Journal: eLife

Article Title: Molecular basis for substrate specificity of the Phactr1/PP1 phosphatase holoenzyme

doi: 10.7554/eLife.61509

Figure Lengend Snippet: ( A ) Rat hippocampal neurons were transfected with plasmids expressing Phactr1 derivatives and GFP, fixed after 1 day and their morphology scored blindly. Scale bar, 10 µm. Statistical significance was assessed by unpaired t-test with Welch's correction (means ± SEM, n = 3–5; **, p<0.01). ( B ) RT-PCR analysis shows that Phactr1 inactivation in brain does not affect expression of other Phactr family members, relative to Rps16 expression. ( C ) Wildtype and Phactr1-null neurons were treated with cytochalasin D (CD) and phosphorylation changes assessed by TMT phosphoproteomics. Top, amino acid frequency table of phosphopeptides showing a statistically significant dependence on Phactr1 (FDR < 0.2), bottom, amino acid frequency table of all phosphopeptides. ( D ) Quantitation of immunoblot data shown in . Lysates from Phactr1-null or wildtype neurons were left untreated or treated with CD or LB for 30' were analysed by immunoblotting with antibodies against IRSp53 pS455 (left); afadin pS1275 (centre); and spectrin αII pS1031 (right). See also and .

Article Snippet: Rabbit anti-IRSp53 pS455, anti-afadin pS1275 and anti-spectrin pS1031 antibodies were custom-made (Covalab); for antigen sequences used see .

Techniques: Transfection, Expressing, Reverse Transcription Polymerase Chain Reaction, Quantitation Assay, Western Blot

Journal: eLife

Article Title: Molecular basis for substrate specificity of the Phactr1/PP1 phosphatase holoenzyme

doi: 10.7554/eLife.61509

Figure Lengend Snippet:

Article Snippet: Rabbit anti-IRSp53 pS455, anti-afadin pS1275 and anti-spectrin pS1031 antibodies were custom-made (Covalab); for antigen sequences used see .

Techniques: Expressing, Construct, Recombinant, Plasmid Preparation, Sequencing, Mutagenesis, Clone Assay, Fractionation, SYBR Green Assay, Software, Protease Inhibitor, Peptide Microarray

Journal: eLife

Article Title: Molecular basis for substrate specificity of the Phactr1/PP1 phosphatase holoenzyme

doi: 10.7554/eLife.61509

Figure Lengend Snippet: ( A ) Phactr1/PP1 dephosphorylation of alanine substitution derivatives of IRSp53 S455 substrate 19mer phosphopeptides. K M 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.

Article Snippet: Rabbit anti-IRSp53 pS455, anti-afadin pS1275 and anti-spectrin pS1031 antibodies were custom-made (Covalab); for antigen sequences used see .

Techniques: De-Phosphorylation Assay, Western Blot, Expressing, Binding Assay, Sequencing

Journal: eLife

Article Title: Molecular basis for substrate specificity of the Phactr1/PP1 phosphatase holoenzyme

doi: 10.7554/eLife.61509

Figure Lengend Snippet: ( A ) Expression of the Phactr1 XXX (464-580), which constitutively binds PP1, induces cytoskeletal rearrangements in NIH3T3 fibroblasts. Scale bar, 20 µm. ( B ) Left, immunoblot analysis of Flag-Phactr1 XXX and Flag-Phactr1 XXX ΔC expression in NIH3T3 cell lines cultured under SILAC labelling conditions. Detection was with Flag antibody. Right, dephosphorylation of endogenous IRSp53 pS455 upon expression of Flag-Phactr1 XXX , detected with anti-IRSp53 p455 antibody. For protein structures, see . ( C ). Phactr1/PP1 phosphatase activity assay data for selected substrates from (data are ± SD, n = 3). ( D, E ) Immunoblot analysis of IRSp53 pS455 and afadin pS1275 in NIH3T3 cells upon overnight serum starvation and 30' serum stimulation ( D ), or 30' treatment with the actin-binding drugs cytochalasin D (CD) or latrunculin B (LB). Quantitation below (data are ± SD, n = 3). See also .

Article Snippet: Rabbit anti-IRSp53 pS455, anti-afadin pS1275 and anti-spectrin pS1031 antibodies were custom-made (Covalab); for antigen sequences used see .

Techniques: Expressing, Western Blot, Cell Culture, De-Phosphorylation Assay, Phosphatase Assay, Binding Assay, Quantitation Assay

Journal: eLife

Article Title: Molecular basis for substrate specificity of the Phactr1/PP1 phosphatase holoenzyme

doi: 10.7554/eLife.61509

Figure Lengend Snippet: ( A, B ) Flexibility in target serine-hydrophobic pocket binding residue spacing, illustrated by ( A ) structure of the Phactr1/PP1-IRSp53(S455E) complex, compared with ( B ) the Phactr1/PP1-IRSp53 wildtype complex. ( C ) Phactr1/PP1 dephosphorylation of derivatives of IRSp53 peptides carrying phosphate at different locations, highlighted in yellow. Phosphatase activity data is shown below (data are mean ± SD, WT n = 15, others n = 2–6) ( D ) Schematic of the PP1-Phactr1 fusion protein PP1-Phactr1(526-580). ( E ) Top, phosphatase activity data for the indicated substrates and enzymes. Bottom, relative catalytic efficiencies for the different substrates (data are mean ± SD, n = from 1 to 15).

Article Snippet: Rabbit anti-IRSp53 pS455, anti-afadin pS1275 and anti-spectrin pS1031 antibodies were custom-made (Covalab); for antigen sequences used see .

Techniques: Binding Assay, De-Phosphorylation Assay, Activity Assay

Journal: eLife

Article Title: Molecular basis for substrate specificity of the Phactr1/PP1 phosphatase holoenzyme

doi: 10.7554/eLife.61509

Figure Lengend Snippet: ( A, B ) Structures of ( A ) the Phactr1/PP1-IRSp53(449-465) and ( B ) the Phactr1/PP1-spectrin(1025–1039) complexes, displayed as in , 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.

Article Snippet: Rabbit anti-IRSp53 pS455, anti-afadin pS1275 and anti-spectrin pS1031 antibodies were custom-made (Covalab); for antigen sequences used see .

Techniques:

Journal: eLife

Article Title: Molecular basis for substrate specificity of the Phactr1/PP1 phosphatase holoenzyme

doi: 10.7554/eLife.61509

Figure Lengend Snippet: ( A ) Rat hippocampal neurons were transfected with plasmids expressing Phactr1 derivatives and GFP, fixed after 1 day and their morphology scored blindly. Scale bar, 10 µm. Statistical significance was assessed by unpaired t-test with Welch's correction (means ± SEM, n = 3–5; **, p<0.01). ( B ) RT-PCR analysis shows that Phactr1 inactivation in brain does not affect expression of other Phactr family members, relative to Rps16 expression. ( C ) Wildtype and Phactr1-null neurons were treated with cytochalasin D (CD) and phosphorylation changes assessed by TMT phosphoproteomics. Top, amino acid frequency table of phosphopeptides showing a statistically significant dependence on Phactr1 (FDR < 0.2), bottom, amino acid frequency table of all phosphopeptides. ( D ) Quantitation of immunoblot data shown in . Lysates from Phactr1-null or wildtype neurons were left untreated or treated with CD or LB for 30' were analysed by immunoblotting with antibodies against IRSp53 pS455 (left); afadin pS1275 (centre); and spectrin αII pS1031 (right). See also and .

Article Snippet: Rabbit anti-IRSp53 pS455, anti-afadin pS1275 and anti-spectrin pS1031 antibodies were custom-made (Covalab); for antigen sequences used see .

Techniques: Transfection, Expressing, Reverse Transcription Polymerase Chain Reaction, Quantitation Assay, Western Blot

Journal: eLife

Article Title: Molecular basis for substrate specificity of the Phactr1/PP1 phosphatase holoenzyme

doi: 10.7554/eLife.61509

Figure Lengend Snippet: Crystallographic data and refinement statistics.

Article Snippet: Rabbit anti-IRSp53 pS455, anti-afadin pS1275 and anti-spectrin pS1031 antibodies were custom-made (Covalab); for antigen sequences used see .

Techniques:

Journal: eLife

Article Title: Molecular basis for substrate specificity of the Phactr1/PP1 phosphatase holoenzyme

doi: 10.7554/eLife.61509

Figure Lengend Snippet: ( A, B ) Structure of the second copy of the Phactr1/PP1-substrate complexes in each asymmetric unit. Each structure is shown alone (top), and superimposed on the first copy (bottom; see ). ( A ) In IRSp53 complex 2, the electrostatic interactions between substrate residues +4, +5 and +6 and Phactr1 basic residues are water-bridged rather than direct. ( B ) In spectrin complex 2, the E1033/N1034 spectrin peptide bond is inverted, losing the mainchain carbonyl and N1034 spectrin sidechain interactions with Phactr1 R576, and the hydrogen bonding interaction between the L1035 spectrin (+4) carbonyl and Phactr1 K550 is water-bridged rather than direct. ( C ) Superposition of the first copy of each complex (shown separately in ). ( D ) Omit map contoured at 3σ of the first copy of the IRSp53 complex.

Article Snippet: Rabbit anti-IRSp53 pS455, anti-afadin pS1275 and anti-spectrin pS1031 antibodies were custom-made (Covalab); for antigen sequences used see .

Techniques:

Journal: eLife

Article Title: Molecular basis for substrate specificity of the Phactr1/PP1 phosphatase holoenzyme

doi: 10.7554/eLife.61509

Figure Lengend Snippet: Left: the PP5-Cdc37(S13E) complex . White surface, PP5; lilac sticks, Cdc37(S13E). Centre, superposition of the PP5-Cdc37(S13E) and Phactr1/PP1-IRSp53 structures. Right, comparison of molecular interactions with substrates at the catalytic site residues of PP5 (lilac) and PP1 (orange). Hydrogen bonds are shown as thick dashed lines: grey for both substrates; colour, for specific substrate.

Article Snippet: Rabbit anti-IRSp53 pS455, anti-afadin pS1275 and anti-spectrin pS1031 antibodies were custom-made (Covalab); for antigen sequences used see .

Techniques:

Journal: eLife

Article Title: Molecular basis for substrate specificity of the Phactr1/PP1 phosphatase holoenzyme

doi: 10.7554/eLife.61509

Figure Lengend Snippet: Left, stick representation of molecular interactions at the catalytic site of PP5-Cdc37(S13E). Centre, superposition of the PP5-Cdc37(S13E) (lilac) and Phactr1/PP1 complex (white) catalytic sites. Note the coincidence of the PP5-Cdc37(S13E) phosphomimetic glutamate with the oxygens of the phosphate present in the Phactr1/PP1 complex. Right, comparison of the PP5-Cdc37(S13E) and Phactr1/PP1-IRSp53 complexes (yellow). Note the inversion of the phosphate and the absence of W1.

Article Snippet: Rabbit anti-IRSp53 pS455, anti-afadin pS1275 and anti-spectrin pS1031 antibodies were custom-made (Covalab); for antigen sequences used see .

Techniques:

Journal: eLife

Article Title: Molecular basis for substrate specificity of the Phactr1/PP1 phosphatase holoenzyme

doi: 10.7554/eLife.61509

Figure Lengend Snippet:

Article Snippet: Rabbit anti-IRSp53 pS455, anti-afadin pS1275 and anti-spectrin pS1031 antibodies were custom-made (Covalab); for antigen sequences used see .

Techniques: Expressing, Construct, Recombinant, Plasmid Preparation, Sequencing, Mutagenesis, Clone Assay, Fractionation, SYBR Green Assay, Software, Protease Inhibitor, Peptide Microarray