Figure 3.
PD1 triggering inhibits binding and phosphorylation of proteins by PKCθ. (A) Western blot monitoring phosphorylation of PKC substrates on serine residues embedded within the pentapeptide motif K/R-X-pS-hydrophobic aa-K/R in lysates of human primary CD4 T cells from 2 independent donors. Cells were left untreated (NT) or triggered for the indicated time points with stimulatory (S) or inhibitory (I) beads. Arrows indicate protein bands that show reduced phosphorylation upon treatment with inhibitory beads. (B,C) Western blot monitoring PKCθ phosphorylation at T538 and total PKCθ levels in lysates of human primary CD8 from 2 independent donors (B) and CD4 (C) T cells. Cells were left NT or triggered for the indicated time points with S or I beads. (D) Quantification of experiments performed as in panels B,C: pT538 PKCθ levels in human primary T cells were normalized to total PKCθ levels for the 30-minute condition for 3 independent experiments. Mean values ± SEM. Statistical analysis was performed with a 2-tailed, unpaired t test. ∗∗P < .01. (E) Venn diagram graphically representing results of the PKCθ interactome and phosphoproteome analyses: 171 proteins underwent a significant PD1-mediated reduction in serine or threonine phosphorylation (dark blue circle), 253 proteins bound less well or did not bind to PKCθ upon PD1 triggering (light blue circle). The overlapping section contains 9 cytoskeleton proteins that show a significant decrease in serine phosphorylation and decreased binding to PKCθ upon PD1 triggering compared with the stimulating condition. (F) Western blot monitoring the effect of pretreatment with the pan-PKC inhibitor BIM8 on the phosphorylation of S5 of L-plastin (pLCP1) in human primary CD4 and CD8 T cells. Cells were pretreated as indicated for 1 hour, followed by triggering with S beads for the indicated times. Two representative blots of 3 independent experiments are shown. Densitometry analysis: the relative ratio of pLCP1 to total LCP in the samples is indicated below. (G) Western blot monitoring phosphorylation of S5 of L-plastin (pLCP1) in human primary CD8 T cells from 2 independent donors, nucleoporated with PKCθ-specific or Ctrl siRNAs. Seventy-two hours after nucleoporation, cells were triggered for 30 minutes with S or I beads. The relative ratio of pLCP1 to total LCP in the samples is indicated below.

PD1 triggering inhibits binding and phosphorylation of proteins by PKCθ. (A) Western blot monitoring phosphorylation of PKC substrates on serine residues embedded within the pentapeptide motif K/R-X-pS-hydrophobic aa-K/R in lysates of human primary CD4 T cells from 2 independent donors. Cells were left untreated (NT) or triggered for the indicated time points with stimulatory (S) or inhibitory (I) beads. Arrows indicate protein bands that show reduced phosphorylation upon treatment with inhibitory beads. (B,C) Western blot monitoring PKCθ phosphorylation at T538 and total PKCθ levels in lysates of human primary CD8 from 2 independent donors (B) and CD4 (C) T cells. Cells were left NT or triggered for the indicated time points with S or I beads. (D) Quantification of experiments performed as in panels B,C: pT538 PKCθ levels in human primary T cells were normalized to total PKCθ levels for the 30-minute condition for 3 independent experiments. Mean values ± SEM. Statistical analysis was performed with a 2-tailed, unpaired t test. ∗∗P < .01. (E) Venn diagram graphically representing results of the PKCθ interactome and phosphoproteome analyses: 171 proteins underwent a significant PD1-mediated reduction in serine or threonine phosphorylation (dark blue circle), 253 proteins bound less well or did not bind to PKCθ upon PD1 triggering (light blue circle). The overlapping section contains 9 cytoskeleton proteins that show a significant decrease in serine phosphorylation and decreased binding to PKCθ upon PD1 triggering compared with the stimulating condition. (F) Western blot monitoring the effect of pretreatment with the pan-PKC inhibitor BIM8 on the phosphorylation of S5 of L-plastin (pLCP1) in human primary CD4 and CD8 T cells. Cells were pretreated as indicated for 1 hour, followed by triggering with S beads for the indicated times. Two representative blots of 3 independent experiments are shown. Densitometry analysis: the relative ratio of pLCP1 to total LCP in the samples is indicated below. (G) Western blot monitoring phosphorylation of S5 of L-plastin (pLCP1) in human primary CD8 T cells from 2 independent donors, nucleoporated with PKCθ-specific or Ctrl siRNAs. Seventy-two hours after nucleoporation, cells were triggered for 30 minutes with S or I beads. The relative ratio of pLCP1 to total LCP in the samples is indicated below.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal