Figure 3: GSK3β binds to and phosphorylates PD-L1 protein in vitro and in vivo.

(a) Schematic diagram of GSK3β phosphorylation and β-TrCP-binding motifs and various mutants of PD-L1 expression constructs. PD-L1 was separated into ECD and ICD. SP, signal peptide; TM, transmembrane domain. The numbers indicate amino-acid positions. (b) In vitro GST pull-down assay of non-glycosylated PD-L1 and GSK3β. (c) Co-immunoprecipitation (co-IP) measuring the interaction of GSK3β and PD-L1 4NQ. Schematic diagram of PD-L1 4NQ deletion or truncation mutants showing on the left. Positions of glycosylation sites were labelled with red colour. The numbers indicate amino-acid positions. (d) Immunocomplex kinase assay measuring PD-L1 phosphorylation by GSK3β. Coomassie blue staining showing equal loading amount of GST-PD-L1. CA, constitutive activation mutant (S9A); KD, kinase dead (K85A) mutant; WT, wild type. (e) Western blot analysis of phosphorylation of PD-L1 protein at T180 and S184 sites by phospho-T180 and -S184 PD-L1 antibodies, respectively. EV, empty vector. (f) Time-lapse microscopy image (at 12 h) showing the dynamic interaction between PD-L1 and PD-1 at the last time point. The kinetic graph showed the quantitative binding of green fluorescent labelled PD-1/Fc protein on PD-L1 WT, 3SA or 4NQ expressing BT549 cells at every hour time point (right). Scale bar, 100 μm. (g) T-cell-meditated tumour cell-killing assay in PD-L1 WT or 3SA-expressing BT549 cells. Representative phase, red fluorescent (nuclear-restricted RFP), and/or green fluorescent (Caspase 3/7 substrate)-merged images of PD-L1 WT- or PD-L1 3SA-expressing cells and activated T-cell co-cultures at 96 h. Green fluorescent cell was counted as dead cell. The quantitative ratio of dead cells showed in bar graph (right). Scale bar, 100 μm. (h) The tumour growth of mouse PD-L1 WT- or PD-L1 3SA-expressing 4T1 cells in BALB/c mice. Quantification of tumour volume is shown on the right and representative images of tumours are shown on the left. n=7 mice per group. Con, vector control; WT, PD-L1 WT; 3SA, PD-L1 3SA. *P<0.05 is statistically significant as shown by Student’s t-test. All error bars are expressed as mean±s.d. of three independent experiments.