Critical Roles of Threonine 187 Phosphorylation in Cellular Stress-induced Rapid and Transient Activation of Transforming Growth Factor-β-activated Kinase 1 (TAK1) in a Signaling Complex Containing TAK1-binding Protein TAB1 and TAB2

Pattama Singhirunnusorn,Shunsuke Suzuki,Noritaka Kawasaki,Ikuo Saiki,Hiroaki Sakurai

doi:10.1074/jbc.m407537200

Abstract

Transforming growth factor-beta-activated kinase 1 (TAK1) mitogen-activated protein kinase kinase kinase has been shown to be activated by cellular stresses including tumor necrosis factor-alpha (TNF-alpha). Here, we characterized the molecular mechanisms of cellular stress-induced TAK1 activation, focusing mainly on the phosphorylation of TAK1 at Thr-187 and Ser-192 in the activation loop. Thr-187 and Ser-192 are conserved among species from Caenorhabditis elegans to human, and their replacement with Ala resulted in inactivation of TAK1. Immunoblotting with a novel phospho-TAK1 antibody revealed that TNF-alpha significantly induced the phosphorylation of endogenous TAK1 at Thr-187, and subsequently the phosphorylated forms of TAK1 rapidly disappeared. Intermolecular autophosphorylation of Thr-187 was essential for TAK1 activation. RNA interference and overexpression experiments demonstrated that TAK1-binding protein TAB1 and TAB2 were involved in the phosphorylation of TAK1, but they regulated TAK1 phosphorylation differentially. Furthermore, SB203580 and p38alpha small interfering RNA enhanced TNF-alpha-induced Thr-187 phosphorylation as well as TAK1 kinase activity, indicating that the phosphorylation is affected by p38alpha/TAB1/TAB2-mediated feedback control of TAK1. These results indicate critical roles of Thr-187 phosphorylation in the stress-induced rapid and transient activation of TAK1 in a signaling complex containing TAB1 and TAB2.

Highlights

Cellular stresses stimulate several intracellular signaling pathways leading to the activation of transcription factors AP-11 and NF-␬B
Drosophila Transforming growth factor-␤-activated kinase 1 (TAK1) and MOM-4, a TAK1 homolog in Caenorhabditis elegans, retained the Thr residue corresponding to Thr-187 of mammalian TAK1
Total DNA amounts were adjusted to 2 ␮g with an empty vector. 24 h after the transfection, IKK␣ (B) and Jun NH2-terminal kinase (JNK) (C) activities were evaluated by anti-I␬B␣, and anti-JNK immunocomplex kinase assay, respectively

Summary

Introduction

Cellular stresses stimulate several intracellular signaling pathways leading to the activation of transcription factors AP-11 and NF-␬B. SB203580 and p38␣ small interfering RNA enhanced TNF-␣-induced Thr-187 phosphorylation as well as TAK1 kinase activity, indicating that the phosphorylation is affected by p38␣/TAB1/TAB2-mediated feedback control of TAK1. These results indicate critical roles of Thr-187 phosphorylation in the stress-induced rapid and transient activation of TAK1 in a signaling complex containing TAB1 and TAB2.

Results

Conclusion