Journal: The Journal of Biological Chemistry

Article Title: A novel gain-of-function phosphorylation site modulates PTPN22 inhibition of TCR signaling

doi: 10.1016/j.jbc.2024.107393

Figure Lengend Snippet: PTPN22 Ser 325 is an inducible GSK3 phosphorylation site in human T cells. A , schematic illustration of 3× FLAG PTPN22 protein purification ( left panel ) and mass spectra ( right panel ) indicating Ser 325 phosphorylation in immunoprecipitated PTPN22 from 3× FLAG PTPN22 Jurkat WT cells cross-linked with antibodies against human CD3/CD28. Data is representative of three independent biological replicates. Peptide MS2 fragmentation pattern shown displaying m/z and peptide spectral match intensity. B , Western blot analysis obtained using a phospho-Ser 325 –specific antibody in immunoprecipitated PTPN22 from the lysates of PTPN22 KO Jurkat cells overexpressing 3× FLAG WT or S325A PTPN22 and treated with antibodies against human CD3/CD28 for the indicated time ( left panel ). Quantification of the phospho-Ser 325 /total PTPN22 ratio normalized to nonstimulated conditions in four independent experiments ( right panel ). Statistical significance was assessed using the two-way ANOVA test followed by Bonferroni’s post hoc test, ∗ p < 0.05. C , endogenous Ser 325 phosphorylation was analyzed by Western blotting in PTPN22 immunoprecipitated from lysates of 3× FLAG WT PTPN22 Jurkat cells stimulated with antibodies against human CD3/CD28 for the indicated times ( left panel ). Quantifications of the phospho-Ser 325 /total PTPN22 ratio normalized to nonstimulated conditions in four independent experiments ( right panel ). Statistical significance was assessed using the Kruskal–Wallis test, ∗ p < 0.05. D , immunoprecipitation analysis of endogenous PTPN22 Ser 325 phosphorylation in lysates of human primary CD4 + effector T cells stimulated with antibodies against human CD3/CD28 for the indicated time ( left panel ). Quantification of the phospho-Ser 325 /total PTPN22 ratio normalized to nonstimulated conditions in five independent experiments ( right panel ). Statistical significance was assessed by using the Kolmogorov–Smirnov test, ∗∗ p < 0.01. E , prediction of potential kinases responsible for PTPN22 Ser 325 phosphorylation in descending order from left to right . F , immunoprecipitation analysis of phospho-PTPN22 Ser 325 in lysates of 3× FLAG PTPN22 WT Jurkat cells with or without incubation with 5 μM GSK3 inhibitor IX and stimulation with antibodies against human CD3/CD28 ( left panel ). Quantifications of phospho-Ser 325 /total PTPN22 ratio from Western blot analysis of four independent experiments ( right panel ). Statistical significance was assessed using the two-way ANOVA test followed by Bonferroni’s post hoc test, ∗∗ p < 0.01. G , immunoprecipitation analysis of PTPN22 and GSK3 interaction in lysates of BioID2 Jurkat cells ( left panel ). Histograms shows quantifications of immunoprecipitated GSK3 α/β based on the Western blot analysis and are representative of four independent experiments ( right panel ). Statistical significance was assessed using the Kolmogorov–Smirnov test, ∗ p < 0.05. CD, cluster of differentiation; GSK3, glycogen synthase kinase 3; PTPN22, protein tyrosine phosphatase nonreceptor type 22.

Article Snippet: Goat anti-human PTPN22 polyclonal antibody was purchased from R&D systems.

Techniques: Protein Purification, Immunoprecipitation, Western Blot, Incubation

Journal: The Journal of Biological Chemistry

Article Title: A novel gain-of-function phosphorylation site modulates PTPN22 inhibition of TCR signaling

doi: 10.1016/j.jbc.2024.107393

Figure Lengend Snippet: Phosphorylation of PTPN22 Ser 325 enhances the inhibitory effect of PTPN22 on T cell receptor signaling. A , immunoprecipitation analysis of PTPN22 Ser 325 phosphorylation in lysates of 3× FLAG PTPN22 WT and CRISPR/Cas9-mediated S325A KI Jurkat cells stimulated with antibodies against human CD3/CD28 for the indicated time by Western blotting ( left panel ). Histogram shows quantifications of the phospho-Ser 325 /total PTPN22 ratio normalized to nonstimulated condition and is representative of four independent experiments ( right panel ). Statistical significance was assessed using the two-way ANOVA followed by Bonferroni’s post hoc test, ∗∗ p < 0.01. B , dual-luciferase reporter assay analysis of full-length PTPN22 inhibition of TCR signaling in PTPN22 KO Jurkat cells overexpressing full-length 3× FLAG WT, S325E, or S325A PTPN22 together with NFAT/AP-1 firefly and Renilla luciferase reporters and stimulated with antibodies against human CD3/CD28. Luciferase activity was measured ( left panel ), and the numbers on the y -axis indicate NFAT/AP-1 firefly luciferase activity normalized first to Renilla luciferase activity in each group (KO, WT, or S325 mutant), then to the amount of PTPN22 relative to that of GAPDH as assessed by Western blotting ( right panel ). Mean ± SEM are shown from three independent experiments each with three replicates per condition. Statistical significance was assessed by using the Kruskal–Wallis test, ∗ p < 0.05. C and D , Western blot analysis of TCR signaling in 3× FLAG PTPN22 WT, and CRISPR/Cas9 mediated S325A ( C ) or S325E ( D ) KI Jurkat cells treated with antibodies against human CD3/CD28 for indicated time, followed by detection of phosphorylated LCK (Tyr 394 ), ZAP70 (Tyr 3 19 ), and PLC-γ (Tyr 783 ) in lysates ( left panels ). Histograms show quantification of phosphorylated LCK, ZAP70, and PLC-γ normalized to relative total protein by four independent experiments ( right panels ). Statistical significance was assessed using the Kolmogorov–Smirnov test, ∗ p < 0.05. E and F , flow cytometry analysis of TCR-induced CD69 expression in 3× FLAG PTPN22 WT, S325A ( E ), or S325E ( F ) KI Jurkat cells treated with (stimulated) or without (mock) antibodies against human CD3/CD28 for 4 h. Histograms show median fluorescent intensity (MFI) from seven independent experiments. Statistical significance was assessed using two-way ANOVA, followed by Bonferroni’s post hoc test, ∗∗ p < 0.01. AP-1, activator protein-1; CD, cluster of differentiation; GSK3, glycogen synthase kinase 3; LCK, lymphocyte-specific protein tyrosine kinase; NFAT, nuclear factor of activated T cells; PLC, phospholipase C; PTPN22, protein tyrosine phosphatase nonreceptor type 22; TCR, T cell receptor; ZAP70, zeta-chain–associated protein kinase 70.

Article Snippet: Goat anti-human PTPN22 polyclonal antibody was purchased from R&D systems.

Techniques: Immunoprecipitation, CRISPR, Western Blot, Luciferase, Reporter Assay, Inhibition, Activity Assay, Mutagenesis, Flow Cytometry, Expressing

Journal: The Journal of Biological Chemistry

Article Title: A novel gain-of-function phosphorylation site modulates PTPN22 inhibition of TCR signaling

doi: 10.1016/j.jbc.2024.107393

Figure Lengend Snippet: In vitro catalytic activity of PTPN22 and its variants. A and B , phosphatase activity assays were performed using immunoprecipitated PTPN22 from PTPN22 WT, or S325A, S325E KI Jurkat cells, and DiFMUP as a substrate. Histograms ( left panel ) show quantification of initial rates of reaction normalized to the amount of PTPN22 WT as assessed by Western blotting ( right panel ). Mean ± SEM are shown from five ( A ) and ten ( B ) independent experiments. Statistical significance was assessed by using the Kolmogorov–Smirnov test, ∗∗ p < 0.01, ∗∗∗ p < 0.001. C and D , dual-luciferase reporter assay analysis of truncated WT and S325E inhibition of TCR signaling in PTPN22 KO Jurkat cells overexpressing 3 × FLAG PTPN22 1-340 ( C ), or PTPN22 1-330 ( D ) WT, and S325E together with NFAT/AP-1 firefly and Renilla luciferase reporters and stimulated with antibodies against human CD3/CD28. Luciferase activity was measured and quantified as described in Figure 2 B . Means ± SEM are shown from three or four independent experiments each with three replicates per condition. Statistical significance was assessed by using the Kruskal–Wallis test, ∗ p < 0.05, ∗∗ p < 0.01. E , representative SDS-PAGE of final purified samples of PTPN22 1-330 WT and S325E recombinant proteins. F and G , phosphatase activity assays were performed by using recombinant PTPN22 1-330 WT or S325E and DiFMUP as a substrate. F , representative Michaelis–Menten curve of eight independent experiments each with three technical replicates and representative SDS PAGE of 1 μM PTPN22 1-330 WT and S325E recombinant proteins. G , dot plot showing k cat and K M . Each data point represents one of eight independent experiments performed as in (F). Statistical significance was assessed using two-tailed Mann–Whitney test, ∗ p < 0.05, ∗∗ p < 0.01. AP-1, activator protein-1; CD, cluster of differentiation; DiFMUP, 6,8-difluoro-4-methylumbelliferyl phosphate; NFAT, nuclear factor of activated T cells; PTPN22, protein tyrosine phosphatase nonreceptor type 22; TCR, T cell receptor.

Article Snippet: Goat anti-human PTPN22 polyclonal antibody was purchased from R&D systems.

Techniques: In Vitro, Activity Assay, Immunoprecipitation, Western Blot, Luciferase, Reporter Assay, Inhibition, SDS Page, Purification, Recombinant, Two Tailed Test, MANN-WHITNEY

Journal: The Journal of Biological Chemistry

Article Title: A novel gain-of-function phosphorylation site modulates PTPN22 inhibition of TCR signaling

doi: 10.1016/j.jbc.2024.107393

Figure Lengend Snippet: Effect of Ser 325 phosphorylation mimic on the deuterium exchange rates of PTPN22 1 to 330. A , rainbow plot showing percent differences in deuterium exchange between PTPN22 1-330 WT and S325E. Deuterium exchange was assessed for five different time points from 10 to 100000 s. The numbering of the polypeptide chain follows the sequence of the protein as used in the experiment, which has a 17-amino acid N-terminal leader preceding the native methionine 1. Data are representative of two independent experiments. B , the averages of number of deuterium exchanged (#D) shown over time for two of the peptides with the greatest changes. C , ribbon representation of PTPN22 (PDB code 2P6X ) annotated with the location of the active site and of polypeptide regions mentioned in the text. For reference, the ribbon color matches the bottom band in the sequence view in A. D , solvent accessible surface representation of the PTP domain in two orthogonal views colored according to the percent difference in the exchange rate at 10,000 s as shown in A. Key residues are indicated. The molecular graphics objects in ( C ) and ( D ) were generated with UCSF Chimera . Ni-NTA, nickel-nitrilotriacetic acid; PTP, protein tyrosine phosphatase; PTPN22, PTP nonreceptor type 22.

Article Snippet: Goat anti-human PTPN22 polyclonal antibody was purchased from R&D systems.

Techniques: Sequencing, Solvent, Generated

Journal: The Journal of Biological Chemistry

Article Title: A novel gain-of-function phosphorylation site modulates PTPN22 inhibition of TCR signaling

doi: 10.1016/j.jbc.2024.107393

Figure Lengend Snippet: Effect of mimicking Ser 325 phosphorylation on the interaction between the PTPN22 interdomain and catalytic domain. A , 2D [ 1 H, 15 N] HSQC spectrum of 15 N-labeled PTPN22 interdomain (residues 299–360, black ) and S325E ( blue ). S325 and E325 residues are annotated. B , 2D [ 1 H, 15 N] HSQC spectrum of 15 N-labeled PTPN22 interdomain (residues 299–360) alone ( black ) and in complex with the PTPN22 catalytic domain (residues 1–299; red ). Peaks with changing intensities are annotated. C , 2D [ 1 H, 15 N] HSQC spectrum of 15 N-labeled PTPN22 S325E interdomain (residues 299–360) alone ( blue ) and in complex with the PTPN22 catalytic domain (residues 1–299; red ). Peaks with changing intensities are annotated. HSQC, heteronuclear single quantum coherence; PTPN22, protein tyrosine phosphatase nonreceptor type 22.

Article Snippet: Goat anti-human PTPN22 polyclonal antibody was purchased from R&D systems.

Techniques: Labeling

Journal: The Journal of Biological Chemistry

Article Title: A novel gain-of-function phosphorylation site modulates PTPN22 inhibition of TCR signaling

doi: 10.1016/j.jbc.2024.107393

Figure Lengend Snippet: Primers used in the study

Article Snippet: Goat anti-human PTPN22 polyclonal antibody was purchased from R&D systems.

Techniques: Sequencing

Journal: bioRxiv

Article Title: Nano-clusters of ligand-activated integrins organize immobile, signalling active, nano-clusters of phosphorylated FAK required for mechanosignaling in focal adhesions

doi: 10.1101/2024.02.25.581925

Figure Lengend Snippet: (a) Kymograph of nanoKymo-FRAP for FAK-GFP for MEFs treated with 10 µM PF-562271 (FAKin) along with the histogram of recovered fraction. The distribution was calculated from fitting intensity vs time plots and plotted along with the Gaussian fit of the population. N=43 from 8 cells. (b) Recovered nanoKymo-FRAP fraction vs Relative population plot comparing the FAKin population with control FAK nanoKymo-FRAP populations. (c) Mean±S.D. plot of half-time of nanoKymo-FRAP control vs FAKin populations. (d) Kymograph of nanoKymo-FLAP for FAKY397F-PAGFP overexpressing MEFs along with histogram immobile nanoKymo-FLAP population and Gaussian fit of the population. N=27 from 5 cells. (e) Immobile nanoKymo-FLAP fraction vs Relative population plot comparing the FAKY397F population with control FAK nanoKymo-FLAP populations. (f) Mean±S.D. plot of half-time of nanoKymo-FLAP control vs FAKY397F populations. (g) Kymograph of nanoKymo-FLAP for FAK-PATagRFP for MEFs treated with 2.5 µM PTP inhibitor (PTPin) along with the histogram of recovered fraction and Gaussian fit of the population. N=20 from 5 cells. (h) Immobile nanoKymo-FLAP fraction vs Relative population plot comparing the PTPin population with control FAK nanoKymo-FLAP populations. (i) Mean±S.D. plot of half-time of nanoKymo-FLAP control vs PTPin populations. P FWHM denotes the fraction of population lying in the full width half maxima of the Gaussian fit. P values were evaluated by Mann-Whitney tests.

Article Snippet: MEFs were treated with either 10 μM FAKin (PF-562271, Sigma-Aldrich, Cat. No. PZ0387), 2.5 μM PTPin (PTP LYP inhibitor, Calbiochem, Cat. No. 540217), 10 μM Y-27632 (Sigma-Aldrich, Cat. No. Y0503), or combinations of these drugs during spreading, as indicated.

Techniques: Control, MANN-WHITNEY

Journal: bioRxiv

Article Title: Nano-clusters of ligand-activated integrins organize immobile, signalling active, nano-clusters of phosphorylated FAK required for mechanosignaling in focal adhesions

doi: 10.1101/2024.02.25.581925

Figure Lengend Snippet: (a) Fluorescence images of MEFs over-expressing FAK-mApple, treated with PTP inhibitor (PTPin) or DMSO for 30 min and Y-27632 or DMSO for another 15 mins, along with the merge. Dashed line represents cell boundary. N=44 from 6 cells. Scale bar, 10 µm. (b) Relative frequency of stable FAK nanoKymo-FLAP population for MEFs overexpressing FAK-PAGFP and treated with 10 µM Y-27632 with PTPin treatment along with the Gaussian fit of the population. P FWHM denotes the fraction of population lying in the full width half maxima of the Gaussian fit. N=37 from 5 cells. (c) Stable nanoKymo-FLAP fraction vs Relative population plot comparing the Y-276329+PTPin population with control and Y-27632 populations. (d) Mean±S.D. plot of half-time of nanoKymo-FLAP control vs Y-27632 vs Y-27632+PTPin treated populations. (e) Control, PTPin treated, Y-27632 treated, and Y-27632 and PTPin treated cells spread and immunostained for FAK. Dashed line represents cell boundary. Scale bar, 5 µm. (f) Quantification of total adhesion area normalized to the cell area. Box plots with data overlay display the upper and lower quartiles and a median, the circle represents the mean, and the whiskers denote the standard deviation values, along with the individual points and P values. P values were evaluated by one-way ANOVA and Tukey’s post hoc test.

Article Snippet: MEFs were treated with either 10 μM FAKin (PF-562271, Sigma-Aldrich, Cat. No. PZ0387), 2.5 μM PTPin (PTP LYP inhibitor, Calbiochem, Cat. No. 540217), 10 μM Y-27632 (Sigma-Aldrich, Cat. No. Y0503), or combinations of these drugs during spreading, as indicated.

Techniques: Fluorescence, Expressing, Control, Standard Deviation