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Identification of PP1-fusion phosphatase substrates

ABSTRACT: This experiments was designed to identify substrates of holophosphatase complexes of PP1 and its specificity-determining regulator proteins. We generated fusions of Flag-tagged Protein Phosphatase 1 fusions with Phactr1-4, Neurabin, Spinophilin, PPPR15A, PPP1R15B, and PNUTS, in each case including known protein interaction domains immediately C-terminal to the PP1-binding sequences. Each fusion protein was stably expressed in 293 Flp-In TRex cells using a tetracycline inducible vector. To investigate the substrate specificities of the PP1-fusion proteins, we performed Tandem-Mass-Tag (TMT) phosphoproteomics. Fractionated peptides were measured in both MS2 and MS3 modes for maximal identification and quantification. Expression of the PP1-fusions revealed numerous phosphorylation sites that were specifically depleted compared with cells expressing PP1 alone.

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ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Permanent Cell Line Cell

SUBMITTER: Roman Fedoryshchak

LAB HEAD: Richard Treisman

PROVIDER: PXD055166 | Pride | 2025-05-28

REPOSITORIES: Pride

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Project description:NIPP1, an established interactor of protein phosphatase 1 (PP1), is implicated in PRC2-mediated regulation of gene expression. Here, we explore whether PP1 associated with NIPP1 is involved in NIPP1-mediated regulation of genes. Therefore, we generated Hela Tet-off (HTO) cell lines that stably and inducibly express a Flag-tagged version of wild-type NIPP1 (HTO-NIPP1wt) or mutant NIPP1 (HTO_NIPP1m). The latter mutant lacks the major PP1 binding site by two point mutations in the RVXF-motif, an established PP1 - binding motif. All cell lines were derived from the same parental HeLa Tet-Off (HTO-PT) cell line which expresses only tTA transactivator and is used as a control. The Flag fusions were only expressed in the absence of doxycyline and at levels that were up to twofold higher than that of endogenous NIPP1. We performed a gene expression profiling of the HTO cell lines, using Whole Human Genome Oligo microarrays from Agilent. A Paired SAM analysis identified 1365 genes with an altered expression (P < 0.01) between the Flag-NIPP1 and parental cell lines. Importantly, only 185 genes were differentially expressed (P<0.01) between the Flag-NIPP1m and parental cell lines. Even more strikingly, only 5% of the genes that were affected by the expression of Flag-NIPP1 also showed a significantly different expression in the Flag-NIPP1m cells. A similar small overlap was noted when the analysis was restricted to the 50 genes that were most upregulated or downregulated by the expression of Flag-NIPP1. Finally, scatter plot analysis revealed no significant correlation between the genes that were affected by the expression of Flag-NIPP1 or Flag-NIPP1m. Collectively, these data demonstrate that a moderate increase in the concentration of NIPP1 affects the expression of numerous genes by a mechanism that depends on associated PP1. In total 12 samples were processed. Three different cell lines were analysed: HTO_parental, HTO_NIPP1wt and HTO_NIPP1m. For each cell line 4 replicates were obtained. The HTO_parental cell line is the control cell line. For the HTO_NIPP1wt the 4 replicates were obtained from two replicates of two different transgenic cell lines expressing FlagNIPP1wt (cell line wt n°1 and 2). Each cell line derived from the same parental control cell line.

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Identification of PP1-fusion phosphatase substrates

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