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IkappaB kinases phosphorylate NF-kappaB p65 subunit on serine 536 in the transactivation domain

H Sakurai1, H Chiba, H Miyoshi

  • 1Discovery Research Laboratory, Tanabe Seiyaku Co., Ltd., 16-89 Kashima 3-chome, Yodogawa-ku, Osaka 532-8505, Japan. hsakurai@tanabe.co.jp

The Journal of Biological Chemistry
|October 16, 1999
PubMed

Insights

The IkappaB kinase (IKK) complex phosphorylates both IkappaB and the p65 subunit of NF-kappaB, specifically at Ser-536. This dual phosphorylation by IKK is crucial for cytokine-induced NF-kappaB activation.

Area of Science:

  • Cellular signaling pathways
  • Molecular biology
  • Immunology

Background:

  • The cytokine-induced nuclear factor-kappaB (NF-kappaB) activation pathway is critical for immune responses and cellular processes.
  • This pathway involves the phosphorylation and degradation of NF-kappaB inhibitors (IkappaBs) by IkappaB kinase (IKK).

Purpose of the Study:

  • To investigate whether the IKK complex phosphorylates the p65 NF-kappaB subunit in addition to IkappaB.
  • To identify the specific phosphorylation site and the kinase responsible for p65 phosphorylation.

Main Methods:

  • Investigated p65 phosphorylation in HeLa cells treated with TNF-alpha.
  • Performed in vitro phosphorylation assays using GST-fused p65 and cytoplasmic fractions.
  • Utilized overexpressed IKKs, recombinant IKKbeta, and NF-kappaB-inducing kinase activation.

Main Results:

  • p65 subunit phosphorylation was observed in the cytoplasm of HeLa cells following TNF-alpha stimulation, mirroring IkappaB phosphorylation kinetics.
  • In vitro assays identified Ser-536 in the p65 carboxyl-terminal transactivation domain as the target residue for phosphorylation.
  • Endogenous IKK complex, overexpressed IKKs, recombinant IKKbeta, and activated NF-kappaB-inducing kinase efficiently phosphorylated p65 at Ser-536 in vivo and in vitro.

Conclusions:

  • The IKK complex phosphorylates the p65 NF-kappaB subunit at Ser-536, in addition to its known role in phosphorylating IkappaB.
  • This dual phosphorylation suggests a more complex regulatory role for the IKK complex in NF-kappaB activation.
  • Identified Ser-536 as a key phosphorylation site on p65 involved in cytokine-induced signaling.

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