PKCθ-Mediated PDK1 Phosphorylation Enhances T Cell Activation by Increasing PDK1 Stability

Abstract

PDK1 is essential for T cell receptor (TCR)-mediated activation of NF-κB, and PDK1-induced phosphorylation of PKCθ is important for TCR-induced NF-κB activation. However, inverse regulation of PDK1 by PKCθ during T cell activation has not been investigated. In this study, we found that PKCθ is involved in human PDK1 phosphorylation and that its kinase activity is crucial for human PDK1 phosphorylation. Mass spectrometry analysis of wild-type PKCθ or of kinase-inactive form of PKCθ revealed that PKCθ induced phosphorylation of human PDK1 at Ser-64. This PKCθ-induced PDK1 phosphorylation positively regulated T cell activation and TCR-induced NF-κB activation. Moreover, phosphorylation of human PDK1 at Ser-64 increased the stability of human PDK1 protein. These results suggest that Ser-64 is an important phosphorylation site that is part of a positive feedback loop for human PDK1-PKCθ-mediated T cell activation.

Keywords: PDK1; PKCθ; T cell; phosphorylation.

Fig. 1. PKCθ kinase activity alters PDK1 mobility via phosphorylation

(A) Jurkat T cells were treated with Ly294002 or OSU03012 and then stimulated with anti-CD3 and anti-CD28 antibodies. Surface expression of CD25 and CD69 on activated Jurkat T cells was analyzed by flow cytometry. (B) Jurkat T cells were stimulated with anti-CD3 and anti-CD28 antibodies, and PDK1 was analyzed by immunoblot analysis with anti-PDK1 antibody. (C) Binding of HA-tagged PKCθ with Myc-tagged PDK1 was analyzed by co-immunoprecipitation analysis. (D) Changes in the mobility of Myc-tagged PDK1 co-immunoprecipitated with HA-tagged PKCθ or HA-tagged PKCθ (K409W) were analyzed by immunoblot analysis with or without λ protein phosphatase treatment. Data are representative of three (A) or two (B–D) independent experiments.

Fig. 2. Ser-64, a novel phosphorylation site on PDK1, is phosphorylated by PKCθ

(A) Experimental scheme of phosphopeptide-specific mass spectrometry analysis. (B) Results of mass spectrometry analysis of PDK1 with PKCθ. Arrows indicate phosphorylated serine. (C) Results of mass spectrometry analysis of PDK1 with kinase activity-deficient PKCθ(K409W). Data are representative of two (B, C) independent experiments.

Fig. 3. Phosphorylation of PDK1 at Ser-64 regulates TCR/CD28-mediated NF-κB activation

(A) NF-κB reporter luciferase assay with wild-type (WT), phosphomimetic (S64D), or phosphorylation-deficient (S64I) PDK1 in HEK293 cells. (B) Protein expression levels of wild-type (WT), phosphomimetic (S64D), or phosphorylation-deficient (S64I) PDK1 in HEK293 cells were assessed by immunoblot analysis. (C) Protein expression levels of wild-type (WT), phosphomimetic (S64D), or phosphorylation-deficient (S64I) PDK1 in Jurkat T cells were assessed by immunoblot analysis. (D) NF-κB reporter luciferase assay with wild-type (WT), phosphomimetic (S64D), or phosphorylation-deficient (S64I) PDK1 in Jurkat T cells after stimulation with anti-CD3 and anti-CD28 antibodies. (E) After stimulation with anti-CD3 and anti-CD28 antibodies, IκBα phosphorylation levels in Jurkat T cells expressing wild-type (WT), phosphomimetic (S64D), or phosphorylation-deficient (S64I) PDK1 were assessed by immunoblot analysis. Data are representative of three (A–E) independent experiments. Results are expressed as the mean ± SD. *p < 0.05; **p < 0.01 (Student’s t- test).

Fig. 4. Ser-64 on PDK1 is important for regulation of protein stability

(A) After treatment with cycloheximide, the protein half-life of wild-type (WT) and phosphomimetic (S64D) PDK1 in HEK293 cells was assessed by immunoblot analysis. (B) After treatment with cycloheximide, the protein half-life of wild-type (WT) and phosphomimetic (S64D) PDK1 in Jurkat T cells was assessed by immunoblot analysis. PDK1 half-life values were calculated by nonlinear regression analysis with GraphPad Prism software. Data are representative of three (A, B) independent experiments.

Fig. 5. Ser-64 on PDK1 is important for regulation of T cell activation

(A) After stimulation with anti-CD3 and anti-CD28 antibodies, IL-2 mRNA expression levels in Jurkat T cells expressing wild-type (WT), phosphomimetic (S64D), or phosphorylation-deficient (S64I) PDK1 were assessed by quantitative RT-PCR. (B) After stimulation with anti-CD3 and anti-CD28 antibodies, IL-2 secretion levels in Jurkat T cells expressing wild-type (WT), phosphomimetic (S64D), or phosphorylation-deficient (S64I) PDK1 were assessed by ELISA. (C) After stimulation with anti-CD3 and anti-CD28 antibodies, surface expression levels of the T cell activation markers CD69 and CD25 in Jurkat T cells expressing wild-type (WT), phosphomimetic (S64D), or phosphorylation-deficient (S64I) PDK1 were assessed by flow cytometry. Data are representative of three (A–C) independent experiments. Results are expressed as the mean ± SD. *p < 0.05; **p < 0.01 (Student’s t-test).