapes a mitotic checkpoint arrest arguing that restricting Cdc20 dephosphorylation to the MCC is important. Collectively our work reveals how Cdc20 can be dephosphorylated in the presence of Cyclin B1-Cdk1 activity without causing premature anaphase onset.

cell biology↗