Fig. 5: Interaction of SHP-2 SH2 domains with two PD-1 ITSM pY248 residues is required for activation of SHP-2 phosphatase activity.

a SHP-2-WT was incubated with 20 µM DiFMUP as substrate in the presence of the indicated phosphopeptides and phosphatase activity was assessed as described in Methods. b Phosphatase activity of SHP-2-WT incubated with bpITSM phosphotyrosyl peptides containing a 4-, 2-, or 1-Ahx spacer; pIRSY727 was used as negative control. c Phosphatase activity of SHP-2-WT, SHP-2-R32A, or SHP-2-R138A incubated with the indicated concentrations of bpITSM phosphotyrosyl peptide containing a 4-Ahx spacer. d Phosphatase activity of SHP-2-WT in the presence of phosphotyrosyl bpITSM peptide (0.01 µM) either alone or with increasing amounts of monophosphorylated pITIM or monophosphorylated pITSM peptide. Results are representative of three independent experiments. e Phosphatase activity of SHP-2-WT in the presence of a phosphopeptide corresponding to the native sequence of PD-1 cytoplasmic tail in which both ITIM-Y223 and ITSM-Y248 tyrosines were phosphorylated (PD-1cyto-pITIM-pITSM) or with a bpITSM phosphotyrosyl peptide containing a 10-Ahx spacer. pIRSY727 was used as negative control. In panels a–e results from one representative of 3–10 independent experiments are shown. f Jurkat T cells stably expressing PD-1-WT (J-PD-1), PD-1-Y223F (J-PD-1-Y223F), or PD-1-Y248F (J-PD-1-Y248F) were co-cultured with Raji-control or Raji-PD-L1 cells loaded with SEE. Where indicated, anti-PD-1 blocking antibody or isotype control was added in the cultures. Culture supernatants were collected at 24 h and IL-2 production was measured. Results are expressed as % of maximum IL-2 production induced in each cell line by Raji-control cells loaded with SEE (****P < 0.0001, n = 3 experiments). g Model for PD-1: PD-1 bridging and activation of SHP-2 phosphatase activity: An “inside-out” regulation of PD-1: PD-1 dimer complex formation is initiated by TCR-mediated activation of TCR proximal Src family kinases, which induce phosphorylation of tyrosines in PD-1 cytoplasmic tail. The stabilization provided by PD-1 ligands allows the appropriate proximity of two phosphorylated PD-1 molecules to induce binding of both SH2 domains of SHP-2 to ITSM-pY248 and activation of SHP-2, leading to inhibition of T-cell responses.