Autoactivation of Transforming Growth Factor β-activated Kinase 1 Is a Sequential Bimolecular Process

Corinne L. Sidler,Peter C.F. Cheung,Roland Scholz,Nicolas Winssinger,Dietbert Neumann,Ramon F. Thali

doi:10.1074/jbc.m109.093468

Abstract

Transforming growth factor-beta-activated kinase 1 (TAK1), an MAP3K, is a key player in processing a multitude of inflammatory stimuli. TAK1 autoactivation involves the interplay with TAK1-binding proteins (TAB), e.g. TAB1 and TAB2, and phosphorylation of several activation segment residues. However, the TAK1 autoactivation is not yet fully understood on the molecular level due to the static nature of available x-ray structural data and the complexity of cellular systems applied for investigation. Here, we established a bacterial expression system to generate recombinant mammalian TAK1 complexes. Co-expression of TAK1 and TAB1, but not TAB2, resulted in a functional and active TAK1-TAB1 complex capable of directly activating full-length heterotrimeric mammalian AMP-activated protein kinase (AMPK) in vitro. TAK1-dependent AMPK activation was mediated via hydrophobic residues of the AMPK kinase domain alphaG-helix as observed in vitro and in transfected cell culture. Co-immunoprecipitation of differently epitope-tagged TAK1 from transfected cells and mutation of hydrophobic alphaG-helix residues in TAK1 point to an intermolecular mechanism of TAB1-induced TAK1 autoactivation, as TAK1 autophosphorylation of the activation segment was impaired in these mutants. TAB1 phosphorylation was enhanced in a subset of these mutants, indicating a critical role of alphaG-helix residues in this process. Analyses of phosphorylation site mutants of the activation segment indicate that autophosphorylation of Ser-192 precedes TAB1 phosphorylation and is followed by sequential phosphorylation of Thr-178, Thr-187, and finally Thr-184. Finally, we present a model for the chronological order of events governing TAB1-induced TAK1 autoactivation.

Highlights

Growth factor-␤ and bone morphogenetic protein-mediated signaling [1]. Because it transduces a multitude of extracellular stimuli, such as those from interleukin-1, tumor necrosis factor-␣, and lipopolysaccharides, the serine/threonine kinase Transforming growth factor-␤-activated kinase 1 (TAK1) represents a key activator of pathways involving I␬B kinase, c-Jun NH2-terminal kinase (JNK), and p38 [2,3,4,5,6,7,8,9]
TAK1-TAB1-mediated AMPK Activation Requires Hydrophobic Residues of the AMPK Kinase Domain ␣G-helix—Recently, we showed that the hydrophobic residues Val-219 and Phe-223 of the AMPK kinase domain ␣G-helix constitute an important distal recognition motif for the AMPK upstream kinases calmodulin-dependent protein kinase kinase-2 (CamKK2) and LKB1 [44]
Because substitution of Thr-178 and Thr-184 with glutamate resulted in some fraction of wild-type activity in TAK1-TAB1 complexes (Fig. 4A), we introduced the same mutations into the activation segment of the lysine-substituted ␣G-helix mutants, hoping to create sufficient basal activity for TAK1 autoactivation

Summary

Introduction

Growth factor-␤ and bone morphogenetic protein-mediated signaling [1]. Because it transduces a multitude of extracellular stimuli, such as those from interleukin-1, tumor necrosis factor-␣, and lipopolysaccharides, the serine/threonine kinase TAK1 represents a key activator of pathways involving I␬B kinase, c-Jun NH2-terminal kinase (JNK), and p38 [2,3,4,5,6,7,8,9]. In contrast to the kinase-deficient mutant TAK1 complex (KWTAK1-TAB1), WT-TAK1-TAB1 efficiently phosphorylated AMPK at Thr-172, as revealed by Western blotting and activity assay (Fig. 2D, compare lanes 3 and 5).

Results

Conclusion