Fig 2: Surface plasmon resonance (SPR) measurements of binding between individual SH2 of SHP1 or SHP2 and immunoreceptor tyrosine-based inhibitory motif (ITIM) or immunoreceptor tyrosine-based switch motif (ITSM) of BTLA.(A) SPR sensorgrams (top) and the derived equilibrium binding curves (bottom) showing the interactions of indicated SH2 and phosphorylated BTLA-ITIM (BTLA FFYF) or BTLA-ITSM (BTLA FFFY) immobilized to Ni sensor chips. Individual SH2 proteins were injected at 20, 50, 100, 200, 500, and 1000 nM. Shown are representative of three independent experiments performed on three different sensorchips on three different days. The calculated Kd values are indicated in the binding curves. (B) A cartoon depicting relative binding affinities of SHP1/SHP2 individual SH2 to BTLA-ITIM/ITSM, with the thickness of arrows matching the relative affinities calculated from the SPR data in (A). (C) Possible interacting modes of SHP1/SHP2-tSH2 with BTLA. Figure 6—source data 1.Raw data for equilibrium binding curves in Figure 6A.

Fig 3: Swapping the cSH2 of SHP1 with that of SHP2 induced PD-1:SHP1 association in T cells.(A) Left: sequence alignment of cSH2 of human and mouse SHP1/SHP2. The underlying dots denote SHP2-cSH2 residues that participate in PD-1-pITSM binding, according to NMR structure of PD-1-pITSM:SHP2-cSH2 complex (PDB code: 6R5G): black dots highlight residues that are conserved in SHP1-cSH2; blue dots highlight residues that are not conserved in SHP1-cSH2. Right: NMR structure of PD-1-pITSM:SHP2-cSH2 complex (PDB code: 6R5G) with PD-1 depicted in a stick model and SHP2-cSH2 showed in a ribbon diagram, in which the blue-dot-denoted residues in the sequence alignment are highlighted in blue sticks. (B) Left: diagram showing the design of mCherry-tagged, SH2-swapped SHP1 variants, with one or both of its SH2 replaced with that of SHP2. Middle: representative TIRF images of PD-1 (GFP) and SHP1 variants (mCherry) in supported lipid bilayer (SLB)-associated SHP1/2 DKO Jurkat cells expressing PD-1-mGFP and mCherry-SHP1 variants. Right: dot plots summarizing mCherry fluorescence intensity (FI) normalized to GFP FI of 40 Jurkat cells under each condition recorded on the same day with the same microscope setting. Error bars: s.d. Scale bars: 5 µm. (C) Representative immunoblots (IBs) of mCherry-SHP1 variants co-precipitated with PD-1-mGFP from indicated cell lysates. IBs of GFP indicate PD-1 input. IBs of phosphotyrosines (pY) indicate PD-1 phosphorylation. Bar graphs summarize optical density (OD) of SHP1 variants normalized to the FI of PD-1, based on flow cytometry data in Figure 4—figure supplement 1. Error bars are s.d. from three independent coculture experiments performed on different days. *p<0.05; **p<0.01; ***p<0.001; ns, not significant; Student’s t-test. Figure 4—source data 1.Uncropped IBs for data shown in Figure 4C. Figure 4—source data 2.Raw data for quantification graphs in Figure 4.

Fig 4: SHP2 expression increased in both human psoriatic patients and IMQ-induced psoriasis-like mice Expression of PTPN11 (gene encoding SHP2) in skin lesions in psoriatic patients compared with skin from healthy donors based on microarray data (No. GSE14905).Expression of PTPN11 in human PBMCs of psoriatic patients (n = 14) and normal controls (n = 16).Representative SHP2 staining in skin sections of psoriatic patients (n = 13) and normal controls (n = 5). Scale bars: 200 µm.Western blot analysis of PBMCs lysates derived from psoriatic patients and normal controls.The catalytic activity of SHP2 was measured in human PBMCs lysates derived from psoriatic patients (n = 25) and normal controls (n = 25).Representative p-ERK staining of skin sections of psoriatic patients and normal controls. Scale bars: 200 µm.Quantitative PCR analysis of Ptpn11 mRNA levels in the IMQ-treated or non-treated dorsal back of C57BL/6 mice at day 5 (n = 6/group). Data were normalized to Gapdh expression.Representative histological sections of IMQ-treated or non-treated dorsal back of C57BL/6 mice at day 5. Scale bar: 100 µm.Quantitative PCR analysis of mRNA levels in human PBMCs derived from psoriatic patients (n = 20) and normal healthy controls (n = 20). Data information: Data are represented as mean ± SEM. P values are determined by two-tailed Mann–Whitney U test (A and B) or two-tailed unpaired Student’s t-test (E, G, and I). *P < 0.05, **P < 0.01. Source data are available online for this figure.

Fig 5: PDGFRß kinase domain directly mediates the phosphorylation of SHP-2.(a) Purified PDGFRß KD was incubated with recombinant human SHP-2 in the presence or absence of ATP and phosphatase inhibitors. Proteins were separated by SDS-PAGE and analyzed by immunoblot analysis for phosphotyrosine (upper panel). Total proteins are shown in the lower panel. As a control, SHP-1, a structurally similar phosphatase to SHP-2, was also included (lane 5). (b) The catalytic activity of SHP-2 was assessed using a malachite green phosphatase assay kit using Src pY529 (TSTEPQ-pY-QPGENL) as the substrate. Recombinant SHP-2 was incubated with pPDGFR KD or sPDGFRr, GST:LRP1-ICD, in the presence ATP and MgCl2. Liberated phosphate complexed with malachite green was measured at 620 nm (*p<0.0001 one way ANOVA5; *p<0.05 Tukey’s multiple comparison post hoc test).