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PDK1 nucleates T cell receptor-induced signaling complex for NF-kappaB activation.

PDK1 nucleates T cell receptor-induced signaling complex for NF-kappaB activation. Lee KY, et al. Science 308:114-118(2005) Chen-Chung Lin, Dep. of Cell Biology. Introduction (TCR-CD3 complex ). TCR-CD3 complex. (Immunoreceptor tyrosine-based activation motifs). Signaling cascade.

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PDK1 nucleates T cell receptor-induced signaling complex for NF-kappaB activation.

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  1. PDK1 nucleates T cell receptor-induced signaling complex for NF-kappaB activation. Lee KY, et al. Science 308:114-118(2005) Chen-Chung Lin, Dep. of Cell Biology

  2. Introduction (TCR-CD3 complex ) TCR-CD3 complex (Immunoreceptor tyrosine-based activation motifs) Signaling cascade T-cell maturation, proliferation, activation (Adapted from Kuby, et al. Immunology text book.)

  3. Introduction (TCR-CD3 signal cascade) (Adapted from Kuby, et al. Immunology text book.)

  4. NF-kB (as a dimer) is a transcription factor. At un-stimulus state, NF-kB dimer inhibited by binding to inhibitor of kB (IkB). At stumulus state, IkB will release from NF-kB by IkB kinase (IKK) complex phosphorylation; the phosphorylation further cause ubiquitination of IkB then induce proteasome degradation of IkB. Regulation of IKK complex is very important for NF-kB activation pathway. Introduction (NF-kB major pathway) (Kane, LP. et al. Trends Immunol. 23, 413)

  5. ZAP-70 tyrosine kinase, which is recruited to the TCR–CD3 complex upon receptor crosslinking and activated. Vav protein phosphorylation is required for IKK activation. Vav protein could regulates PKCq. Vav protein could be upstream regulator for PI3K and Akt pathway. Bcl10-MALT1 complex can ubiquitinate NEMO (IKKg). Introduction (Proximal signaling pathway: TCR-NF-kB) ? ? (Kane, LP. et al. Trends Immunol. 23, 413)

  6. Aims • Study the possible signaling proteins to link between TCR and PKCq pathway.

  7. PDK-1 and PKCq • 3-phosphoinositide-dependent kinase-1 (PDK-1) regulate many PKC isotypes by phosphorylayion of activation loop of PKCs; those PKCs including PKCq. • PDK-1 activity could partially regulated by PI3K signaling. • Hypothesize that PDK1 play a proximal role in TCR mediated NFkB activation in T cells. ?

  8. Association between PDK1 and PKCq TCRPDK1?PKCqNFkB activation Jurkat T Cells Primary Cells

  9. Requirement of PDK1 for TCR-mediated NF-kB activation TCRPDK1?PKCq (activated by phosphorylation??) NFkB activation Primary Cells Jurkat T Cells

  10. Requirement of PDK1 for TCR-mediated NF-kB activation TCRPDK1?PKCq (activated by phosphorylation??) NFkB activation PDK1 knockout  lethal Use si(h)RNA to study PDK1 function

  11. Generation and characterizationof PDK1-knockdown Jurkat T cells. TCRPDK1?PKCq (activated by phosphorylation) IkB degradation NFkB activation Stable Knockdown of PDK-1 Ctl shRNA

  12. Generation and characterizationof PDK1-knockdown Jurkat T cells. TCRPDK1?PKCq (activated by phosphorylation) IkB degradation NFkB activation Effector responses (IL-2 gene expression level)

  13. Generation and characterizationof PDK1-knockdown Jurkat T cells. PKCq would involve in regulating TCR induced Ca2+ response. (Pfeifhofer C et al., J. Exp. Med.197, 1525) TCRPDK1?PKCq (activated by phosphorylation) IkB degradation NFkB activation Effector responses (Ca2+ mobilization) PDK1-mediated phosphorylation of PKCq may be required for functional activation of PKCq after TCR stimulation.

  14. Phosphorylation level of PKCq is decreased after PDK1 knockdown. Phosphorylation of PKCq only occurs in TCR stimulated cells. PDK1 functions in activation of PKCq. ? TCRPDK1PKCq (activated by phosphorylation) IkB degradation NFkB activation Effector responses (IL-2 gene expression level)

  15. Recruitment of PKCq to lipid rafts depend on PDK1 After TCR stimulation, PDK1 goes to lipid raft. After TCR stimulation, PKCq and IKKb goes to lipid raft, but not in PDK1 kockdown cells.

  16. PKCq physically interact with IKK complex. After TCR stimulation, the IKK complex association with PKCq increased. Knock down PDK1 would impair the association of PKCq and IKK complex. Recruitment of PKCq to lipid rafts depend on PDK1

  17. CARD11, Bcl10-MALT1 and IKK • Activation of IKK required ubiquitination of NEMO (IKKg) by Bcl10-MALT1 complex. • Recruitment of PKCq to the TCR is not effected in CARD11 knockout T cells.

  18. Association of PKCq with IKKb and NEMO (IKKg) was not effected by CARD11 deficiency. Recruitment of PKCq-IKK to the receptor is independent of CARD11 but dependent on PDK1. Requirement of PDK1 for the recruitmentof CARD11 into the lipid rafts after TCRstimulation.

  19. PDK1 and CARD11 protein association CARD11 and PDK1 are membrane-associated proteins. Test whether activated PDK1 could recruit CARD11 to lipid raft and serve as nucleating role in this pathway

  20. Requirement of PDK1 for the recruitmentof CARD11 into the lipid rafts after TCRstimulation. GUK (guanylate kinase) domain of CARD11 appeared to be responsible for this specific interaction with PDK1. PDK1 plays an essential role in recruitment of CARD11 and Bcl10 complex to lipid raft.

  21. Model

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