PP1-PIP fusion proteins.

A. Structures of fusion proteins. N-terminally Flag-tagged PP1α(7-304) is linked to sequences from each of the four families of RVxF-ΦΦ-R-W PIPs, shown as an open box. Each fusion contains sequences immediately C-terminal to the PP1 interaction motif (coloured lines) including known protein interaction domains previously implicated in potential substrate interactions (coloured blocks). For PIP sequences in each fusion see Figure S1A and Methods.

Middle, sequences of the RVxF-ΦΦ-R-W string in each PIP, with motifs coloured. Each fusion contains the sequences C-terminal to the dashed line, represents the position of PP1-SGSGS linker insertion.

Bottom, structures of PP1/PIP complexes. Crystal structures of different PIP/PP1 complexes superimposed, aligned on PP1. Grey: PP1 (PDB: 4MOV), with PIP sequences as follows: green, Phactr1 (PDB: 6ZEE); magenta, Neurabin (PDB: 3HVQ); orange, R15A (PDB: 7NZM); blue, PNUTS (PDB: 4MOY). Dashed line, GSGSG linker.

B. Activity of PP1-Phactr1 expressed in Flp-In T-Rex 293 cells. PP1-Phactr1 expression was induced by tetracycline as indicated. Phosphorylation of Phactr1/PP1 substrates IRSp53 S455 and Afadin S1275 is shown below.

C. Analysis of Phactr1/PP1 substrate Afadin pS1275 phosphorylation in Flp-In TRex 293 cells expressing PP1 and PP1-fusion proteins.

PP1-PIP fusion phosphoproteomics.

A. TMT phosphoproteomics workflow.

B. Average sample-to-sample correlations between triplicates from cells expressing the different fusion proteins, PP1α(7-304)-SGSGS alone, or empty vector. For the same fusion-expressing cell lines, the average of Pearson coefficients of correlation within a triplicate are shown.

C. Phosphorylation sites enriched in PP1 samples as opposed to PP1-Phactr1 fusion samples. Purple - hits conforming to the Phactr1 substrate motif S/T-x2,3-Φ-L. Red – top hits identified previously (Fedoryshchak et al., 2020). Dashed line, 5% false-discovery rate cutoff.

D. Enrichment of hits conforming to the Phactr1 substrate motif S/T-x2,3-Φ-L and of hits identified in the previous study in all Phactr samples calculated using Fisher’s exact test.

E. Venn diagram showing overlap between hits identified as potential Phactr1-4 substrates.

Phosphoproteomics of PP1-Neurabin and PP1-Spinophilin.

A-B. Identification of PP1-Neurabin (A) and PP1-Spinophilin (B) substrates by depletion of phosphorylation sites in the corresponding samples compared to average abundance in the dataset (excluding PP1-Neurabin and PP1-Spinophilin). Dashed line, 5% false-discovery threshold; significantly depleted sites shown in red.

C. Sequences of significantly depleted phosphorylation sites identified in A and B.

D. Immunoblot analysis of 4E-BP1 phosphorylation sites in 293 Flp-In T-Rex cells upon expression of PP1-Neurabin or empty vector.

E. Protein synthesis quantification assay. 293 Flp-In T-Rex cells expressing vector alone, PP1-Neurabin, or PP1, were induced with tetracycline (50 nM) and/or treated with rapamycin (50 nM) for 16h as indicated before treatment with O-propargyl puromycin to label nascent polypeptides, which were conjugated to AlexaFluor-488 azide and quantified by flow cytometry. Fluorescence intensities were normalized to untreated cells.

4E-BP1 is a substrate of PP1-Neurabin.

A. mCherry-tagged wild-type 4E-BP1 or 4E-BP1(118+A) were expressed and purified from 293 cells, incubated with increasing amounts of recombinant PP1-Neurabin. Phosphorylation of the indicated sites was analysed by immunoblotting.

B. Quantification of A.

C. Left, sequence alignment of potential Neurabin/Spinophilin PDZ domain ligands.

Grey shading, hydrophobic residues; pink, acidic residues; cyan, basic residues; orange, hydrophilic residues. Underlining shows sequences N-terminally linked to 6-carboxyfluorescein (FAM) for use in fluorescence polarisation (FP) assay. Right, binding affinities for the Neurabin and Spinophilin PDZ domains as determined in the FP assay.

D. FP assay. FAM-labelled peptides (see C) were titrated with increasing concentrations of recombinant Neurabin PDZ domain and affinity estimated from change in fluorescence anisotropy. For Spinophilin data see Figure S4B.

Substrate specificity determinants of PP1-Neurabin.

A. Top, synthetic substrate peptides contain either the 4E-BP1 T70 or IRSp53 S455 phosphorylation sites, joined by a GSG linker to the Neurabin PDZ-binding C-terminal sequences. PBM, PDZ-binding motif. Below, sequences of the different peptides analysed; highlights indicate the dephosphorylation site (yellow), the +4/+6 region (orange), and the PDZ-binding sequence (cyan), with alanine and other substitutions indicated in red. Right, KM and catalytic efficiencies; for catalytic efficiency quantification see Figure S5A.

B-E. Peptides were treated with recombinant PP1-Neurabin, PP1-Phactr1 or PP1 in the presence of the phosphate sensor, and KM and catalytic efficiencies determined. Panels show relative catalytic efficiencies as determined from data displayed in Figure S5B-S5E. B. Comparison of Neurabin-PP1 and Phactr1-PP1 substrates 4E-BP1 and IRSp53. C. Role of the +4/+6 region in 4E-BP1 substrate recognition. D. Role of the +5 residue in IRSp53 substrate recognition. E. Role of 4E-BP1 +1/+2 residues.

Structural analysis of 4E-BP1 interactions with PP1-Neurabin.

A. Schematic of the PP1-4E-BP1 chimera and of Neurabin PP1-interacting and PDZ domain sequences.

B. Crystal structure of the PP1-4E-BP1/Neurabin complex. PP1 in white surface representation, Neurabin in lilac surface representation, 4E-BP1 in blue stick representation, with unresolved sequences indicated by dashed line. PP1 active site presumptive Mn2+ ions in purple.

C. Comparison of PP1-4E-BP1/Neurabin complex structure with previous Neurabin/PP1 holophosphatase structure (Ragusa et al., 2010). PP1 in white surface representation, Neurabin in ribbon representation (lilac, PP1-4E-BP1/Neurabin; red, Neurabin/PP1). 4E-BP1 in blue stick representation, unresolved sequences not shown. Structures are superimposed on PP1 residues 7-298 (rmsd=0.21 Å, 277 alpha carbons).

D. Close-up view of interactions between 4E-BP1 C-terminal sequences (blue sticks) with the Neurabin PDZ domain (lilac cartoons).

E. AlphaFold3 model of the phosphorylated PP1-4E-BP1 chimera / Neurabin(423-593) interaction. A close-up view of predicted interaction of pT70 with the PP1 catalytic site is shown. For PAE and pLDDT plots, see Figure S6A. PP1 and Neurabin are shown respectively in white and lilac surface representation, with PP1 active site Mn2+ ions in purple. 4E-BP1 sequences are in stick representation, colour-coded according to the AlphaFold3 pLDDT score (inset). See also Figure S6B, S6C.

F. AlphaFold3 modelling of the Neurabin(423-593)/PP1 - 5x phospho-4E-BP1 interaction. PP1 and Neurabin are shown respectively in white and lilac surface representation, with PP1 active site Mn2+ ions in purple. 4E-BP1 sequences are in ribbon and stick representation, colour-coded according to the AlphaFold3 pLDDT score (inset), with the 4E-BP1 phosphorylations at T37, T46, S65, T70 and S101 shown in spheres. For PAE and pLDDT plots, see Figure S6F.

Crystallographic data and refinement statistics.

A. PIP sequences in each fusion. Sequences of the PP1-binding and C-terminal sequences in each RVxF-ΦΦ-R-W PIP fusion are shown. Blue line indicates fusion point. Known interaction domains are overlined.

B. IRSp53 WT or L460A mutant were transfected into 293 Flp-In T-Rex cells expressing the different fusion proteins, induced by tetracycline. Immunoblotting for total and S455-phosphorylated IRSp53 is shown. Flag tag indicates expression of the fusion phosphatases. Quantification is at right.

A. Phosphorylation sites depleted in PP1-expressing samples compared with control empty-vector samples. Dashed line, 1% false-discovery rate cutoff.

B. Frequency plots for residues identified as PP1 hits in (A) and for all phosphorylation sites in the analysis. Enrichment is broadly consistent with published findings (Hoermann et al., 2020).

C-E. Phosphorylation sites depleted in PP1-Phactr2 (C), PP1-Phactr3 (D) and PP1-Phactr4 (E) fusion samples compared with PP1 samples. Purple - hits conforming to the Phactr1 substrate motif S/T-x2,3-Φ-L. Red – top Phactr1/PP1 hits identified previously (Fedoryshchak et al., 2020).

A-C. Identification of PP1-R15A (A), PP1-R15B (B) and PP1-PNUTS (C) substrates by depletion of phosphorylation sites in the corresponding samples as opposed to average abundance in the dataset. Dashed line, 5% false-discovery threshold; red, significantly depleted phosphorylation sites; blue, PNUTS phosphorylation sites arising from overexpression of PP1-PNUTS.

D. Sequences of significantly depleted phosphorylation sites from PP1-PNUTS samples.

E. Comparison of total protein levels in cells PP1-Neurabin cells with or without induction. Neurabin and 4E-BPs are highlighted in red. Dashed line, 5% false-discovery threshold.

F. Specificity analysis of the commercial anti-phospho-S65 antibody.

G. mTORC1 pathway schematic (see (Hoeffer and Klann, 2010; Liu and Sabatini, 2020)).

A. Immunoblotting analysis of wildtype mCherry-4E-BP1 or mutants either lacking the 6 C-terminal residues (ΔCter), or containing an additional C-terminal alanine (118+A) upon expression in 293 cells with or without PP1-Neurabin expression as indicated.

B. Left, sequence alignment of potential Neurabin/Spinophilin PDZ domain ligands.

Grey shading, hydrophobic residues; pink, acidic residues; cyan, basic residues; orange, hydrophilic residues. Underlining shows sequences N-terminally linked to 6-carboxyfluorescein (FAM) for use in fluorescence polarisation (FP) assay. FAM-labelled peptides were titrated with increasing concentrations of recombinant Spinophilin PDZ domain and affinity estimated from change in fluorescence anisotropy (for summary see Figure 4C).

C. Immunoblotting analysis of S6K phosphorylation 293 Flp-In T-Rex cells upon expression of PP1-Neurabin or empty vector.

A. Catalytic efficiencies for the various peptide dephosphorylation reactions by PP1-Neurabin, PP1-Phactr1 and PP1 are shown.

B-E. Dephosphorylation reaction rates plotted against substrate concentration for different sets of phosphopeptides with PP1-Neurabin, PP1-Phactr1 or PP1.

A, B. AlphaFold3 models of the phosphorylated (A) and unphosphorylated (B) PP1-4E-BP1 chimera / Neurabin(423-593) interaction. Left, PAE plots; right, pLDDT plots, with confidence boundaries indicated by dashed lines (>90%, very high (side-chains); 70-90%, high (main-chain); 50-70%, low).

C, D. AlphaFold3 models of the phosphorylated (C) and unphosphorylated (D) PP1-4E-BP1 chimera / Neurabin(423-593) interaction. PP1 and Neurabin are shown respectively in white and lilac surface representation with PP1 active site Mn2+ ions in purple. 4E-BP1 sequences are in stick representation, colour coded according to the AlphaFold3 pLDDT score (inset), with pT70 and T70 in space-fill; linker residues are in black. Below are shown close-up views of predicted interactions with the PP1 catalytic site. For PAE and pLDDT plots, see A, B.

E. Comparison of crystal structure and AlphaFold3 model of 4E-BP1/PDZ interactions in phosphorylated and unphosphorylated PP1-4E-BP1 chimera / Neurabin(423-593) interaction. Predicted structures are oriented by superposition of the PDZ domain, shown in lilac ribbon representation. 4E-BP1 sequences are in stick representation, colour coded according to the AlphaFold3 pLDDT score (inset)

F. AlphaFold3 modelling of the Neurabin(423-593)/PP1 - 5x phospho-4E-BP1 interaction. Left, PAE plots; right, pLDDT plots, with confidence boundaries indicated by dashed lines (>90%, very high (side-chains); 70-90%, high (main-chain); 50-70%, low).