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

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

• Lee KY, et al. Science 308114-118(2005)
• Chen-Chung Lin, Dep. of Cell Biology

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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.)
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Introduction (TCR-CD3 signal cascade)
(Adapted from Kuby, et al. Immunology text book.)
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Introduction (NF-kB major pathway)

• 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.

(Kane, LP. et al. Trends Immunol. 23, 413)
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Introduction (Proximal signaling pathway
TCR-NF-kB)

• ZAP-70 tyrosine kinase, which is recruited to the
  TCRCD3 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).

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(Kane, LP. et al. Trends Immunol. 23, 413)
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Aims

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

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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.

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Association between PDK1 and PKCq
TCR??PDK1??PKCq???NFkB activation
Jurkat T Cells
Primary Cells
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Requirement of PDK1 for TCR-mediated NF-kB
activation
TCR?PDK1??PKCq (activated by phosphorylation??)
???NFkB activation
Primary Cells
Jurkat T Cells
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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
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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
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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)
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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.
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PDK1 functions in activation of PKCq.
?
TCR?PDK1?PKCq (activated by phosphorylation)
??IkB degradation ?NFkB activation? Effector
responses (IL-2 gene expression level)

• Phosphorylation level of PKCq is decreased after
  PDK1 knockdown.
• Phosphorylation of PKCq only occurs in TCR
  stimulated cells.

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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.
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Recruitment of PKCq to lipid rafts depend on PDK1

• 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.

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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.

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Requirement of PDK1 for the recruitmentof CARD11
into the lipid rafts after TCRstimulation.

• 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.

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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
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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.
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Model