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. 2023 May 19;14:2859. doi: 10.1038/s41467-023-38605-3

Fig. 3. ERK phosphorylates PD-1 at Thr234 to stabilize PD-1.

Fig. 3

a–c IB analysis of WCL derived from HEK293T cells co-transfected PD-1-cFlag or PD-1-cHA with different kinases as indicated. d Representative mIHC images of CD3 (pink), PD-1 (red), phosphor-ERK at Thr202/Try204 (p-ERK, green), CK (cyan), and DAPI nuclear staining (blue) in human colon tumor sections. White arrows indicate positive cells for PD-1 and p-ERK colocalization. Yellow color is considered overlapping for PD-1 and p-ERK staining. n = 5. Scale bars, 50 μm. e IB analysis of WCL derived from Jurkat cells treated with PHA (150 ng/mL) for 3 days and Trametinib (1 or 3 μM) for 24 h before harvesting. f, g Cell surface PD-1 on shGFP- or shERK1/2-treated Jurkat cells with pre-stimulation of PHA (150 ng/mL) for 3 days was analyzed by flow cytometry (f). The relative mean fluorescence intensity (MFI) of PD-1 on the surface of was quantified (g). Data were presented as mean ± S.D. n = 3 biologically independent samples per group. Two-tailed unpaired t-test. h A schematic illustration and sequence alignment of a potential ERK binding D-domain on PD-1 protein sequence, (K/R)0-2-(X)1-6-Φ-X-Φ, where Φ is a hydrophobic residue and X is any amino acid. i, j IB analysis of WCL and anti-HA IPs (i) or GST pull-down precipitates (i) from HEK293T cell lysates transfected with indicated constructs. k, l In vitro phosphorylation assays of bacterially purified recombinant GST, GST-PD-1 truncations or T234A mutant by ERK1 kinase. m, n IB analysis of WCL and anti-Flag or anti-HA IPs derived from HEK293T cells transfected with indicated constructs. o–q IB analysis of WCL and anti-PD-1 IPs from Jurkat pre-treated with PHA (150 ng/mL) for 3 days (o, p) or MOLT-4 (q) cells. Cells were treated with indicated Trametinib (0.5 or 1 μM) for 24 h (p, q). All IB data are representative of two independent experiments. Source data are provided as a Source data file.