Fig 2: Substrate interactions with the Phactr1/PP1 holoenzyme.(A, B) Structures of (A) the Phactr1/PP1-IRSp53(449-465) and (B) the Phactr1/PP1-spectrin(1025–1039) complexes, displayed as in Figure 1, with IRSp53 and spectrin displayed in orange and magenta sticks, respectively. (C) Summary of substrate interactions. Hydrogen bonds are shown as thick dashed lines: grey for both substrates; colour, for specific substrate. Composite hydrophobic surface residues are highlighted in blue (see F). (D) Inversion of the recruited phosphate. Phosphate and metal ion contacts in the Phactr1/PP1 and in Phactr1/PP1-IRSp53 structures are shown. Metal coordination bonds, solid continuous lines; hydrogen bonds, dashed lines; W1 and W2, water molecules. (E) Potential catalytic mechanism. Left, a hypothetical substrate complex, based on the Phactr1/PP1 complex, assuming that its phosphate corresponds to that of IRSp53 pS455. Right, the observed Phactr1/PP1-IRSp53 product complex. W1 and W2, water molecules; grey bars, metal coordination bonds; dashes, hydrogen bonds. Proposed nucleophilic attack by activated W1 results in phosphate inversion. (F) Docking of the SxxxLL motif (sticks) with the Phactr1/PP1 hydrophobic pocket. Phactr1/PP1 in surface representation, with the composite hydrophobic surface in light blue, and other Phactr1 and PP1 surfaces in green and white, respectively.

Fig 3: Efficient dephosphorylation involves substrate interaction with the Phactr1/PP1 composite surface.(A) Phactr1/PP1 dephosphorylation of alanine substitution derivatives of IRSp53 S455 substrate 19mer phosphopeptides. KM values are highlighted: green,<40 µM; yellow, 40–80 µM; red,>80 µM. (B) Immunoblot analysis of total IRSp53 and IRSp53 phospho-S455 levels after expression of wild-type IRSp53 or IRSp53 L460A in NIH3T3 cells with 30' CD or LB treatment as indicated. (C,D) Overlay binding affinity assay of IRSp53 (C) and spectrin αII (D). Arrays contained the variants of the wild-type sequence, in which each amino acid is systematically changed to each other amino acid as indicated vertically, with wild-type sequence circled in green. Yellow line, position of the invariant unphosphorylated target serine.

Fig 4: 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 1—figure supplement 1A 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, representing 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 T-REx 293 cells expressing PP1 and PP1-fusion proteins. Figure 1—source data 1.Sequence alignments related to Figure 1A, Figure 1—figure supplement 1A. Figure 1—source data 2.Original files for western blot analysis displayed in Figure 1B. Figure 1—source data 3.Full-size western blots indicating the relevant bands and treatments related to Figure 1B. Figure 1—source data 4.Original files for western blot analysis displayed in Figure 1C. Figure 1—source data 5.Full-size western blots indicating the relevant bands and treatments related to Figure 1C.

Fig 5: 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 (FEMDI); MUT, mutated PBM (FEsgs). 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. Peptides were treated with recombinant PP1-Neurabin, PP1-Phactr1, or PP1 in the presence of the phosphate sensor, and KM and catalytic efficiencies determined. KM are shown at the right; for catalytic efficiency quantification, see Figure 5—figure supplement 1A. For raw and processed data, see Supplementary file 3. (B–E) Panels show relative catalytic efficiencies as determined from data displayed in Figure 5—figure supplement 1B–E. Each panel shows different subsets of the data to highlight comparison between different enzymes and/or substrates. For raw and processed data, see Supplementary file 3. (B) Comparison of Neurabin-PP1 and Phactr1-PP1 substrates 4E-BP1 and IRSp53 to assess the role of the Neurabin PDZ domain in substrate recognition. (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. Figure 5—source data 1.Activity assay data related to Figure 5, Figure 5—figure supplement 1.