Positive Regulation of IκB Kinase Signaling by Protein Serine/Threonine Phosphat

ase 2A

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 37232RUNNING 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

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

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NF-κB, nuclear factor-kappa B; IκB, inhibitor of kappa B; IKK, IκB kinase; PP2A,protein serine/threonine phosphatas

e 2A; TNF, tumor necrosis factor α; TNF-R1, TNF-receptor type 1; MEFs, murine embryonic fibroblasts.

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

Second, PP2A forms stable complexes wit

h IKK in untransfected mammalian cells.

Third, deletion of the PP2A binding site in I

KKγ.

okadaic acid , fostriecin

amino acid residue 121-179 of the IKKγ

attenuates T loop phosporylation and catalytic activation

Materials and Methods B lymphocytes, cell culture and transfections Generation of T7-tagged IKK γ internal deletion mutant of IKKγ lacki

ng amino acids 121-179 Generation of MEF cell lines stably expressing T7-tagged IKK γ con

structs 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

Effects of PP2A inhibitors on I κB degradation PP2A associates with IKK complexes Identification of a putative PP2A binding site in I

KK γ Role of PP2A in IKK signaling Rescue studies of IKK γ-deficient cells

Effects of PP2A inhibitors on I κB degradation

Despite thecapacity of PP2A to inactivate IKK in vitro,exposure of TNF-treated cells to an antagonist ofPP2A impairs rather than enhances thedegradation of IκBα.

Conclusion:

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 wereefficiently integrated into endogenous IKK complexes containing the IKKα and IKKβcatalytic 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 These functional results, together with data shown, furth

er 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