Tumor Necrosis Factor-α-induced IKK Phosphorylation of NF-κB p65 on Serine 536 Is Mediated through the TRAF2, TRAF5, and TAK1 Signaling Pathway

Abstract

The activation of NF-kappaB has been shown to be regulated by multiple phosphorylations of IkappaBs and the NF-kappaB p65 subunit. Here, we characterized the intracellular signaling pathway leading to phosphorylation of p65 on Ser-536 using a novel anti-phospho-p65 (Ser-536) antibody. The Ser-536 of endogenous p65 was rapidly phosphorylated in response to a wide variety of NF-kappaB stimulants including TNF-alpha in the cytoplasm and rapidly dephosphorylated in the nucleus. The TNF-alpha-but not IL-1beta-induced Ser-536 phosphorylation was severely impaired in murine embryonic fibroblasts derived from traf2-/-traf5-/- mice. Bay 11-7082, an inhibitor of IkappaB phosphorylation, inhibited the TNF-alpha-induced phosphorylation in vivo. In addition, overexpression of TGF-beta-activated kinase 1 (TAK1), IKKalpha and IKKbeta stimulated the phosphorylation, and their dominant negative mutants blocked the TNF-alpha-induced phosphorylation. Moreover, small interfering RNAs (siRNAs) against TAK1, IKKalpha and IKKbeta blocked the phosphorylation of endogenous p65. On the other hand, calyculin-A, a protein phosphatase inhibitor, blocked the dephosphorylation in the nucleus in vivo. These results indicate that similar signaling pathways were utilized for the phosphorylations of IkappaBalpha and p65, which further support the idea that both IkappaB and NF-kappaB are substrates for the IKK complex in the activation of NF-kappaB.

TNAP, a Novel Repressor of NF-κB-inducing Kinase, Suppresses NF-κB Activation

“…5B, left panel). TNF-␣ stimulation induces rapid phosphorylation of p65, consistent with several reports (65)(66)(67). Knockdown of TNAP increases TNF-␣-induced phosphorylation of p65 (Fig.…”

“…Several kinases that have been implicated in p65 phosphorylation include protein kinase A (Ser-276) (91,92), mitogen-and stressactivated protein kinase-1 (Ser-276) (93), casein kinase II (Ser-529) (94), protein kinase C (Ser-471), phosphatidylinositol 3-kinase/Akt (Ser-536) (95), p90 ribosomal S6 kinase-1 (Ser-536) (96), IKK2 (Ser-536) (64,65,70), NIK, and IKK1(Ser-536) (35,68). TNF-␣ has been shown to induce p65 phosphorylation at Ser-529 via casein kinase II (94), and IKK1 (68) and IKK2 (64,65,70) phosphorylates p65 on Ser-536. Our data confirm that TNF-␣ induces rapid phosphorylation of p65 on Ser-536 and show for the first time that TNAP regulates TNF-␣-induced and NIK-mediated p65 phosphorylation.…”

Nod1 acts as an intracellular receptor to stimulate chemokine production and neutrophil recruitment in vivo

“…A possible explanation is that diff erences in gene expression refl ect diff erential activation of intracellular signaling molecules. For example, RICK, a downstream factor of Nod1, has been shown to activate NF-kB through TRAF6, whereas TRAF2 is critical for TNFα signaling (31)(32)(33).…”

Human T-cell Lymphotropic Virus Type 1 Tax Induction of Biologically Active NF-κB Requires IκB Kinase-1-mediated Phosphorylation of RelA/p65

“…These two highly related kinases appear to act in concert to transmit the activation signal in a directional manner, leading to the phosphorylation of I B␣ (20,21). The second phase in the activation of NF-B involves the post-translational modification of the NF-B subunits themselves by both phosphorylation and acetylation (22)(23)(24)(25)(26)(27)(28)(29)(30)(31). Although the former phosphorylation events on I B␣ are required for release of NF-B, the latter modifications on RelA/ p65 appear to enhance its transcriptional activity and the duration of the response (32).…”