Positive regulation of IkappaB kinase signaling by protein serine/threonine phosphatase 2A.
Transcription factor NF-kappaB plays a key regulatory role in the cellular response to pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF). In the absence of TNF, NF-kappaB is sequestered in the cytoplasm by inhibitory IkappaB proteins. Phosphorylation of IkappaBby the beta-catalytic subunit of IKK, a multicomponent IkappaB kinase, targets the inhibitor for proteolytic destruction and facilitates nuclear translocation of NF-kappaB. This pathway is initiated by TNF-dependent phosphorylation of T loop serines in IKKbeta, which greatly stimulates IkappaB kinase activity. 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. Here we provided several in vivo lines of evidence indicating that PP2A plays a positive rather than a negative role in the regulation of IKK. First, TNF-induced degradation of IkappaB is attenuated in cells treated with okadaic acid or fostriecin, two potent inhibitors of PP2A. Second, PP2A forms stable complexes with IKK in untransfected mammalian cells. This interaction is critically dependent on amino acid residues 121-179 of the IKKgamma regulatory subunit. Third, deletion of the PP2A-binding site in IKKgamma attenuates T loop phosphorylation and catalytic activation of IKKbeta in cells treated with TNF. Taken together, these data provide strong evidence that the formation of IKK.PP2A complexes is required for the proper induction of IkappaB kinase activity in vivo.
Kray
Arlene E
AE
Department of Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee 37232-6600, USA.
Carter
Robert S
RS
Pennington
Kevin N
KN
Gomez
Rey J
RJ
Sanders
Laura E
LE
Llanes
Joan M
JM
Khan
Wasif N
WN
Ballard
Dean W
DW
Wadzinski
Brian E
BE
D021581:Active Transport, Cell Nucleus; D000255:Adenosine Triphosphate / Q000737:chemistry; D000475:Alkenes / Q000494:pharmacology; D000818:Animals; D001402:B-Lymphocytes / Q000378:metabolism; D002384:Catalysis; D002460:Cell Line; D002853:Chromatography, Liquid; D003593:Cytoplasm / Q000378:metabolism; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005347:Fibroblasts / Q000378:metabolism; D017353:Gene Deletion; D006801:Humans; D051550:I-kappa B Kinase / Q000378:metabolism*; D015151:Immunoblotting; D047468:Immunoprecipitation; D007249:Inflammation; D019169:Jurkat Cells; D051379:Mice; D008810:Mice, Inbred C57BL; D009154:Mutation; D019319:Okadaic Acid / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D010766:Phosphorylation; D011090:Polyenes; D054648:Protein Phosphatase 2; D011753:Pyrones; D012685:Sepharose / Q000737:chemistry; D012694:Serine / Q000737:chemistry*; D015398:Signal Transduction; D013154:Spleen / Q000166:cytology; D013912:Threonine / Q000737:chemistry*; D013997:Time Factors; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D000475:Alkenes; D004791:Enzyme Inhibitors; D011090:Polyenes; D011753:Pyrones; D014409:Tumor Necrosis Factor-alpha; D019319:Okadaic Acid; D013912:Threonine; D012694:Serine; D000255:Adenosine Triphosphate; D012685:Sepharose; D051550:I-kappa B Kinase; D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2; C040313:fostriecin