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

J Immunol. 2004 May 15;172(10):6336-44. doi: 10.4049/jimmunol.172.10.6336.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Active Transport, Cell Nucleus / genetics
  • Active Transport, Cell Nucleus / immunology
  • Adaptor Proteins, Signal Transducing
  • Amino Acid Sequence
  • Animals
  • CD28 Antigens / pharmacology
  • CD3 Complex / pharmacology
  • Cell Nucleus / genetics
  • Cell Nucleus / immunology
  • Cell Nucleus / metabolism*
  • Cytosol / enzymology
  • Cytosol / metabolism
  • Humans
  • I-kappa B Kinase
  • Isoenzymes / physiology
  • Jurkat Cells
  • Kinetics
  • Lymphocyte Activation* / genetics
  • MAP Kinase Kinase Kinases / physiology
  • Mice
  • Molecular Sequence Data
  • NF-kappa B / antagonists & inhibitors
  • NF-kappa B / deficiency
  • NF-kappa B / genetics
  • NF-kappa B / metabolism*
  • Peptide Fragments / metabolism
  • Phosphatidylinositol 3-Kinases / physiology
  • Phosphoproteins / deficiency
  • Phosphoproteins / genetics
  • Phosphoproteins / physiology
  • Phosphorylation
  • Protein Kinase C / physiology
  • Protein Kinase C-theta
  • Protein-Serine-Threonine Kinases / metabolism
  • Protein-Serine-Threonine Kinases / physiology*
  • Proteins / genetics
  • Proteins / physiology
  • Proto-Oncogene Proteins / physiology
  • Proto-Oncogene Proteins c-akt
  • Receptor-Interacting Protein Serine-Threonine Kinases
  • Serine / metabolism*
  • Signal Transduction / genetics
  • Signal Transduction / immunology
  • T-Lymphocyte Subsets / enzymology
  • T-Lymphocyte Subsets / immunology
  • T-Lymphocyte Subsets / metabolism*
  • Transcription Factor RelA
  • Transcriptional Activation* / immunology

Substances

  • Adaptor Proteins, Signal Transducing
  • CD28 Antigens
  • CD3 Complex
  • Isoenzymes
  • NF-kappa B
  • Peptide Fragments
  • Phosphoproteins
  • Proteins
  • Proto-Oncogene Proteins
  • SLP-76 signal Transducing adaptor proteins
  • Transcription Factor RelA
  • Serine
  • Phosphatidylinositol 3-Kinases
  • AKT1 protein, human
  • Protein-Serine-Threonine Kinases
  • Proto-Oncogene Proteins c-akt
  • RIPK1 protein, human
  • Receptor-Interacting Protein Serine-Threonine Kinases
  • Ripk1 protein, mouse
  • CHUK protein, human
  • Chuk protein, mouse
  • I-kappa B Kinase
  • IKBKB protein, human
  • IKBKE protein, human
  • Ikbkb protein, mouse
  • Ikbke protein, mouse
  • PRKCQ protein, human
  • Prkcq protein, mouse
  • Protein Kinase C
  • Protein Kinase C-theta
  • MAP Kinase Kinase Kinases
  • MAP3K8 protein, human
  • NF-kappa B kinase