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A Protein Phosphatase 1 specific phosphatase targeting peptide (PhosTAP) to identify the PP1 phosphatome

ABSTRACT: Most eukaryotic proteins are regulated by phosphorylation. Phosphoprotein phosphatases (PPPs) are the key serine/threonine phosphatases that regulate all essential signaling cascades. In particular, Protein Phosphatase 1 (PP1) dephosphorylates a predicted ~80% of all ser/thr phosphorylation sites. Here, we developed a phosphatase targeting peptide (PhosTAP) that binds all PP1 isoforms and does so with a stronger affinity than any other known cognate PP1 regulator. This PhosTAP can be used as a PP1 recruitment tool for PhosTAC-type recruitment in in vitro and cellular experiments, as well as in phosphoproteomics experiments to identify PP1-specific substrates and phosphosites. The latter is especially important to further our understanding of cellular signaling, as the identification of substrates and especially phosphosites that are targeted by specific phosphatases, like PP1, lags far behind that of their kinase counterparts. This is due to a lack of chemical/biological tools that are specific for individual phosphatases and a lack of established active site recognition sequences. Using PhosTAP-based proteomics studies, we show that, counter to our current understanding, many PP1 regulators are also substrates; that the number of residues between regulator PP1-binding and phosphosites vary significantly; and that PP1 also counteracts the activities of mitotic kinases. Finally, we also discovered that Haspin kinase is a direct substrate of PP1 and that its PP1-dependent dephosphorylation modulates its activity during anaphase. Taken together, we show that PP1-specific PhosTAPs are a powerful tool for studying PP1 activity in vitro and in cells.

INSTRUMENT(S): Orbitrap Fusion Lumos, Q Exactive Plus

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Arminja Kettenbach

PROVIDER: MSV000094153 | MassIVE | Thu Feb 22 07:44:00 GMT 2024

SECONDARY ACCESSION(S): PXD050038

REPOSITORIES: MassIVE

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Project description:Protein phosphatase 1 (PP1) is a Ser/Thr phosphatase that has been implicated in many key cellular functions including transcriptional regulation. Due to its involvement these many processes, it becomes difficult to directly link PP1 to transcriptional regulation on the chromatin level as no direct genomic binding sites have been identified. Previous work has failed to address this as the most common method used, namely chromatin immunoprecipitation (ChIP), is an antibody-dependent technique and currently no ChIP-grade PP1 antibodies have been developed. Using DamID, an alternative to ChIP, we have identified PP1 isoform-specific binding sites on the promoter regions of genes. We also identified the binding sites of three main PP1 regulatory subunits (R-subunits) in order to identify potential PP1 holo-enzymes binding sites. Our study revealed the full extent of PP1 isoform specific binding an allowed us to investigate the dependency of the R-subunits on PP1 for chromatin targeting. This data establishes PP1 as a chromatin interactor and allow for the identification of direct effects PP1 can have on the regulation of the genes on whose promoter it is bound. HeLa stable cell lines were created using constructs derived from pIND-(V5)-EcoDam. These constructs express trace amounts of Dam or C-terminal fusions with PP1α, PP1β, PP1γ and three R-subunits, PNUTS, NIPP1 and RepoMan, both wildtype (WT) and their PP1-binding mutants (RATA). Two independent stable cell lines were set up for each of the constructs. DamID-DNA was labeled and hybridized to a GeneChip Human Promoter 1.0R Array (Affymetrix, Santa Clara, CA, USA). The tiling array readouts were analyzed with the “model-based analysis of tiling arrays” (MAT) algorithm (version 1.0.0) against the hg19 reference genome. We normalized two biological replicates of each Dam-fusion over two Dam-only biological replicates. Each biological repeat consisted of 2 technical repeats pooled together prior to hybridization to the tiling array.

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A Protein Phosphatase 1 specific phosphatase targeting peptide (PhosTAP) to identify the PP1 phosphatome

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