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PDK1 Nucleates T Cell Receptor-Induced Signaling Complex for NF-κB Activation

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Science  01 Apr 2005:
Vol. 308, Issue 5718, pp. 114-118
DOI: 10.1126/science.1107107

Abstract

Activation of the transcription factor NF-κB after engagement of the T cell receptor (TCR) is important for T cell proliferation and activation during the adaptive immune response. Recent reports have elucidated a signaling pathway that involves the protein kinase C θ (PKCθ), the scaffold protein CARD11 (also called CARMA-1), the caspase recruitment domain (CARD)–containing protein Bcl10, and the paracaspase (protease related to caspases) MALT1 as critical intermediates linking the TCR to the IκB kinase (IKK) complex. However, the events proximal to the TCR that initiate the activation of this signaling pathway remain poorly defined. We demonstrate that 3-phosphoinositide-dependent kinase 1 (PDK1) has an essential role in this pathway by regulating the activation of PKCθ and through signal-dependent recruiting of both PKCθ and CARD11 to lipid rafts. PDK1-associated PKCθ recruits the IKK complex, whereas PDK1-associated CARD11 recruits the Bcl10-MALT1 complex, thereby allowing activation of the IKK complex through Bcl10-MALT1–dependent ubiquitination of the IKK complex subunit known as NEMO (NF-κB essential modifier). Hence, PDK1 plays a critical role by nucleating the TCR-induced NF-κB activation pathway in T cells.

Stimulation of T cells by engagement of the TCR-CD3 complex along with the coreceptor CD28 initiates signal transduction cascades that lead to the activation of the IκB kinase (IKK) complex (1). The canonical IKK complex is composed of three subunits: IKKα, IKKβ, and NEMO (also called IKKγ). IKKα and IKKβ are catalytic kinase subunits, whereas NEMO is a structural and regulatory subunit (26). Upon TCR-mediated signaling, the IKK complex is recruited into lipid rafts, and protein kinase C θ (PKCθ) is thought to have an important role in this recruitment event (79). However, the mechanism responsible for the activation of PKCθ and its recruitment to lipid rafts remains unclear. TCR stimulation also requires recruitment of CARD11, Bcl10, and MALT1 to lipid rafts. Knockout of genes encoding these proteins causes a block in signaling to NF-κB from both T cell and B cell receptors (10). However, the mechanism by which these proteins initiate the NF-κB activation pathway has remained obscure. A recent report has provided some insight into this question by demonstrating that the Bcl10-MALT1 complex can ubiquitinate the NEMO subunit of the IKK complex, thereby causing the activation of IKK, and hence NF-κB, in T cells (11). However, it remains unclear how the IKK and Bcl10-CARD11-MALT1 complexes interact and what role, if any, PKCθ recruitment and activation has in this process.

The activity of kinases that belong to the PKC family is regulated by the activation loop, which retains the kinase in an inactive conformation. Phosphorylation of the activation loop leads to its displacement from the active site and to stabilization of the catalytically competent conformation of the enzyme (12). One kinase upstream of both atypical and conventional PKC isoforms is 3-phosphoinositide-dependent kinase (PDK)–1, whose activity is partly regulated by phosphoinositide 3-kinase (PI3K). PI3K-generated lipids activate PDK1, which then phosphorylates and activates several members of the conserved AGC kinase superfamily including protein kinases A (PKA), G (PKG), and C (PKC) (12). Activation of PDK1 by the PI3K pathway provides a possible explanation for why the lipid products of PI3K, namely phosphatidylinositol-3,4-bisphosphate (PIP2) and phosphatidylinositol-3,4-5-trisphosphate (PIP3) stimulate the activation of protein kinase-dependent signaling pathways (1214). The PI3K pathway also augments signaling to NF-κB, probably through its effect on Akt (15). However, PDK1 knockouts exhibit early embryonic lethality, thus preventing straightforward analysis of PDK1 function in T cell activation. In addition, conditional knockout of PDK1 in the T cell lineage results in a block early in thymocyte development, and hence, PDK1-deficient T cells suitable for studying TCR signaling cannot be isolated (16). Therefore, the importance of PDK1 in T cell signaling remains unclear.

To assess the role of PDK1 in TCR signaling through PKCθ, we tested whether endogenous PDK1 interacted with PKCθ in primary T cells that were either unstimulated or stimulated with antibodies against CD3 and CD28 (anti-CD3 anti-CD28). Efficient interaction between endogenous PKCθ and PDK1 was observed only in stimulated cells (Fig. 1A). An inducible interaction between endogenous PDK1 and PKCθ was also observed in stimulated Jurkat T cells (Fig. 1B). PDK1 regulates the activity of PKCs by phosphorylating a specific threonine residue (Thr538 for PKCθ) in their activation loop (17). We used a phospho-specific antibody that recognizes Thr538-phosphorylated PKCθ to confirm that stimulation of primary T cells (Fig. 1C) and Jurkat T cells (Fig. 1D) with anti-CD3 and anti-CD28 led to phosphorylation of Thr538 of PKCθ.

Fig. 1.

Requirement of PDK1 for TCR-mediated NF-κB activation. (A and B) Interaction between endogenous PDK1 and PKCθ. Primary (A) and Jurkat (B) T cells (5 × 107) were stimulated with or without anti-CD3 and anti-CD28. Proteins from cell lysates were immunoprecipitated (IP) and immunoblotted (IB) with antibodies against PKCθ (anti-PKCθ) and PDK1 (anti-PDK1) as indicated. (C and D) Phosphorylation of PKCθ at Thr538. Primary (C) and Jurkat (D) T cells (5 × 107) were stimulated with or without anti-CD3 and anti-CD28. Cell lysates were immunoprecipitated with anti-PKCθ and immunoblotted with anti-phospho-PKCθ (Thr538). (E) Confirmation of shRNA-induced suppression of PDK1 expression. Myc-tagged PDK1 was transfected into HEK293 with shRNA1, shRNA2, shRNA3, or sihCARD11-1, a control shRNA targeting CARD11. The expression of Myc-PDK1 was evaluated by immunoblotting with antibody to Myc (anti-Myc). GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (F and G) Role of PDK1 in TCR-mediated NF-κB activation (F) but not TNFα-mediated NF-κB activation (G). Jurkat T cells were transfected with the pBllx κB reporter construct and the indicated shRNA or control. Cells were stimulated with TNFα or anti-CD3 and anti-CD28 or left untreated. Luciferase activity was quantified as outlined in (19).

The lack of appropriate PDK1 knockout cells prompted us to knock down PDK1 with use of RNA interference. We designed three short hairpin RNAs (shRNAs) targeting PDK1 (fig. S1, A and B) and cloned them into the pSUPER.Retro vector (18, 19). Suppression of PDK1 expression was tested in 293 cells by cotransfecting the shRNAs along with a vector expressing PDK1. ShRNA2 and shRNA3 but not shRNA1 efficiently suppressed the expression of PDK1 (Fig. 1E) in a dose-dependent manner (20). To test the role of PDK1 in TCR-mediated NF-κB activation, we transfected the shRNAs along with an NF-κB–dependent reporter construct into Jurkat cells. After transfection, the cells were stimulated with either anti-CD3 and anti-CD28 (Fig. 1F) or with tumor necrosis factor α (TNF-α) (Fig. 1G). Both shRNA2 and shRNA3 inhibited the activation of the immunoglobulin κ2 (Igκ2)-luciferase (LUC) reporter by CD3-CD28 cross-linking, whereas the control shRNA1 had no effect. The inhibitory effect was TCR-specific, because the shRNAs had no effect on NF-κB activation by TNF-α. Similar effects were seen with the use of shCARD11-1 as a positive control (Fig. 1F) (21), suggesting that PDK1 is required for NF-κB activation during TCR-mediated signaling.

Stable knockdown of PDK1 (PDK1kd) recapitulated the blockade of TCR-mediated NF-κB activation observed in transient transfection experiments (Fig. 1F). Retroviruses encoding both green fluorescent protein (GFP) and shRNA were produced (fig. S1C) and used to generate T cell lines stably expressing PDK1-specific shRNAs (fig. S1F) (19). PDK1kd Jurkat cells showed decreased cell proliferation but normal cell cycle distribution (fig. S1, F and G) (19), as did a glioblastoma cell line (U87-MG) with decreased PDK1 (22). In PDK1kd Jurkat cells generated with use of shRNA3 (and shRNA2 but not control shRNA1), both degradation of IκBα (Fig. 2E) and activation of NF-κB (Fig. 2C) were severely inhibited. Furthermore, loss of PDK1 led to impairment of functional NF-κB signaling as assessed by intracellular interleukin-2 (IL-2) quantification by flow cytometry (Fig. 2D) (19). In PDK1kd Jurkat cells, Ca2+ mobilization was decreased compared with that in control cells (Fig. 2E) (19). PKCθ knockout T cells also have impaired Ca2+ mobilization after TCR stimulation (23). Therefore, these results suggest PDK1-mediated phosphorylation of PKCθ may be required for functional activation of PKCθ after TCR stimulation.

Fig. 2.

Generation and characterization of PDK1-knockdown Jurkat T cells. (A) Confirmation of suppression of exogenously expressed PDK1 by pSUPER.retro/GFP-containing shRNAs. Myc-tagged PDK1 was transfected into HEK293 along with pSUPER, pSUPER.retro/GFP/shRNA1, shRNA2, or shRNA3. The expression of Myc-PDK1 was evaluated by immunoblotting with anti-Myc. (B) IκB-α degradation in PDK1-knockdown Jurkat cells. Jurkat and PDK1-knockdown Jurkat cells were stimulated with anti-CD3 and anti-CD28 for the indicated times and immunoblotted with antibody to IκBα. wt, wild type. (C) Effect of PDK1 knockdown on NF-κB luciferase assay. Igκ2-LUC (pBIIx) vector (2 μg) or equal amount of pcDNA3 was transfected into wild-type, irrelevant knockdown, or PDK1kd Jurkat cells. After 48 hours, cells were stimulated with anti-CD3 and anti-CD28, and luciferase assays were performed following the manufacturer's instructions. Error bars indicate standard error of the mean. (D) Decreased IL-2 production in PDK1-knockdown Jurkat cells after stimulation of cells with anti-CD3 and anti-CD28. Cells were stimulated or unstimulated, incubated for 5 hours with brefeldin-A, stained for intracellular IL-2, and analyzed by flow cytometry. Error bars indicate standard error of the mean. (E) Impaired Ca2+ mobilization in PDK1-knockdown Jurkat cells. Jurkat T cells were loaded with 2 μM fluo-3AM for 30 min in Hanks' balanced salt solution (HBSS) buffer containing 0.01% Pluronic F-127 (Sigma). The cells were then washed three times with HEPES buffer, resuspended in the same buffer, and analyzed after 15 min at room temperature.

To determine whether PDK1 functions in the activation of PKCθ, we tested the phosphorylation of PKCθ after stimulation with anti-CD3 and anti-CD28. Phosphorylation of PKCθ was substantially decreased in the two PDK1kd Jurkat cell lines (Fig. 3A). Treatment of cells with TNF-α did not lead to phosphorylation of PKCθ (Fig. 3A, lanes 5 to 7), indicating that PDK1-mediated PKCθ phosphorylation is specific to TCR signaling. Knockdown of PDK1 did not lead to a discernible alteration in the level of PKCθ protein (Fig. 3A).

Fig. 3.

Requirement of PDK1 for activation of NF-κB through the activation and recruitment of PKCθ. (A) Impairment of the phosphorylation of PKCθ in PDK1 knockdown Jurkat cells. Wild-type Jurkat or PDK1 knockdown cells were stimulated for 30 min with either anti-CD3 and anti-CD28 or with TNF-α. Lysates were immunoblotted with anti-phospho-PKCθ (Thr538) or an antibody that recognizes all forms of PKCθ. (B) Redistribution of PDK1 after stimulation of cells with anti-CD3 and anti-CD28. Jurkat or PDK1kd cells (5 × 107) were then lysed with 1% Triton X-100 (Sigma) and subjected to sucrose density gradient centrifugation to isolate lipid rafts. The distribution of PDK1 in each fraction was determined by immunoblotting with a specific antibody. (C) The recruitment of PKCθ and IKKβ to lipid rafts is defective in PDK1-knockdown Jurkat cells. Jurkat or PDK1kd Jurkat cells (5 × 107) were stimulated with or without anti-CD3 and anti-CD28. Proteins from equal volumes of fraction generated as in (A) were analyzed by immunoblotting with antibodies specific for PKCθ and IKKβ (anti-IKKβ). CTX, cholera toxin B subunit. (D) Failure of PKCθ to interact with the IKK complex in PDK1 knockdown cells. Wild-type and shRNA3-transduced Jurkat T cells were stimulated with or without anti-CD3 and anti-CD28. Cell lysates were prepared and immunoprecipiated with anti-PKCθ. Immunoblotting was performed with antibodies to PKCθ, IKKα, IKKβ, and NEMO, respectively.

To assess whether the recruitment of PKCθ to lipid rafts depends on PDK1, we prepared the detergent-insoluble membrane fractions of wild-type and PDK1kd cells by centrifugation in a discontinuous sucrose gradient (7, 19). In unstimulated cells, PDK1 was detected in both the cytoplasm and lipid rafts; however, after exposure of cells to anti-CD3 and anti-CD28, the fraction of PDK1 in lipid rafts increased (Fig. 3B). Almost no PKCθ was detected in lipid rafts after stimulation of PDK1kd Jurkat cells with anti-CD3 and anti-CD28, whereas prominent recruitment of PKCθ was observed in wild-type Jurkat cells (Fig. 3C). Immunofluorescence analysis also showed the recruitment of PKCθ to the membrane to be decreased in PDK1kd cells (fig. S2A).

The results presented above suggest that PDK1 plays an essential role in linking T cell stimulation to NF-κB activation, probably through phosphorylation of PKCθ and recruitment of PKCθ into lipid rafts. PKCθ physically interacts with IKK complexes in lipid rafts (9), and hence PDK1, through PKCθ, may recruit IKK complexes to the TCR-signaling complex. We observed signal-dependent recruitment of IKKβ into lipid rafts in wild-type and control shRNA1-expressing cells (Fig. 3C) (7). However, this recruitment was abolished in the PDK1kd cells, supporting the hypothesis that PDK1, through its ability to activate and recruit PKCθ, regulates the recruitment of IKKβ. We also detected association of endogenous PKCθ with components of the IKK complex by co-immunoprecipitation from TCR-stimulated cells (Fig. 3D), and this interaction was dramatically diminished in cells lacking PDK1.

Although the results presented above indicate that PDK1 and PKCθ recruit IKK to the TCR-signaling complex, they do not address how the recruited IKK becomes activated to signal to NF-κB. Activation of IKK in response to TCR signaling depends on the regulatory ubiquitination of NEMO by the Bcl10-MALT1 complex (11). However, recruitment of PKCθ to the TCR is unaffected in CARD11 knockout T cells (24), and the association of PKCθ with IKKβ and NEMO was not affected by CARD11 deficiency (Fig. 4A), suggesting that recruitment of PKCθ-IKK to the receptor is independent of CARD11 but dependent on PDK1. Because PDK1 and CARD11 are both membrane-associated proteins, we tested the possibility that activated PDK1 might also recruit CARD11 into lipid rafts and thereby serve a critical nucleating role in the assembly of this signaling pathway. Association between endogenous PDK1 and CARD11 was observed in cells stimulated with antibodies to CD3 and CD28 (Fig. 4B). In addition, overexpressed PDK1 selectively interacted with CARD11 but not with Bcl10, consistent with the hypothesis that PDK1 recruits CARD11, whereas Bcl10 and MALT1 might be recruited to the membrane via CARD11 (Fig. 4B). The GUK (guanylate kinase) domain of CARD11 appeared to be responsible for this specific interaction with PDK1 (Fig. 4C). The recruitment of CARD11 and Bcl10 after TCR stimulation was completely abolished in PDK1kd cells, strongly suggesting that PDK1 plays an essential role in this process (Fig. 4C). Immunofluorescence analysis also showed a reduction in membrane recruitment of CARD11 in PDK1kd, but not in control, Jurkat cells (fig. S2B) (19).

Fig. 4.

Requirement of PDK1 for the recruitment of CARD11 into the lipid rafts after TCR stimulation. (A) Interaction between PKCθ and IKKs. CARD11-deficient Jurkat T cells (5 × 107) were stimulated with or without anti-CD3 and anti-CD28. Proteins from lysates were immunoprecipitated with anti-PKCθ, anti-NEMO, and anti-IKKβ, and immunoblotting was done with anti-IKKβ and anti-PKCθ. CARMA1, CARD11. (B) Interaction of CARD11 with PDK1. Wild-type Jurkat cells (5 × 107) stimulated with or without anti-CD3 and anti-CD28. The cells were lysed, and proteins were immunoprecipitated with anti-PDK1. Interaction of exogenously expressed proteins was examined in HEK293 cells transfected with Myc-tagged PDK1 and Myc-tagged CARD11. Transfected cells were lysed and immunoprecipitated with antibody to CARD11 (anti-CARD11), and immunoblotting was done with anti-Myc. For the interaction between PDK1 and Bcl10, Myc-tagged PDK1 was transfected into HEK293 cells along with Bcl10. After immunoprecipitation with anti-Myc, immunoblotting was done with anti-Bcl10 and anti-Myc. IgH, immunoglobulin heavy chain. (C) Requirement of GUK domain for interaction with PDK1. Myc-tagged PDK1 was transfected into HEK293 cells along with Myc-tagged CARD11, ΔCARD (caspase recruitment domain), ΔCC (coiled-coil), ΔPDZ (PSD-95, DLG, and ZO-1), ΔSH3 (src homology 3), or ΔGUK (guanylate kinase). Proteins immunoprecipitated with anti-PDK1 were immunoblotted with anti-CARD11 and anti-Myc, respectively. (D) Defective recruitment of CARD11 to lipid rafts in PDK1 knockdown cells. Wild-type or PDK1kd Jurkat cells (5 × 107) were stimulated with or without anti-CD3 and anti-CD28. Proteins from equal volumes of fractions prepared as in Fig. 3A were separated by SDS–polyacrylamide gel electrophoresis and analyzed by immunoblotting with anti-CARD11 and anti-Bcl10.

We propose the following model to explain the sequence of events leading from the TCR to NF-κB activation (Fig. 5). Upon TCR engagement, PI3K is activated, thereby producing the lipid effectors PIP2 and PIP3. These lipid effectors help activate membrane-associated PDK1 and lead to its enrichment in rafts. PDK1 then recruits and activates PKCθ by phosphorylation, and the active PKCθ subsequently recruits the IKK complex into the lipid raft (Fig. 5). PDK1 simultaneously recruits the CARD11-Bcl10-MALT1 complex, and the membrane-bound Bcl10 complex then activates IKK through ubiquitination of NEMO. PDK1 therefore plays a central nucleating role by linking the TCR to two signaling cassettes, PKCθ-IKK and CARD11-MALT1-Bcl10, leading ultimately to the activation of NF-κB in response to signaling from the TCR.

Fig. 5.

A model of TCR signaling to NF-κB. Stimulation of cells through TCR complexes leads to phosphorylation of cytoplasmic immunoreceptor tyrosine-based activation motifs (ITAMs) and recruitment and activation (gray arrows) of PI3K and, subsequently, PDK1. Activated PDK1 phosphorylates (bold arrow) PKCθ, which is able to recruit (double-headed arrows) IKKs to lipid raft domains. Meanwhile, PDK1 also recruits the CARD11-Bcl10-MALT1 complex to the lipid raft through its GUK domain. This signaling complex (PDK1-CARD11-Bcl10-MALT1) is able to ubiquitinate (dashed arrow) NEMO (which was recruited to lipid rafts by the phospho-PKCθ-PDK1 complex), leading to activation of the IKK complex and, lastly, activation of NF-κB.

Supporting Online Material

www.sciencemag.org/cgi/content/full/308/5718/114/DC1

Materials and Methods

Figs. S1 to S3

References and Notes

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