The cDNAs for kinase active and kinase inactive forms of Fyn, Lck, and ZAP-70 were previously described32C35

The cDNAs for kinase active and kinase inactive forms of Fyn, Lck, and ZAP-70 were previously described32C35. function but how SHP-2 enzymatic activation is mechanistically regulated by one PD-1 phosphotyrosine remains a puzzle. We found that after PD-1 Propylparaben phosphorylation, SHP-2 can Propylparaben bridge phosphorylated ITSM-Y248 residues on two PD-1 molecules via its amino terminal (N)-SH2 and carboxyterminal (C)-SH2 domains forming a PD-1: PD-1 dimer in live cells. The biophysical ability of SHP-2 to interact with two ITSM-pY248 residues was documented by isothermal titration calorimetry. SHP-2 interaction with two ITSM-pY248 phosphopeptides induced robust enzymatic activation. Our results unravel a mechanism of PD-1: SHP-2 interaction that depends only on ITSM-Y248 and explain how a single docking site within the PD-1 cytoplasmic tail can activate SHP-2 and PD-1-mediated inhibitory function. at room temperature and placed immediately at 37?oC for the indicated time points. Preparation of Dynabeads M-450 (Thermo Scientific) tosylactivated magnetic beads using anti-CD3 (UCHT1, Biolegend) and anti-CD28 (CD28.2, Biolegend) mAb was done as previously described29. Preparation of Dynabeads coated with anti-mouse antibodies CD3 (clone 145-2C11, Biolegend) and Propylparaben anti-CD28 (clone 37.51, Biolegend) was done by the same method. For Raji-mediated stimulation cells were resuspended at 1??106?cells/ml in RPMI complete medium and loaded with 0.5?ng/ml SEE (Toxin Technologies) by 30?min rotation at 37?oC followed by three washes to remove excess SEE. Jurkat T cells or primary Rabbit Polyclonal to SGK (phospho-Ser422) human T cells were cultured in 96-well tissue culture plates, at 105 cells/well with equal numbers of Raji cells (with or without SEE loading) in a final volume of 100?l. When indicated, a PD-1 blocking antibody (clone EH12) or an isotype control IgG was added in the cultures. Fyn KO mice (pp59fyn KO; JAX stock #002271)61 and wild-type control mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Mice of either sex at 6C8-weeks of age were used. All procedures were in accordance with National Institutes of Health Guidelines for the Care and Use of Animals and a relevant protocol had been approved by the Institutional Animal Care and Use Committee. Activation of mouse T cells was performed with 1?g/ml a-CD3 (clone 145-2C11, Biolegend) and 1?g/ml a-CD28 (clone 37.51, Biolegend). For staining of mouse T cells anti-mouse PD-1-PE (clone RMP1-30), anti-mouse CD4-Pacific blue (clone GK1.5) and anti-mouse CD8a-APC (clone 53.6.7) antibodies (Biolegend) were used followed by flow cytometry. Cell transfection For transfection experiments, COS cells were transfected by GeneJuice transfection reagent (EMD Millipore Corp., Billerica, MA) according to the manufacturers instructions. Primary human T cells were transfected using the Nucleofector system and human primary transfection kit (Lonza, VPA-1002) according to the manufacturers instructions. Immunoprecipitation and immunoblotting Cell lysates were prepared in lysis buffer containing 50?mM Tris-HCl, pH 7.4, 150?mM NaCl, 2?mM MgCl2, 10% glycerol and 1% NP-40 supplemented with 2?mM sodium orthovanadate, 1?mM sodium fluoride, 1?mM phenylmethylsulfonyl fluoride (PMSF), and protease Inhibitor Cocktail (Thermo Scientific) and were resolved by sodium dodecyl sulfateCpolyacrylamide gel electrophoresis (SDS-PAGE) followed by western blotting with the following antibodies: SHP-2 cat# sc-280, Santa Cruz Biotechnology; Fyn Cat# sc-16, Santa Cruz Biotechnology; Lck Cat# 06-583, EMD Millipore; ZAP-70 (clone 2F3.2), Cat# 05-253, EMD Millipore; FLAG (clone M2) Cat#F3165, Sigma; anti-pY (clone 4G10) Cat# 05-321, EMD Millipore. The mouse monoclonal anti-PD-1 antibodies clones EH12 and EH33 have been previously described62. The rabbit polyclonal anti-phospho-Y248 (ITSM) PD-1 antibody was developed in our laboratory31. Immunoprecipitations were performed with PD-1 mAb clone EH12 covalently conjugated to Dynabeads protein G (Thermo Scientific). Antibody-coated beads were washed in IP buffer (lysis buffer without NP-40) and subsequently incubated with 500?g of cell lysates overnight at 4?oC with gentle rotation. After SDS-PAGE, proteins were transferred to a nitrocellulose membrane, followed by western blotting with the indicated antibodies, and images were captured with digital imager FluorChem E (Proteinsimple, San Jose, CA). DNA constructs, cloning, and mutagenesis SHP-2 cDNA (Addgene, Cambridge, MA) was used to generate glutathione-S-transferase (GST) fusions to SHP-2 wild-type full-length (SHP-2-WT-FL) using the pGEX 4T-3 vector (GE Healthcare Life Sciences, Marlborough, MA) and Flag-tagged SHP-2 using the p3xFLAG-CMV10 vector (Addgene, Cambridge, MA). Human PD-1 and PD-L1 cDNAs were expressed in pEF6 vector. For NanoBiT experiments in HEK-293 cells, human PD-1 cDNA was cloned in Large BiT pBit1.1C[TK/LgBit] and in Small BiT pBit2.1C[TK/SmBit] vectors (Promega). For NanoBiT experiments in human T cells PD-1 cDNA was cloned in Large BiT pBiT1.3-C [CMV/LgBiT/Hyg] and in Small BiT pBiT2.3-C [CMV/SmBiT/Blast] vectors (Promega). For confocal microscopy experiments in HEK-293 cells, human PD-1 full-length was cloned in frame with VN173 or VC155 of Venus protein39. hPD-1 VN173 and hPD-1 VC155 were synthesized by gBlock (IDT) and each one was cloned into pBit1.1C[TK/LgBit] vector (Promega) after removing LgBiT. For mutagenizing the human PD-1 tyrosine residues Y223 (within the ITIM motif) and Y248 (within the ITSM motif), and the arginine residues of Propylparaben SHP-2 R32 (within the N-SH2 domain) and R138 (within the C-SH2 domain) the QuickChange Lightning Site-Directed Mutagenesis kit from Agilent Technologies was used. All mutations.