1. Biochemistry and Chemical Biology
  2. Structural Biology and Molecular Biophysics
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Molecular basis for substrate specificity of the Phactr1/PP1 phosphatase holoenzyme

  1. Roman O Fedoryshchak
  2. Magdalena Přechová
  3. Abbey M Butler
  4. Rebecca Lee
  5. Nicola O'Reilly
  6. Helen R Flynn
  7. Ambrosius P Snijders
  8. Noreen Eder
  9. Sila Ultanir
  10. Stephane Mouilleron  Is a corresponding author
  11. Richard Treisman  Is a corresponding author
  1. Signalling and Transcription Laboratory, The Francis Crick Institute, United Kingdom
  2. Structural Biology Science Technology Platform, The Francis Crick Institute, United Kingdom
  3. Peptide Chemistry Science Technology Platform, The Francis Crick Institute, United Kingdom
  4. Proteomics Science Technology Platform, The Francis Crick Institute, United Kingdom
  5. Kinases and Brain Development Laboratory The Francis Crick Institute, United Kingdom
Research Article
Cite this article as: eLife 2020;9:e61509 doi: 10.7554/eLife.61509
6 figures, 2 tables and 6 additional files

Figures

Figure 1 with 2 supplements
Phactr1 binds PP1 using an extended RVxF-ϕϕ-R motif.

(A) Domain structure of Phactr family proteins. RPEL motifs, red; PP1-binding domain, green; nuclear localisation sequences, black. Below, Phactr1 C-terminal sequence, indicating RVxF-ϕϕ-R-W string, RPEL consensus, secondary structure elements, and mutations known to impair Phactr/PP1 interaction (Allen et al., 2004; Kim et al., 2007). (B) Structure-based alignment of Phactr1 PP1-binding sequences with other PP1 cofactors. (C) Bio-layer interferometry assay of PP1α(7-300) binding to Phactr1(517-580), its derivatives, and analogous sequences from Phactr2-4 (data are means ± SD, n = 3). (D) Structure of the Phactr1(516-580)/PP1α(7-300) complex. Phactr1, green ribbon, with RPEL motif highlighted in red, RVxF-ϕϕ-R-W sidechains as sticks; PP1, white surface, with acidic groove in red, hydrophobic groove in dark grey and C-terminal groove in gold; manganese ions, purple spheres; phosphate, orange sticks. (E–H) Detail of the individual interactions, with important residues highlighted (Phactr1, green bold; PP1, black). For comparison with other PIPs, see Figure 1—figure supplement 2A.

Figure 1—figure supplement 1
Crystallisation of Phactr1/PP1 complexes.

(A) Sequence-based alignment of Phactr-family C-terminal sequences, with differences highlighted in red. The extended Phactr1 RVxF-ϕϕ-R-W string is shown above, with presumed structurally equivalent residues indicated in blue on each sequence. Note that the sequence alignment introduces a gap into the atypical RVxF motif. (B) Structure of Phactr(507-580)/PP1α(7-300) solved at pH 8.5 (green) compared with that of Phactr1(516-580)/PP1α(7-300) solved at pH 5.25 (orange). The Phactr1 sequences C-terminal to the amphipathic α-helix (residue 467) adopt different conformations. In the pH 5.25 structure, residues 576–580 were not resolved in the density, and residues 567–575 were poorly resolved. (C) Omit map contoured at 3σ of the Phactr1/PP1 holoenzyme catalytic centre (metal ion/phosphate coordinating residues, white sticks; phosphate, orange sticks; water molecules, red spheres; coordination bonds, solid lines; hydrogen bonds, dashed lines). (D) Omit map contoured at 3σ of Phactr1 516–580 (green cartoon and sticks) across the PP1 surface (white).

Figure 1—figure supplement 2
Phactr1 binds PP1 using an extended RVxF-ϕϕ-R string.

(A) Top, structure-based alignment of PIPs that bind PP1 through extended RVxF motifs including PPP1R15B (Chen et al., 2015), spinophilin (Ragusa et al., 2010), PNUTS (Choy et al., 2014), Repoman, (Kumar et al., 2016), PPP1R15A (Choy et al., 2015), NIPP1 (O'Connell et al., 2012), KNL1 (Bajaj et al., 2018), GL and GM (Yu et al., 2018). Below, comparison of the trajectories of Phactr1 (green), PPP1R15B (yellow), spinophilin (magenta; with PDZ domain), PNUTS (blue), Repoman (grey), PPP1R15A (orange), NIPP1 (brown), KNL1 (purple), GL (light blue) and GM dark green. Boxes show detail of interactions by the RVxF, Arg, and Trp motifs in each PIP. (B) Phactr1 RPEL3 (salmon) overlaps the RVxF motif LIRF(519-522) (green) and adopts different conformations when bound to G-actin (left) and PP1 (right). L519, I520 and F522 make critical but distinct hydrophobic contacts in both the G-actin and PP1 complexes.

Figure 2 with 1 supplement
Phactr1/PP1 interaction remodels the PP1 hydrophobic groove.

(A) Molecular interactions by the Phactr1 C-terminal sequences (green; residues involved in intrachain interactions or contacting PP1 are shown as sticks). (B) The novel composite surface formed by Phactr1/PP1 interaction, showing the deep hydrophobic pocket, adjacent narrow amphipathic cavity with associated waters (red spheres) and glycerol (cyan sticks), and residues constituting the Phactr1-derived basic rim (blue). (C) PP1 surface electrostatics are transformed in the Phactr1/PP1 complex. Left, surface representation of the Phactr1/PP1 complex; centre, electrostatic surface potential representation of Phactr1/PP1 complex. Right, electrostatic surface potential representation of PP1 (positive, blue; negative, red).

Figure 2—figure supplement 1
Spinophilin remodels the PP1 hydrophobic groove in a different way to Phactr1.

Top, the spinophilin/PP1 complex shown in surface representation (magenta, spinophilin; grey, PP1); bottom, the analogous region of the Phactr1/PP1 complex in the same orientation from Figure 2B, repeated for comparison.

Figure 3 with 2 supplements
Identification of Phactr1/PP1 substrates.

(A–D), NIH3T3 cells, (EF) neuronal cells. (A) SILAC phosphoproteomics in NIH3T3 cells. NIH3T3 cell lines conditionally expressed Phactr1XXX (constitutively binds PP1, not G-actin), Phactr1XXXΔC (binds neither PP1 nor G-actin), or vector alone (Wiezlak et al., 2012). Forward and reverse SILAC phosphoproteomics were used to generate a dephosphorylation score, quantified below (red highlights, hits also detected as Phactr1-dependent in neurons). (B) Annotation enrichment analysis of the entire SILAC phosphoproteomics dataset for GO Biological Process terms, showing terms with dephosphorylation score >1, FDR < 0.02. (C) Candidate Phactr1/PP1 substrates ranked by dephosphorylation score (blue, cytoskeletal structural or regulatory proteins; asterisks, proteins with multiple dephosphorylation sites). Michaelis constant (KM, µM), specificity constant (kcat/KM, µM*min−1*U−1) and sequence context (blue, basic; red, acidic; black, hydrophobic) are shown. (*), KM could not be reliably determined; (nd), not done. (D) Amino acid frequency among phosphorylation sites with dephosphorylation score >2.5 (top) compared with all phosphorylation sites (bottom). (E) Phactr1-dependent protein dephosphorylation in cortical and hippocampal neurons treated with cytochalasin D (CD). Differential Z-score, the difference between the phosphorylation change observed in Phactr1-wildtype and Phactr1-null neurons, plotted versus statistical significance. Red highlights, peptides also observed in the NIH3T3 SILAC phosphoproteomics. (F) Validation of TMT phosphoproteomics data in primary cortical neurons treated for 30' with CD or latrunculin B (LB). For quantitation, see Figure 3—figure supplement 2D. See also Supplementary file 3.

Figure 3—figure supplement 1
Substrates of the Phactr1/PP1 complex in NIH3T3 fibroblasts.

(A) Expression of the Phactr1XXX(464-580), which constitutively binds PP1, induces cytoskeletal rearrangements in NIH3T3 fibroblasts. Scale bar, 20 µm. (B) Left, immunoblot analysis of Flag-Phactr1XXX and Flag-Phactr1XXXΔC 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-Phactr1XXX, detected with anti-IRSp53 p455 antibody. For protein structures, see Figure 3A. (C). Phactr1/PP1 phosphatase activity assay data for selected substrates from Figure 3D (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 Supplementary file 1.

Figure 3—figure supplement 2
Neuronal substrates of the Phactr1/PP1 complex.

(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 Figure 3F. 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 Supplementary files 1 and 3.

Figure 4 with 3 supplements
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.

Figure 4—figure supplement 1
Substrate interactions in the Phactr1/PP1 complex.

(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 Figure 4). (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/N1034spectrin peptide bond is inverted, losing the mainchain carbonyl and N1034spectrin sidechain interactions with Phactr1 R576, and the hydrogen bonding interaction between the L1035spectrin(+4) carbonyl and Phactr1 K550 is water-bridged rather than direct. (C) Superposition of the first copy of each complex (shown separately in Figure 4A and B). (D) Omit map contoured at 3σ of the first copy of the IRSp53 complex.

Figure 4—figure supplement 2
Opposite polarity of substrate binding to PP5 and Phactr1/PP1.

Left: the PP5-Cdc37(S13E) complex (Oberoi et al., 2016). 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.

Figure 4—figure supplement 3
Comparison of the PP5-Cdc37(S13E), Phactr1/PP1 and Phactr1/PP1-IRSp53 structures gives insight into catalytic mechanism.

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.

Figure 5 with 1 supplement
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.

Figure 5—figure supplement 1
Binding of candidate Phactr1/PP1 substrates in the peptide array overlay assay.

19-mer unphosphorylated peptides from the indicated substrates were arrayed in triplicate as shown and tested for their ability to recruit GST-Phactr1(516-580)/PP1 from solution. Visualisation was with anti-GST antibody.

Figure 6 with 1 supplement
Flexible substrate interactions and substrate specificity of the Phactr1/PP1 complex.

(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).

Figure 6—figure supplement 1
Flexible substrate interactions and substrate specificity of the Phactr1/PP1 complex.

(A) Gel filtration profiles of the Phactr1/PP1 (green line) and spinophilin/PP1 complexes (magenta line) used for the substrate-specificity analysis. (B) Structure of the PP1-Phactr1 fusion protein, solved at 1.78 Å resolution. Phactr1 residues 526–580, dark blue cartoon; PP1, white surface; unresolved SGSGS linker, dashes; trajectory of Phactr1 residues 516–580 in the Phactr1/PP1 complex, green cartoon. PP1-Phactr1 exhibited 0.25 Å RMSD over 2395 atoms compared with the equivalent region of the Phactr1/PP1 holoenzyme complex. (C) Top, phosphatase activity data for 19mer peptides containing glycogen phosphorylase pS15 (top) and GluR1 pS863 (bottom). Relative catalytic efficiencies are shown in Figure 6E (glycogen phosphorylase pS15) or at right (GluR1 pS863) (data are ±half range, n = 2).

Tables

Table 1
Crystallographic data and refinement statistics.
Phactr1/PP1
(pH 8.5)
Phactr1/PP1
(pH 5.25)
Phactr1/PP1-IRSp53
(pH 8.5)
Phactr1/PP1-spectrin-αII
(pH 8.5)
Phactr1/PP1-IRSp53-S455E
(pH 8.5)
PP1-Phactr1(526-580)
(pH 8.5)
PDB ID6ZEE6ZEF6ZEG6ZEH6ZEI6ZEJ
Resolution range238.79–1.90
(1.93–1.90)
56.05–1.94
(2.01–1.94)
35.12–1.09
(1.13–1.09)
48.47–1.30
(1.35–1.30)
69.37–1.39
(1.44–1.39)
59.48–1.78
(1.84–1.78)
Space groupP65P 1P 21P 21P 21P 65
Unit cell a, b, c137.7 137.7 238.847.5 57.5 89.648.6 122.3 69.048.5 122.3 69.348.7 122.3 69.4137.37 137.37 238.02
α, β, γ90 90 12078.0 74.6 81.690 92.2 9090 92.1 9090 92.2 9090 90 120
Total reflections2 420 272 (99 345)522 449 (32 609)664 254 (62 356)1 278 289 (123 745)1 058 251 (79 018)5 001 498 (505 725)
Unique reflections200 820 (9 938)65 136 (6 061)332 349 (31 383)196 809 (19 297)162 314 (15 982)241 814 (24 087)
Multiplicity12.1 (10.0)8.0 (5.4)2.0 (2.0)6.5 (6.4)6.5 (4.9)20.7 (21.0)
Completeness (%)100 (100)98.5 (92.0)99.3 (93.7)99.1 (96.8)99.6 (98.3)99.7 (99.6)
Mean I/sigma(I)7.5 (1.1)9.7 (2.7)15.4 (1.8)5.9 (1.1)12.5 (1.1)12.9 (1.1)
Wilson B-factor33.13010.811.116.426.4
R-merge0.14 (2.1)0.13 (0.57)0.01 (0.44)0.13 (1.78)0.06 (1.05)0.179 (3.19)
R-meas0.16 (2.3)0.14 (0.64)0.02 (0.62)0.15 (1.94)0.07 (1.18)0.18 (3.26)
R-pim0.04 (0.75)0.04 (0.26)0.01 (0.44)0.05 (0.75)0.02 (0.52)0.04 (0.70)
CC1/20.99 (0.70)0.99 (0.88)1.0 (0.65)0.99 (0.47)1.0 (0.54)1.0 (0.54)
Reflections used in refinement199 843 (6 288)65 085 (6 061)332 111 (31 289)195 791 (19 135)162 184 (15 972)241 401 (24 054)
Reflections used for R-free10 138 (347)3 132 (295)16 696 (1 543)9 611 (927)1 999 (197)12 175 (1 276)
R-work0.23 (0.34)0.18 (0.22)0.11 (0.23)0.13 (0.23)0.12 (0.24)0.24 (0.36)
R-free0.26 (0.37)0.21 (0.24)0.14 (0.24)0.16 (0.26)0.15 (0.27)0.27 (0.40)
Number of non-hydrogen atoms18 2015 9627 2256 7786 82217 414
Macromolecules174285 6836 3396 1046 09916 682
Ligands333742193258
Solvent740242844655691674
Protein residues21607157547427422 094
RMS(bonds)0.0110.0050.010.010.0110.011
RMS(angles)0.850.841.171.151.290.84
Ramachandran favoured (%)9695.6297.1596.9996.9995.85
Ramachandran allowed (%)44.382.853.012.884.15
Ramachandran outliers (%)00000.140
Clashscore2.512.654.73.053.872.7
Average B-factor44.44116.21921.741
Macromolecules44.940.814.617.620.141.1
Ligands48.746.723.614.923.744.7
Solvent41.643.12832.835.437.1
Appendix 1—key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional
information
Gene Mus musculusAfdnUniProtQ9QZQ1Encodes Afadin, also known as AF6, MLLT4
Gene Mus musculusBaiap2UniProtQ8BKX1Encodes IRSp53, also known as BAIAP2
Gene Mus musculusSptan1UniProtP16546Encodes Spectrin, also known as SPTA2, Fodrin, SPTAN1
Gene Mus musculusPhactr1UniProtQ2M3X8
Strain E. coli5-alpha competent E. coliNEBC2992I
Strain E. coliProtein expression BL21 (DE3)NEBC2527H
Cell line Mus musculusNIH3T3ATCCCRL-1658
Strain, strain background Mus musculusPhactr1tm1d/tm1dM. PrechovaConstructed from Phactr1tm1a(KOMP)Wtsihttps://www.komp.org/
M. Přechová, PhD thesis (University College London)
AntibodyRabbit polyclonal anti-FlagSigmaF7425, RRID:AB_439687WB 1:500
AntibodyGoat polyclonal anti-IRSp53Abcamab15697
RRID:AB_302045
WB 1:500
AntibodyMouse monoclonal anti-Afadin (B-5)Santa Cruzsc-74433
RRID:AB_1118734
WB 1:200
Antibodyanti-GST HRP ConjugateVWRRPN1236
RRID:AB_771429
WB 1:1000
AntibodyMouse monoclonal anti-spectrin (D8B7)Abcamab11755
RRID:AB_298540
WB 1:500
AntibodyMouse monoclonal anti-Gapdh (G-9)Santa-Cruzsc-365062
RRID:AB_10847862
WB 1:1000
AntibodyRabbit polyclonal anti-IRSp53 pS455This paperWB 1:500
See Materials and methods;
Figure 3F
AntibodyRabbit polyclonal anti-afadin pS1282This paperWB 1:500
See Materials and methods;
Figure 3F
AntibodyRabbit polyclonal anti-spectrin pS1031This paperWB 1:500
See Materials and methods;
Figure 3F
AntibodyIRDye 680RD Secondary AntibodiesLicor925-68073
RRID:AB_2716687
WB 1:10000
AntibodyIRDye 800CW Secondary AntibodiesLicor925-32214
RRID:AB_2687553
WB 1:10000
Recombinant DNA reagent (plasmid)pcDNA3.1 IRSp53Dr. Eunjoon KimPMID:15673667
Recombinant DNA reagent (plasmid)pcDNA3.1 IRSp53 L460AThis paperSee Materials and methods;
Figure 5B
Recombinant DNA reagent (plasmid)pEF Phactr1Wiezlak et al., 2012
Recombinant DNA reagent (plasmid)pEF Phactr1(464-580)This paperSee Materials and methods;
Figure 3—figure supplement 1A
Recombinant DNA reagent (plasmid)pEF Phactr1XXX(464-580)This paperSee Materials and methods;
Figure 3—figure supplement 1A
Recombinant DNA reagent (plasmid)pTRIPZDiring et al., 2019
Recombinant DNA reagent (plasmid)pTRIPZ Phactr1XXXThis paperSee Wiezlak et al., 2012;
Materials and methods;
Figure 3—figure supplement 1B
Recombinant DNA reagent (plasmid)pTRIPZ Phactr1XXXΔCThis paperSee Wiezlak et al., 2012;
Materials and methods;
Figure 3—figure supplement 1B
Recombinant DNA reagent (plasmid)pGEX 6P2GE Healthcare27-4598-01
Recombinant DNA reagent (plasmid)pGEX Phactr1(517-580)This paperSee Materials and methods;
Figure 1
Recombinant DNA reagent (plasmid)pGEX Phactr1(523-580)This paperSee Materials and methods
Figure 1
Recombinant DNA reagent (plasmid)pET28 PP1(7-300)Dr. Wolfgang PetiRRID:Addgene_26566
Recombinant DNA reagent (plasmid)pET28 PP1-(SG)9-IRSp53 S455EThis paperSee Materials and methods;
Figure 6
Recombinant DNA reagent (plasmid)pET28 PP1-(SG)9-IRSp53This paperSee Materials and methods;
Figure 4
Recombinant DNA reagent (plasmid)pET28 PP1-(SG)9-spectrinThis paperSee Materials and methods;
Figure 4
Recombinant DNA reagent (plasmid)pET28 PP1-Phactr1 fusionThis paperSee Materials and methods;
Figure 6
Recombinant DNA reagentpGro7 plasmidTakara3340
Sequence-based reagentOligonucleotidesThis paperSee Materials and methods;
Supplementary file 4
Sequence-based reagentPeptidesThis paperSee Materials and methods;
Supplementary file 4
Commercial assay or kitQ5 Site-Directed Mutagenesis KitNEBe0552s
Commercial assay or kitNEBuilder HiFi DNA Assembly Cloning KitNEBe5520s
Commercial assay or kitTMT10plex Isobaric Label Reagent Set, 0.8mgThermo90111
Commercial assay or kitHigh-Select Fe-NTA Phosphopeptide Enrichment KitThermoA32992
Commercial assay or kitHigh-Select TiO2 Phosphopeptide Enrichment KitThermoA32993
Commercial assay or kitHigh pH Reversed Phase Fractionation KitPierce84868
Commercial assay or kitGenElute mammalian total RNA kitSigmaRTN350-1KT
Commercial assay or kitTranscriptor First Strand cDNA Synthesis kitRoche04897030001
Commercial assay or kitSYBR green RT-qPCR mixLife TechnologiesA25742
Commercial assay or kitBiomol Green reagentEnzo Life SciencesBML-AK111-1000
Chemical compounddoxycyclineSigmaD9891-100G1 µg/ml
Chemical compoundcytochalasin DCalbiochemA25742
Chemical compoundlatrunculin BVWR428020-5MG
Chemical compoundLipofectamine 2000Invitrogen11668-019
Chemical compoundTexas Red-phalloidinInvitrogenT7471
Chemical compoundpNPPEnzoSV-30770-02
Chemical compoundSuperSignal West Pico Plus reagentThermo34577
SoftwareOctet software version 7.0ForteBiohttps://www.fortebio.com/
SoftwareXcaliburThermohttps://www.thermofisher.com/order/catalog/product/OPTON-30965
SoftwareMaxQuantCox and Mann, 2008http://coxdocs.org/doku.php?id=maxquant:start
SoftwarePerseusTyanova et al., 2016https://www.maxquant.org/perseus/
SoftwareWeblogoUniversity of California, Berkeleyhttps://weblogo.berkeley.edu/logo.cgi/
SoftwareGraphPad PrizmGraphPadhttps://www.graphpad.com/scientific-software/prism/
SoftwareImage Studio Lite 5.2LI-CORhttps://www.licor.com/bio/image-studio-lite/
SoftwareSnapGene softwareInsightful Sciencesnapgene.com
Chemical compoundManganese ChlorideFluka221279-500G
Chemical compoundArabinoseBiosynth limitedMA02043
Chemical compoundIPTGNeo BiotechNB-45-00030-25G
Chemical compoundChloramphenicolAcros organic227920250
Chemical compoundTrisSDS10708976001
Chemical compoundImidazoleSigma-AldrichI2399-100G
Chemical compoundSodium ChlorideSigma AldrichS9888-1KG
Chemical compoundTriton X100Sigma AldrichX100-100ML
Chemical compoundTCEPFluorochemM02624
Chemical compoundAEBSFMelfordA20010-5.0
Chemical compoundBenzamidineMelfordB4101
Chemical compoundComplete EDTA Free Protease Inhibitor tabletRoche05056489001
Chemical compoundGlutathione Sephanrose 4BGE Healthcare17-0756-05
Chemical compoundNi-NTA AgaroseQiagen30230
Chemical compoundTween 20Sigma-AldrichP1379-100ML
Chemical compoundBSASigma-AldrichA2153-100G
Chemical compoundLithium ChlorideHampton researchHR2-631
Chemical compoundTri Sodium CitrateHampton researchHR2-549
Chemical compoundPEG 6000Hampton researchHR2-533
Chemical compoundPEG 3350Hampton researchHR2-527
Chemical compoundSodium BromideHampton researchHR2-699
Chemical compoundPotassium citrateHampton researchHR2-683
Chemical compoundBis-Tris-PropaneSigma-AldrichB6755-500G
Chemical compoundSodium IodideSigma-Aldrich383112-100G
Chemical compoundGlycerolSDSG7893-2L
Chemical compoundEthylene GlycolSigma-Aldrich324558-1L
OtherAmino-PEG500-UC540 membraneIntavisPeptide array membrane; see Materials and methods, and Figure 5

Additional files

Supplementary file 1

Phosphoproteomics data.

(A, B) SILAC phosphoproteomics in NIH3T3 cells. (A) NIH3T3 cells expressing doxycycline-inducible Phactr1XXX, Phactr1XXXΔC or vector alone were cultured in R0K0 or R10K8 SILAC media, protein expression induced with doxycycline, and cell lysates analysed by MS proteomics. Phosphorylation sites are annotated with dephosphorylation score, raw H/L values listed. (B) 1D enrichment analysis of Gene Ontology terms and kinase motifs based on the dataset from A, table A. Terms with Benjamini-Hochberg FDR < 0.02 are shown. Gene Ontology Biological Process terms are in bold and those with positive mean value are reported in Figure 3B. (C-E) Wildtype (WT) or Phactr1-null (KO) cortical and hippocampal neurons DIV10 were treated with DMSO vehicle, CD or LB for 30' and then analysed by TMT phosphoproteomics in MS2 and MS3 modes. Reporter ion intensities were normalised using Z-score function. (C) Raw and Z-score values for MS2 mode. (D) Raw and Z-score values for MS3 mode. (E) t-test was applied to (DMSO - CD) differences in WT neurons vs KO neurons. Phosphorylation sites are described and annotated with t-test difference and significance.

https://cdn.elifesciences.org/articles/61509/elife-61509-supp1-v2.xlsx
Supplementary file 2

Phosphatase activity source data.

https://cdn.elifesciences.org/articles/61509/elife-61509-supp2-v2.xlsx
Supplementary file 3

Immunoblot source data.

https://cdn.elifesciences.org/articles/61509/elife-61509-supp3-v2.pdf
Supplementary file 4

Peptides and oligonucleotides.

https://cdn.elifesciences.org/articles/61509/elife-61509-supp4-v2.xlsx
Supplementary file 5

Summary of modelled residues for the different structures.

https://cdn.elifesciences.org/articles/61509/elife-61509-supp5-v2.docx
Transparent reporting form
https://cdn.elifesciences.org/articles/61509/elife-61509-transrepform-v2.docx

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