Transient and selective NF-kappa B p65 serine 536 phosphorylation induced by T cell costimulation is mediated by I kappa B kinase beta and controls the kinetics of p65 nuclear import.
Full transcriptional activity of the nuclear, DNA-bound form of NF-kappaB requires additional posttranslational modifications. In this study, we systematically mapped the T cell costimulation-induced phosphorylation sites within the C-terminal half of the strongly trans-activating NF-kappaB p65 subunit and identified serine 536 as the main phosphorylation site. The transient kinetics of serine 536 phosphorylation paralleled the kinetics of IkappaBalpha and IkappaB kinase (IKK) phosphorylation and also mirrored the principle of T cell costimulation. The TCR-induced pathway leading to serine 536 phosphorylation is regulated by the kinases Cot (Tpl2), receptor interacting protein, protein kinase Ctheta, and NF-kappaB-inducing kinase, but is independent from the phosphatidylinositol 3-kinase/Akt signaling pathway. Loss-of-function and gain-of-function experiments showed phosphorylation of p65 serine 536 by IKKbeta, but not by IKKalpha. Phosphorylation occurs within the cytoplasmic and intact NF-kappaB/IkappaBalpha complex and requires prior phosphorylation of IkappaBalpha at serines 32 and 36. Reconstitution of p65(-/-) cells either with wild-type p65 or a p65 mutant containing a serine to alanine mutation revealed the importance of this phosphorylation site for cytosolic IkappaBalpha localization and the kinetics of p65 nuclear import.
Mattioli
Ivan
I
University of Bern, Department of Chemistry and Biochemistry, Bern, Switzerland.
Sebald
Andrea
A
Bucher
Cyril
C
Charles
Roch-Philippe
RP
Nakano
Hiroyasu
H
Doi
Takahiro
T
Kracht
Michael
M
Schmitz
M Lienhard
ML
D021581:Active Transport, Cell Nucleus / Q000235:genetics / Q000276:immunology; D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000818:Animals; D018106:CD28 Antigens / Q000494:pharmacology; D017252:CD3 Complex / Q000494:pharmacology; D002467:Cell Nucleus / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D003600:Cytosol / Q000201:enzymology / Q000378:metabolism; D006801:Humans; D051550:I-kappa B Kinase; D007527:Isoenzymes / Q000502:physiology; D019169:Jurkat Cells; D007700:Kinetics; D008213:Lymphocyte Activation / Q000235:genetics; D020930:MAP Kinase Kinase Kinases / Q000502:physiology; D051379:Mice; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000502:physiology; D010750:Phosphoproteins / Q000172:deficiency / Q000235:genetics / Q000502:physiology; D010766:Phosphorylation; D011493:Protein Kinase C / Q000502:physiology; D000074121:Protein Kinase C-theta; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism / Q000502:physiology*; D011506:Proteins / Q000235:genetics / Q000502:physiology; D011518:Proto-Oncogene Proteins / Q000502:physiology; D051057:Proto-Oncogene Proteins c-akt; D053422:Receptor-Interacting Protein Serine-Threonine Kinases; D012694:Serine / Q000378:metabolism*; D015398:Signal Transduction / Q000235:genetics / Q000276:immunology; D016176:T-Lymphocyte Subsets / Q000201:enzymology / Q000276:immunology / Q000378:metabolism*; D051996:Transcription Factor RelA; D015533:Transcriptional Activation / Q000276:immunology; D000097782:NF-kappaB-Inducing Kinase
D048868:Adaptor Proteins, Signal Transducing; D018106:CD28 Antigens; D017252:CD3 Complex; D051550:I-kappa B Kinase; D007527:Isoenzymes; D020930:MAP Kinase Kinase Kinases; D016328:NF-kappa B; D010446:Peptide Fragments; D019869:Phosphatidylinositol 3-Kinases; D010750:Phosphoproteins; D011493:Protein Kinase C; D000074121:Protein Kinase C-theta; D017346:Protein Serine-Threonine Kinases; D011506:Proteins; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D053422:Receptor-Interacting Protein Serine-Threonine Kinases; D012694:Serine; D051996:Transcription Factor RelA; D000097782:NF-kappaB-Inducing Kinase; C092947:SLP-76 signal Transducing adaptor proteins; C494918:AKT1 protein, human; C506249:RIPK1 protein, human; C506251:Ripk1 protein, mouse; C496559:CHUK protein, human; C496564:Chuk protein, mouse; C496560:IKBKB protein, human; C496565:IKBKE protein, human; C496562:Ikbkb protein, mouse; C496566:Ikbke protein, mouse; C076233:PRKCQ protein, human; C489593:Prkcq protein, mouse; C069937:MAP3K8 protein, human