A protein phosphatase 1 specific phos phatase ta rgeting p eptide (PhosTAP) to identify the PP1 phosphatome

Abstract

Phosphoprotein phosphatases (PPPs) are the key serine/threonine phosphatases that regulate all essential signaling cascades. In particular, Protein Phosphatase 1 (PP1) dephosphorylates ~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 PP1 regulator. This PhosTAP can be used as a PP1 recruitment tool for Phosphorylation Targeting Chimera (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 lags behind that of their kinase counterparts. Using PhosTAP-based proteomics, 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 counteracts the activities of mitotic kinases. Finally, we also found that Haspin kinase is a direct substrate of PP1 and that its PP1-dependent dephosphorylation modulates its activity during anaphase. Together, we show that PP1-specific PhosTAPs are a powerful tool for +studying PP1 activity in vitro and in cells.

Keywords: phosphatase targeting peptide (PhosTAP); phosphoprotein phosphatases (PPP); protein engineering; protein phosphatase 1 (PP1); protein–protein interactions.

Fig. 1.

Engineering a PP1 panspecific PhosTAP_PP1. (A) Optimization of PhosTAP_PP1. PP1 SLiMs are bolded. Modified amino acid residues are colored in red. (B) Upper panel, 2Fo-Fc (σ, 1.0) electron density for the bound PhosTAP_PP1. Lower panel, the PhosTAP_PP1:PP1α complex: PhosTAP_PP1 shown as a blue/orange surface and PP1 as a gray surface (bound Mn²⁺ ions shown as spheres in pink). PP1-specific SLiMs, which bind at the expected PP1 SLiM binding sites, are annotated. (C) ITC thermograms of PhosTAP_PP1 with PP1α and PP1γ. (D) GFP-PhosTAP_PP1 cellular distribution in HeLa cells. White bar = 20 µm. (E) GFP-Trap pulldown followed by immunoblotting showing the binding of PhosTAP_PP1 to endogenous PP1α, PP1β, and PP1γ in HEK293T cells. (F) MS analysis of GFP-PhosTAP_PP1 and GFP-PhosTAP_PP1ff to PP1α, PP1β, and PP1γ (n = 3; mean ± SD). (G) Quantification of mitotic duration (left) and mitotic cell death (right) in control HeLa cells and HeLa cells expressing low or high levels of EGFP-PhosTAP_PP1.

Fig. 2.

Displacement of PP1 endogenous interactors by PhosTAP_PP1. (A) Schematic of competitive PIB-mass spectrometry using PhosTAP_PP1 (and PhosTAP_PP1ff). (B) Heat map showing the displacement of PP1 interacting proteins by PhosTAP_PP1/PhosTAP_PP1ff. Each box corresponds to the mean of three replicates. Color intensity indicates log₂ area. (C) Heat map showing the PP2Ac, PP4c, PP5c, PP6c, and their regulatory subunits detected by PIB-MS. (D) Correlation of competitive PIB assay of PhosTAP_PP1 and PhosTAP_PP1ff.

Fig. 3.

PhosTAP_PP1 phosphoproteomics reveals PP1 substrates and site-specific dephosphorylation motif preference. (A) Schematic describing the effect of PhosTAP_PP1/PhosTAP_PP1ff on PP1 dephosphorylation activity by competitively displacing endogenous PP1 interactors. (B) Schematic of the phosphoproteomic experiment with PhosTAP_PP1 and PhosTAP_PP1ff. (C and D) Volcano plot of quantified phosphosites. Significantly regulated phosphosites that increase in the presence of PhosTAP_PP1 (C) and PhosTAP_PP1ff (D) are shown in green. (E) WebLogos of all phospho (all, Top), phospho-threonine (pT, Middle), and phospho-serine (pS, Bottom) sites of all phosphosites regulated by leveraging PhosTAP_PP1/PhosTAP_PP1ff. (F) Icelogos of all phospho (all, Top), phospho-threonine (pT, Middle), and phospho-serine (pS, Bottom) sites of all phosphosites regulated by leveraging PhosTAP_PP1/PhosTAP_PP1ff.

Fig. 4.

Haspin is a direct substrate of PP1. (A) Quantification of Haspin pT97 abundance in the presence of PhosTAP_PP1. (B) 2D [¹H, ¹⁵N] HSQC spectrum of ¹⁵N-labeled Haspin_197-293 (black) and ¹⁵N-labeled Haspin_197-293 in complexed with PP1α_7-330 (red). (C) In vitro dephosphorylation assay of Haspin pT97 by PP1 (n = 3; mean ± SD). (D) Aligned sequence of the RVxF motif on Haspin depicting conservation between various species and mutation of Haspin RSVLF to ASALA. (E) In vitro kinase assay of synthetic Haspin substrate Hasptide by Haspin WT and 3A (n = 3; mean ± SD). (F) Control HeLa cells and HeLa cells transiently transfected with Flag-Haspin WT and Flag-Haspin 3A were stained for FLAG signal and phospho-H3T3. The figure shows confocal immunofluorescence images of metaphase and anaphase cells. (Scale bars represent 10 µm). (G) Quantification of GFP (FLAG) and RFP (pH3T3) fluorescence intensities of immunofluorescence images from F (n = 5; mean ± SD).