Supplementary Figure 1 CD4 + T cells from PKC-θ null mice are defective in NF-κB activation during T cell receptor signaling. CD4 + T cells were

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1 Supplementary Figure 1 CD4 + T cells from PKC-θ null mice are defective in NF-κB activation during T cell receptor signaling. CD4 + T cells were isolated from wild type (PKC-θ- WT) or PKC-θ null (PKC-θ-KO) mice by negative selection. Isolated T cells were stimulated with anti-cd3 anti-cd28 and then protein lysates were made and were analyzed by immunoblot analysis. Data are representative of two independent experiments.

2 Supplementary Figure 2 Activation loop phosphorylation of AKT is impaired in PDK1 knockout T-cells following stimulation with anti-cd3 anti-cd28, but hydrophobic motif phosphorylation of AKT is unaffected. Primary CD4 + T cells were purified from the lymph nodes of CD4-Cre Pdk1 flox/flox and CD4-Cre Pdk1 +/flox mice and stimulated with anti-cd3 anti- CD28 for 15 or 60 min. Phosphorylation of AKT at T308 and at S473 were analyzed by immunoblotting with anti-phospho T308 and anti-phospho S473 of AKT (Cell signaling). Data are representative of two independent experiments.

3 Supplementary Figure 3 PDK1 is not involved in stability of IL-2 mrna in cells treated with anti-cd3 anti-cd28. IL-2 mrna stability was analyzed by semiquantitative (a) and quantitative PCR analysis with Quantitect SYBR Green PCR kit (Qiagen) (b). Primary CD4 + T cells were purified from the lymphnodes of CD4-Cre Pdk1 flox/flox (n = 2) and CD4-Cre Pdk1 +/flox (n = 2) mice and stimulated with anti-cd3 anti-cd28 for 6 h. Actinomycin D (final concentration at 10 µg/ml) was then added to the culture media. At each time point, total RNA was isolated from the cells by using Trizol reagent and reverse transcribed with Superscript III (Invitrogen). Error bars, s.d. of triplicate samples.

4 Supplementary Figure 4 Cre expressing retrovirus-mediated Pdk1 gene deletion in effector CD4 + T cells impairs IL-2 production during the T cell receptor signaling. Total splenocytes were prepared from Pdk1 flox/flox or Pdk1 +/+ mice and the prepared cells were treated with ConA for 24 h. Following ConA stimulation, the cells were infected with the retrovirus (MSCV-Cre IRES GFP) and were cultured for 3 d later from the infection. GFP-positive (GFP + ) and GFP-negative (GFP ) CD4 + T cells were sorted and then stimulated with anti-cd3 anti- CD28 for 24 h in 96 well plates. IL-2 production was analyzed by ELISA. Data are representative of two independent experiments. Error bars, s.d. of triplicate samples.

5 Supplementary Figure 5 ER-Cre-mediated Pdk1 gene deletion in effector CD4+ T cell impairs Iκ κbα α degradation and IL-2 production during the T cell receptor signaling. Total splenocytes were prepared from ER-Cre Pdk1flox/flox or ER-Cre Pdk1+/+ mice and were treated with ConA for 24 h. The cells were then treated with 4-OHT (100 nm) for 84 h for deletion of Pdk1 gene. After deletion of Pdk1 gene, CD4+ T cells were isolated through negative selection and the isolated T cells were stimulated with anti-cd3 anti-cd28 for 20 min (a) and 24 h (b). Data are representative of two independent experiments (error bars (b), s.d. of triplicate samples).

6 Supplementary Figure 6 PDK1 is dispensible for effector T cell survival. (a) and (b) As in the supplementary Fig. 4, total splenocytes (Pdk1 flox/flox or Pdk1 +/+ ) were infected with retrovirus and 3 d later, the ratio of GFP-positive to GFP-negative CD4 + T cell or CD8 + T cell was set to 1. Each following day the ratios were checked by flow cytometry. These data are representative data from 3 independent experiments. (c) As in the supplementary Fig. 5, total splenocytes (ER- CRE Pdk1 flox/flox or ER-Cre Pdk1 +/+ ) were treated with 4-OHT (100 nm). And then, flow cytometry was performed after culturing for 3 d and 6 d in the presence (+) or absence ( ) of exogenous IL-7, followed by staining with annexin V 7-amino-actinomycin D (7AAD) and gating on CD4 + CD8 cells. Numbers beside boxed areas indicate percent 7AAD and annexin V double positive cells (top), annexin V positive cells (lower right) or 7AAD annexin V negative cells (lower left). Data are representative of two (d) to three (a,b) experiments.

7 Supplementary Figure 7 CD28 coligation is needed for NF-κB activation and IL-2 production during T cell receptor signaling. (a) CD4 + T cells were isolated from spleen, purified by negative selection, stimulated with either anti-cd3, anti-cd28, or anti-cd3 anti- CD28 for 20 min and lysates were made and analyzed by immunoblot analysis. (b) Total splenocytes were isolated and treated with ConA and 48hrs later were restimulated with either anti-cd3, anti-cd28, or anti-cd3 anti-cd28 for 6 h with in the presence of Brefeldin A. Intracellular IL-2 was analyzed with BD Cytofix/Cytoperm Plus and APC-conjugated anti-il-2. Data are representative of two (b) to three (a) independent experiments.

8 Supplementary Figure 8 PDK1 and PKC-θ are recruited by engagement of antigen receptor with antigen-mhc complex. CD4 + T cells were isolated from TCR transgenic mice specific for pigeon cytochrome c (PCC) peptide and the isolated T cells were incubated with CH27 B cells that had been prepulsed with PCC (1 ng/ml) for 24 h. After 30 min incubation at 37 C on cover slips coated with poly-l-lysine, localization of PDK1 and PKC-θ was analyzed by immunofluorescent microscopy.

9 Supplementary Figure 9 PDK1 binds to PKC-θ in primary T cell upon antigen receptor stimulation and phosphorylates PKC-θ in vitro. (a) CD4 + T cells were isolated from spleen and the isolated T cells were stimulated with anti-cd3 anti-cd28. PDK1 was immunoprecipitated from whole cell lysates using rabbit anti-pdk1. Coimmunoprecipitation of PKC-θ was analyzed by immunoblotting with goat anti-pkc-θ. (b) In vitro kinase assays were performed with GST-PKC-θ and PDK1 immunoprecipitated using mouse anti-myc from whole cell lystae of HEK293 cells transfected with pcmv/myc-pdk1. Data are representative of two independent experiments

10 Supplementary Figure 10 Coligation of CD3 and CD28 is required for activation loop phosphorylation of PKC-θ θ and Iκ κbα α degradation. Jurkat T cells were stimulated with antihuman CD3 (a) or anti-human CD28 (b) and cell lysates from the stimulated cells were analyzed by immunoblot analysis. Data are representative of three independent experiments.

11 Supplementary Figure 11 Mobility shift of PDK1 is caused by PDK1 phosphorylation. Jurkat T cells were stimulated with anti-human CD3/28 for 4 h and PDK1 was immunoprecipitaed with rabbit anti-pdk1. The immunoprecipiated PDK1 was treated with lamda protein phosphatase for 1 h at 30 C with or without phosphatase inhibitor and the mobility of PDK1 was analyzed by immunoblot analysis. Data are representative of two independent experiments.

12 Supplementary Figure 12 T513 is a crucial regulatory site for PDK1 PKC-θ θ binding. HEK293 cells were transfected with pegz-ha/pkc-θ and pcmv-myc/pdk1 or pcmvmyc/pdk1 point mutant. HA-PKC-θ was immunoprecipitated with mouse anti-ha and coimmunoprecipitated Myc-PDK1 was analyzed by immunoblot analysis. Data are representative of five independent experiments

13 Supplementary Figure 13 between PDK1 and AKT. The three-dimensional structure around T513 conserved

14 Supplementary Figure 14 PDK1 is essential for integration of TCR and CD28-mediated signaling to NF-κ κb activation. CD28 induced PI3K activation leads to PIP2 phosphorylation to PIP3 and recruitment of PDK1. PDK1 binding to PIP3 promotes auto-phosphorylation at T513. PDK1 phosphorylation renders it competent for binding to both PKC-θ, recruited by TCR ligation, and to CARMA1. PKC-θ is phosphorylated by PDK1 and this PKC-θ may activate NF-κB through phosphorylation of CARMA1. Yellow arrows, phosphorylation; Dashed arrow, recruitment or binding; Black arrow, conversion of state.