Abstract
The c-MYC (MYC) oncoprotein is deregulated in over 50% of cancers, yet regulatory mechanisms controlling MYC remain unclear. To this end, we interrogated the MYC interactome using BioID mass spectrometry (MS) and identified PP1 (protein phosphatase 1) and its regulatory subunit PNUTS (protein phosphatase-1 nuclear-targeting subunit) as MYC interactors. We demonstrate that endogenous MYC and PNUTS interact across multiple cell types and that they co-occupy MYC target gene promoters. Inhibiting PP1 by RNAi or pharmacological inhibition results in MYC hyperphosphorylation at multiple serine and threonine residues, leading to a decrease in MYC protein levels due to proteasomal degradation through the canonical SCFFBXW7 pathway. MYC hyperphosphorylation can be rescued specifically with exogenous PP1, but not other phosphatases. Hyperphosphorylated MYC retained interaction with its transcriptional partner MAX, but binding to chromatin is significantly compromised. Our work demonstrates that PP1/PNUTS stabilizes chromatin-bound MYC in proliferating cells.
Highlights
To this end, our goal was to better understand the posttranslational modifications (PTMs) and regulators of MYC by interrogating the MYC interactome using BioID
proximity ligation assay (PLA) was used to validate the endogenous MYC-phosphatase-1 nucleartargeting subunit (PNUTS) interaction and sequential Chromatin immunoprecipitation (ChIP)-re-ChIP of MYC followed-by PNUTS revealed that both proteins bind to MYCbound gene promoters
MYC can induce PP1/PNUTS expression, suggesting a feed-forward loop in which MYC drives PP1/PNUTS expression to enable their interaction and regulate gene transcription. Consistent with these results, recent genome-wide DamID analysis demonstrated that PP1 and PNUTS promoterbinding significantly overlaps with MYC ChIP-seq datasets from the ENCODE consortium39
Summary
Our goal was to better understand the posttranslational modifications (PTMs) and regulators of MYC by interrogating the MYC interactome using BioID. We describe here the interaction of MYC with the PP1/PNUTS holoenzyme protein complex. MYC can induce PNUTS expression, suggesting a feed-forward co-operative regulatory loop. This is further supported by the co-localization of MYC and PNUTS to the promoters of MYC target genes. Inhibition of PP1/PNUTS triggers hyperphosphorylation of MYC, leading to chromatin eviction and degradation by the canonical SCFFBXW7 pathway. PP1/PNUTS is amplified in multiple cancer types, suggesting a model in which elevated PP1/PNUTS expression confers a growth advantage by increasing MYC protein stability
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