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A chemical-genetic system to rapidly inhibit the PP2A-B56 phosphatase

ABSTRACT: Serine-threonine phosphatases have been challenging to study because of the lack of specific inhibitors. Their catalytic domains are druggable, but these are shared or very similar between individual phosphatase complexes, precluding their specific inhibition. Instead, phosphatase complexes achieve specificity by interacting with short-linear motifs (SLiMs) in substrates or their binding partners. We develop here a chemical-genetic system to rapidly inhibit these interactions within the PP2A-B56 family. Drug-inducible recruitment of ectopic SLiMs (“directSLiMs”) is used to rapidly block the SLiM-binding pocket on the B56 regulatory subunit, thereby displacing endogenous interactors and inhibiting PP2A-B56 activity within seconds. We use this system to characterise PP2A-B56 substrates during mitosis and to identify a novel role for PP2A-B56 in maintaining kinetochore microtubule attachments at metaphase. The directSLiMs approach can be used to inhibit any other phosphatase, enzyme or protein that uses a critical SLiM-binding interface, providing a powerful strategy to inhibit and characterise proteins once considered “undruggable”.

INSTRUMENT(S): Orbitrap Eclipse

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell

SUBMITTER: Juan Manuel Valverde

LAB HEAD: Adrian Saurin

PROVIDER: PXD056390 | Pride | 2025-02-06

REPOSITORIES: pride

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			Action	DRS
	260224_JMV074_F10_Phospho.raw	Raw
	260224_JMV074_F11_Phospho.raw	Raw
	260224_JMV074_F12_Phospho.raw	Raw
	260224_JMV074_F13_Phospho.raw	Raw
	260224_JMV074_F14_Phospho.raw	Raw

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PP2A-Rts1 antagonizes Rck2-mediated hyperosmotic stress signaling in yeast

Project description:In Saccharomyces cerevisiae impairment of protein phosphatase PP2A-Rts1 leads to temperature and hyperosmotic stress sensitivity, yet the underlying mechanism and the scope of action of the phosphatase in the stress response remain elusive. Using quantitative mass spectrometry-based approaches we have identified a set of putative substrate proteins that show both, hyperosmotic stress- and PP2A-Rts1-dependent changes in their phosphorylation pattern. A comparative analysis with published MS-shotgun data revealed that the phosphorylation status of many of these sites is regulated by the MAPKAP kinase Rck2, suggesting a node of regulation. Detailed gel mobility shift assays and protein-protein interaction analysis strongly suggest that Rck2 activity is directly regulated by PP2A-Rts1 via a SLiM B56-family interaction motif, uncovering a previously unknown mechanism of how PP2A influences the response to hyperosmotic stress in Yeast.

2022-05-16 | PXD019647 | Pride

Structural mechanism for inhibition of PP2A-B56 and oncogenicity by CIP2A

Project description:Abstract The protein phosphatase 2A (PP2A) heterotrimer PP2A-B56 is a human tumor suppressor. However, the molecular mechanisms inhibiting PP2A-B56in cancer are poorly understood. Here, we report molecular level details and structural mechanism of PP2A-B56inhibition by an oncoprotein CIP2A. Upon direct binding to PP2A-B56 trimer, CIP2A replaces PP2A-A subunit and thereby hijacks both the B56 and the catalytic PP2Ac subunit to form a CIP2A-B56-PP2Ac pseudotrimer. Further, CIP2A competes with B56 substrate binding by blocking the LxxIxE-motif substrate binding pocket on B56. Relevant to oncogenic activity of CIP2A across human cancers, the N-terminal head domain-mediated interaction with B56 stabilizes CIP2A protein. Functionally, CRISPR/Cas9-mediated single amino acid mutagenesis of the head domain blunted MYC expression and MEK phosphorylation, and abrogated triple-negative breast cancer in vivo tumor growth. Collectively, we discover a unique multi-step “hijack and mute” mechanism of protein complex regulation inhibiting tumor suppressor PP2A-B56. Further, the results unfold single structural determinant for CIP2A´s oncogenic activity, potentially facilitating therapeutic modulation of CIP2A in cancer and other diseases.

2023-03-11 | PXD030297 | Pride

A dynamic charge:charge interaction modulates PP2A:B56 interactions

Project description:The recruitment of specific substrates by ser/thr protein phosphatase 2A (PP2A) regulatory B-subunits is poorly understood, limiting our understanding of PP2A-regulated signaling. Recently, the first PP2A:B56 consensus binding motif, LxxIxE, which confers substrate specificity to PP2A:B56 holoenzymes, was identified. However, most validated LxxIxE motifs interact with PP2A:B56 with low micromolar affinities, suggesting that additional recognition motifs exist that function cooperatively to enhance PP2A:B56 binding. Here, we report the requirement of a positively charged motif in a subset of PP2A:B56 interactors that facilitates B56 binding via dynamic charge:charge interactions. Using molecular and cellular approaches, we show that a highly conserved, negatively charged groove on B56 mediates this dynamic interaction. We also show that this motif is required for efficient binding and dephosphorylation of KIF4A in cells, confirming the functional relevance of this motif for PP2A:B56 dephosphorylation. Collectively our results provide an important framework for understanding PP2A regulation of cellular signaling.

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Determinants of site-specific dephosphorylation by PP2A-B56

Project description:The tumor suppressor PP2A is a major cellular protein phosphatase that regulates numerous cellular processes through the formation of holoenzymes containing distinct regulatory B-subunits. However, the determinants of differential substrate dephosphorylation by PP2A are poorly understood. Here, we develop a specific genetically encoded inhibitor of PP2A-B56 and perform global phosphoproteomic studies to identify hundreds of regulated phosphorylation sites. We show that PP2A-B56 substrate specificity is controlled by affinity and position of B56 binding motifs and that PP2A active site preference is determined by the B-subunit. These insights uncover PP2A-B56 as a novel negative regulator of ADAM17 mediated growth factor signalling and tumor development. Collectively our work identifies basic principles of PP2A-B56 specificity with broad implications for understanding signalling in eukaryotes.

2019-08-28 | MSV000084245 | MassIVE

To be or not to be phosphorylated: Understanding the role of Ebola virus nucleoprotein in the dynamic interplay with the transcriptional activator VP30 and the host phosphatase PP2A-B56

Project description:Ebola virus (EBOV) transcription is essentially regulated via dynamic dephosphorylation of its viral transcription activator VP30 by the host phosphatase PP2A. The nucleoprotein NP has emerged as a third key player in the regulation of this process by recruiting both the regulatory subunit B56 of PP2A and its substrate VP30 to initiate VP30 dephosphorylation and hence viral transcription. Both binding sites are located in close proximity to each other in NP`s C-terminal disordered region. This study investigates NP's role in VP30 dephosphorylation and transcription activation, focusing on the spatial requirements of NP’s binding sites. Increasing the distance between PP2A-B56 and VP30 at the NP interface revealed that close spatial and orientational contact is necessary for efficient VP30 dephosphorylation and viral transcription. Longer distances were lethal for recombinant EBOV except when a compensatory mutation, NP T603I, occurred. This mutation, located between the NP binding sites for PP2A-B56 and VP30, fully restored functionality. Mass spectrometry showed that T603 is phosphorylated in recEBOV-NPwt virions. Mutational analysis indicated that T603I facilitates VP30 dephosphorylation in otherwise lethal recEBOV and that dynamic phosphorylation of NP T603 is important for efficient primary viral transcription in the WT context. These findings emphasize the critical and evolutionarily pressured interplay between VP30 and PP2A-B56 within the NP C-terminal disordered region and highlight the important role of NP on the regulation of viral transcription during the EBOV life cycle.

2025-01-14 | PXD056320 | Pride

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

Project description: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.

2021-03-04 | MSV000087003 | MassIVE

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

Project description:Mutations in the tumour suppressor BRCA2 are associated with predisposition to breast and ovarian cancers. BRCA2 has a central role in genome integrity by facilitating the repair of toxic DNA double-strand breaks (DSBs) by homologous recombination. BRCA2 acts by promoting RAD51 nucleofilament 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 DNA 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.

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The yeast PP2A-CDC55 phosphatase regulates the transcriptional response to hyperosmolarity stress by regulating Msn2 and Msn4

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Project description:Short linear motifs (SLiMs) form dynamic protein-protein interactions essential for signaling; however, sequence degeneracy and low binding affinities make them difficult to identify. In this study, we employed multiple, unbiased systematic approaches for SLiM discovery to elucidate the regulatory network of calcineurin (CN), the Ca2+-regulated phosphatase that recognizes LxVP and PxIxIT motifs. In vitro proteome-wide detection of CN-binding peptides, in situ SLiM-dependent proximity labeling, as well as in silico modeling of motif determinants uncovered unanticipated CN interactors, including centrosomal and nuclear pore complex (NPC) proteins – structures where Ca2+ signaling is largely uncharacterized. Here, we show that CN binds to and dephosphorylates NPC proteins (nucleoporins) in vitro and promotes the accumulation of a nuclear transport reporter in HeLa cells. Furthermore, IP/MS analyses on HeLa cells treated with DMSO or the CN inhibitor, Cyclosporin A (CsA), reveal multiple CN-dependent phosphosites on key transport nucleoporins in vivo. Together, these data suggest that CN positively regulates nuclear transport by dephosphorylating key transport nucleoporins. In summary, the CN network assembled here provides a resource to investigate Ca2+ and CN signaling and the demonstrated synergy between experimental and computational methods establishes a blueprint for examining SLiM-based networks.

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