SGK1 Phosphorylation of IκB Kinase α and p300 Up-regulates NF-κB Activity and Increases N-Methyl-D-aspartate Receptor NR2A and NR2B Expression

Derek J.C. Tai,Yun-Li Ma,Chia-Chen Su,Eminy H.Y. Lee

doi:10.1074/jbc.m805055200

Abstract

Serum- and glucocorticoid-inducible kinase 1 (SGK1) is a downstream target of phosphatidylinositol 3-kinase signaling, and it regulates various cellular and physiological functions, but the SGK1 substrate proteins and genes regulated by SGK1 are less known. Here we have identified IkappaB kinase alpha (IKKalpha) as a novel substrate of SGK1 by using biochemical and bioinformatic approaches. SGK1 directly phosphorylates IKKalpha at Thr-23 and indirectly activates IKKalpha at Ser-180. Furthermore, SGK1 enhanced nuclear factor kappaB (NF-kappaB) activity and up-regulated N-methyl-d-aspartate receptor NR2A and NR2B expression through activation of IKKalpha at Thr-23 and Ser-180, and these two residues play an equally important role in mediating these effects of SGK1. Although SGK1 does not phosphorylate IKKbeta, IKKbeta activity is still required for IKK complex activation and for SGK1 phosphorylation and activation of NF-kappaB. In addition, SGK1 increased the acetylation of NF-kappaB through phosphorylation of p300 at Ser-1834, and this also leads to NF-kappaB activation and NR2A and NR2B expression. Moreover, an endogenous stimulus of SGK1, insulin, increased IKKalpha and NF-kappaB phosphorylation as well as NF-kappaB acetylation and NF-kappaB activity, but SGK1 small interfering RNA transfection blocked these effects of insulin. In examination of the functional significance of the SGK1-IKKalpha-NF-kappaB signaling pathway, we found that transfection of the IKKalpha double mutant (IKKalphaT23A/S180A) to rat hippocampus antagonized SGK-1-mediated spatial memory facilitation. Our results together demonstrated novel substrate proteins of SGK1 and novel SGK1 signaling pathways. Activation of these signaling pathways enhances NR2A and NR2B expression that is implicated in neuronal plasticity.

Highlights

Serum- and glucocorticoid-inducible kinase 1 (SGK1) is a downstream target of phosphatidylinositol 3-kinase signaling [11]
Our results revealed that I␬B kinase ␣ (IKK␣), but not IKK␤, is a novel substrate of SGK1 and that SGK1 phosphorylation of IKK␣ increases nuclear factor ␬B (NF-␬B) activity and up-regulates the expression of the N-methyl-D-aspartate (NMDA) receptor subunit NR2A and NR2B
We have found that SGK1 indirectly increased IKK␣ phosphorylation at Ser-180, and that IKK␣ phosphorylation at Ser-180 is important as IKK␣ phosphorylation at Thr-23 in SGK1-mediated NF-␬B phosphorylation, NF-␬B activation, and NR2A and NR2B expression

Summary

Introduction

SGK1 is a downstream target of phosphatidylinositol 3-kinase signaling [11]. SGK1 is first phosphorylated at Ser-422 by 3-phosphoinositide-dependent PDK2, which enables SGK1 to be further phosphorylated at Thr-256 by PDK1 [12]. Further experiments in HEK293T cells revealed that phosphorylation of IKK␣ at Thr-23 and Ser-180 was both increased after co-transfection of the constitutively active SGK1, SGKS422D, and IKK␣ (Fig. 2D). Results from Western blot revealed that SGK1 increased the phosphorylation of p65 at both Ser-529 and Ser-536, and this effect is dependent on the activity of IKK␣ (Fig. 3B).

Results

Conclusion