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

Abstract

Transforming growth factor-␤-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. Coexpression 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 ␣G-helix as observed in vitro and in transfected cell culture. Co-immunoprecipitation of differently epitopetagged TAK1 from transfected cells and mutation of hydrophobic ␣G-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 ␣G-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.Transforming growth factor-␤-activated kinase 1 (TAK1) was first identified as an MAP3K 4 involved in transforming 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 IB kinase, c-Jun NH 2 -terminal kinase (JNK), and p38 (2-9). Furthermore, a regulatory function of TAK1 has also been described in the Wnt signaling pathway (10, 11). TAK1 activity is regulated by association of TAK1-binding proteins, namely TAB1, TAB2, TAB3, and TAB4 (12-17). TAB1, a "pseudophosphatase" that shows close structural similarity with the Mg 2ϩ -or Mn 2ϩ -dependent protein phosphatase family member protein phosphatase 2C␣ (PP2C␣) (18), was originally found in a yeast two-hybrid screen as a protein, which directly triggers TAK1 activity (13). Both structural and functional studies revealed that an evolutionarily conserved motif in the carboxyl-terminal region of TAB1 (residues 480 -504) is sufficient to bind to the catalytic domain of TAK1. However, the presence of a slightly extended TAB1 carboxyl-terminal domain (residues 437-504) is indispensable for full activation of TAK1 in HeLa cells (19 -2...

TAK1 control of cell death

“…TAB1 mediates oligomerization, autophosphorylation and activation of TAK1. 16,33,34 TAB1 is essential for TAK1 activation in response to osmotic stress but dispensable for cytokinemediated TAK1 activation. 16 O-linked glycosylation of TAB1 on S395 is implicated in activation of TAK1 activation in response to osmotic stress.…”

TAK1, more than just innate immunity

“…The mechanism by which TAK1 is activated when unanchored K63 polyubiquitin chains bind to TAB2 and TAB3 remains unclear (10). However, it triggers conformational changes in TAK1 that lead to autophosphorylation primarily on Thr187 and also on Thr178, Thr184, and Ser192 (2,11,12). There is evidence to suggest that K63 polyubiquitin chains sequester TAK1 complexes, facilitates their oligomerization, and may also function as a scaffold that aids TAK1 phosphorylation of the IKK complex, which consists of IKKa, IKKb, and IKKc [also known as NF-jB essential modulator (NEMO)].…”

“…TAK1 was identified as an upstream kinase of AMPK in a screen using yeast mutants deficient in upstream kinases of Snf1 (the yeast ortholog of mammalian AMPK) (60). In vitro, TAK1 has been shown to directly activate AMPK (11). Around the same time, a study was published in which a transgenic mouse expressing a dominant negative TAK1 had cardiac abnormalities resembling the Wolff-Parkinson-White syndrome, which is caused by mutations in AMPK (61).…”

Transforming Growth Factor (TGF)-β-activated Kinase 1 (TAK1) Activation Requires Phosphorylation of Serine 412 by Protein Kinase A Catalytic Subunit α (PKACα) and X-linked Protein Kinase (PRKX)

“…Numerous studies have suggested that site-specific ubiquitination and phosphorylation on TAK1 regulate TAK1 kinase activation (3). In particular, the phosphorylation of residues Thr-184 and Thr-187 located at the TAK1 kinase domain is required for TAK1 kinase activity (10,11). However, a few researchers have suggested that the phosphorylation of TAK1 Thr-187 is insufficient for its activation (12)(13)(14)(15).…”