Determinants of site-specific dephosphorylation by PP2A-B56
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Orbitrap Fusion Lumos, Orbitrap Fusion, Q Exactive Plus
ORGANISM(S): Homo Sapiens (ncbitaxon:9606)
SUBMITTER: 
Arminja Kettenbach
PROVIDER: MSV000084245 | MassIVE | Wed Aug 28 14:13:00 BST 2019
SECONDARY ACCESSION(S): PXD015205
REPOSITORIES: MassIVE
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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.
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Project description:PP2A is an abundant ser/thr protein phosphatase that act as a tumor suppressor by antagonizing multiple oncogenic kinases. For this reason, compounds able to activate PP2A holoenzymes are attractive anticancer agents. DT-061 and iHAP are phenothiazine derivatives that have recently been claimed to activate specific PP2A-B56 complexes to mediate cell killing. Here we show that iHAP and DT-061 do not activate PP2A-B56 complexes in vitro and that CRISPR removal of B56 regulatory subunits does not affect cellular toxicity of the compounds. Through genome-wide CRISPR screens we uncover the cell killing mechanism of iHAP and DT-061. We find that iHAP is a microtubule poison and removal of mitotic regulators causes hypersensitivity. In contrast, DT-061 disrupts both Golgi and ER consistent with an impact on cellular lipid composition. Indeed, we directly visualize DT-061 in cytoplasmic granules that co-localize with Golgi markers shortly after addition of the compound. Our work uncovers that well known side-effects of phenothiazine derived compounds cause cell killing by iHAP and DT-061 arguing that these compounds cannot be used for dissecting PP2A biology.
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Project description:RAS-mediated human cell transformation requires inhibition of the tumor suppressor Protein Phosphatase 2A (PP2A). Both RAS and PP2A mediate their effects by phosphoregulation, but phosphoprotein targets and cellular processes in which RAS and PP2A activities converge in human cancers have not been systematically analyzed. Here, based on mass spectrometry phosphoproteome data we discover that phosphosites co-regulated by RAS and PP2A are enriched on proteins involved in epigenetic gene regulation. As examples, RAS and PP2A co-regulate the same phosphorylation sites on HDAC1/2, KDM1A, MTA1/2, RNF168 and TP53BP1. Mechanistically, we validate co-regulation of NuRD chromatin repressor complex by RAS and PP2A. Consistent with their known synergistic effects in cancer, RAS activation and PP2A inhibition resulted in epigenetic reporter de-repression and activation of oncogenic transcription. Notably, transcriptional de-repression by PP2A inhibition was associated with increased euchromatin and decrease in global DNA methylation. Further, targeting of RAS- and PP2A-regulated epigenetic proteins decreased viability of KRAS-mutant human lung cancer cells. Collectively the results indicate that epigenetic protein complexes involved in oncogenic gene expression constitute a significant point of convergence for RAS hyperactivity and PP2A inhibition in cancer. Further, this work provides a resource for future studies focusing on phosphoregulation as a previously unappreciated layer of regulation of epigenetic gene regulation in cancer, and in other RAS/PP2A-regulated cellular processes.
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