Transcript Positive Regulation of IκB Kinase Signaling by Protein

Positive Regulation of IκB Kinase
Signaling by Protein Serine/Threonine
Phosphatase 2A
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Arlene E. Kray1, Robert S. Carter2, etc. From the
1Department of Pharmacology and the 2Department of
Microbiology and Immunology,Vanderbilt University
Medical Center, Nashville, TN 37232
RUNNING TITLE: Regulation of IKK by associated PP2A
系級:銘傳大學生物科技系4年級
學生:劉永偉
日期:2005.10.11
Introduction
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Transcription factor NF-κB plays a key
regulatory role in the cellular response to
proinflammatory cytokines such as tumor
necrosis factor-α (TNF-α).
http://www-ermm.cbcu.cam.ac.uk/ 03006756h.htm
http://www.devbio.com/ image.php?id=125
NF-κB, nuclear factor-kappa B;
 IκB, inhibitor of kappa B;
 IKK, IκB kinase;
 PP2A,protein serine/threonine
phosphatase 2A;
 TNF, tumor necrosis factor α;
 TNF-R1, TNF-receptor type 1;
 MEFs, murine embryonic fibroblasts.
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Prior in vitro mixing experiments indicate
that protein serine/threonine phosphatase
2A (PP2A) can dephosphorylate these T
loop serines and inactivate IKK,suggesting
a negative regulatory role for PP2A in IKK
signaling.
First, TNF-induced degradation of IκB
okadaic acid , fostriecin
 Second, PP2A forms stable complexes
with IKK in untransfected mammalian cells.
amino acid residue 121-179 of the IKKγ
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Third, deletion of the PP2A binding site in
IKKγ.
attenuates T loop phosporylation and
catalytic activation
Materials and Methods
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B lymphocytes, cell culture and transfections
Generation of T7-tagged IKK γ internal deletion mutant of IKKγ
lacking amino acids 121-179
Generation of MEF cell lines stably expressing T7-tagged IKK γ
constructs
Immunoblotting
Fast Performance Liquid Chromatography
ATP-Sepharose and microcystin-Sepharose
affinity purifications
Immunoprecipitations
In vitro kinase assays
Microcystin-Sepharose affinity isolations
Add reagents and treat
with mammalian cell
Co-immunoprecipitate extracts
Fractionate by chomatography
Immunoblotting or SDS-PAGE
Results and Discussion
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Effects of PP2A inhibitors on I κB degradation
PP2A associates with IKK complexes
Identification of a putative PP2A binding site in
IKK γ
Role of PP2A in IKK signaling
Rescue studies of IKK γ-deficient cells
Effects of PP2A inhibitors on I κB degradation
Conclusion:
Despite the
capacity of PP2A to
inactivate IKK in vitro,
exposure of TNFtreated cells to an
antagonist of
PP2A impairs rather
than enhances the
degradation of IκBα.
PP2A associates with IKK complexes
JTCE: Jurkat T cell extracts
ATP→MC: ATP eluate was
then incubated with
microcystin-Sepharose
Conclusion:
The PP2A catalytic subunit (PP2AC)
co-eluted with these IKK subunits in
the presence of ATP.
Conclusion:
These data provide multiple
independent lines of evidence
indicating that PP2A forms
stable complexes with IKK in
untransfected mammalian cells.
Identification of a putative PP2A
binding site in IKK γ
Role of PP2A in IKK signaling
IKKβ T loop
phosphorylation
Rescue studies of IKK γ-deficient
cells
Conclusion:
These data demonstrate that
the wild type and PP2A-binding
defective IKKγ constructs were
efficiently integrated into
endogenous IKK complexes
containing the IKKα and IKKβ
catalytic subunits.
Conclusion:
These data with
reconstituted MEFs
provide strong evidence
that PP2A plays a positive
regulatory role in IKK
signaling following cellular
stimulation with the pro
inflammatory cytokine TNF.
Conclusion
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These functional results, together with data shown,
further reinforce the model that PP2A binds to IKK,
facilitating the induction of IκB kinase activity, targeted
degradation of IκB, and release of NF-κB to its nuclear
site of action.
Instead, our studies support the idea that this region of
IKKγ is a novel protein-protein interaction domain, which
facilitates PP2A’s interaction with the IKK complex,
allowing this phosphatase to function as a positive
regulator of IKK signaling.
Thank you