Fig. 4: ERK-mediated Thr234 phosphorylation of PD-1 promotes its interaction with USP5.
From: ERK and USP5 govern PD-1 homeostasis via deubiquitination to modulate tumor immunotherapy
a–c IB analysis of WCL and IPs from HEK293T cells transfected with indicated constructs. Cells were treated with 10 μM MG132 for 12 h. d, e IB analysis of WCL and anti-HA IPs from HEK293T cells transfected with indicated constructs (d) or anti-PD-1 IPs derived from MOTL-4 cells (e). Cells were treated with indicated Trametinib (0.5 or 1 μM) for 24 h and 10 μM MG132 for 12 h (d) or 4 h (e). f IB analysis of WCL and GST pull-down precipitates from HEK293T cell lysates with ectopic expression of PD-1-cHA incubated with recombinant GST-USP5 protein. Cells were treated with 1 μM Ulixertinib for 24 h. g IB analysis of WCL and anti-HA IPs from HEK293T cells transfected with indicted USP5, PD-1 WT, or T234A mutant. Cells were treated with 10 μM MG132 for 12 h. h IB analysis of WCL and GST pull-down precipitates from HEK293T cell lysates with ectopic expression of PD-1-cHA WT or PD-1-cHA T234A incubated with bacterially purified recombinant GST-USP5 protein. i Bacterially purified recombinant GST-PD-1 (192-288) WT or T234A mutant was phosphorylated by ERK1 in vitro. Subsequently, GST pull-down assay was performed with purified recombinant His-USP5. IB analysis with indicated antibodies. j, k 3 μg of indicated biotin-labeled synthetic PD-1 peptides were incubated with 4 μg purified recombinant GST-USP5 (j) or different domains (k), respectively. Streptavidin beads were added to perform pull-down assays and precipitations were analyzed with IB as indicated. Dot blot was used to identify biotin-labeled synthetic PD-1 peptides. l, m IB analysis of WCL and Ni-NTA pull-down products from lysates of HEK293T cells transfected with the indicated constructs and treated with/without Trametinib (1 or 3 μM) for 12 h (l). Cells were treated with 10 μM MG132 for 12 h. n IB analysis of WCL and anti-PD-1 denatured-IPs from lysates of shGFP- or shERK1/2-treated Jurkat cells using indicated antibodies. Cells were pre-treated with PHA (150 ng/mL) for 3 days and 5 μM MG132 for 6 h. All data are representative of two independent experiments. Source data are provided as a Source data file.
