Homo-oligomerization and Activation of AMP-activated Protein Kinase Are Mediated by the Kinase Domain αG-Helix

Scholz, Roland; Suter, Marianne; Weimann, Théodore; Polge, Cécile; Konarev, Petr V.; Thali, Ramon F.; Tuerk, Roland; Viollet, Benoı̂t; Wallimann, Theo; Schlattner, Uwe; Neumann, Dietbert

doi:10.1074/jbc.m109.047670

Cited by 25 publications

(39 citation statements)

References 89 publications

(83 reference statements)

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“…Plasmids, Enzymes, and Inhibitors-Bacterial expression plasmids coding for mammalian nontagged AMPK ␣1␤1␥1 (WT-AMPK) (45,46), nontagged ␣1(V219E,F223E)␤1␥1 (VEFE-AMPK) (44), and mammalian expression constructs coding for Myc-tagged AMPK ␣1 and ␣1(V219E,F223E) subunits, respectively (44,47), were described earlier. The bacterial expression plasmid encoding nontagged kinase-deficient mutant ␣1(D157A)␤1␥1 (DA-AMPK) was generated analogous to WT-AMPK (45,46).…”

Section: Methodsmentioning

confidence: 99%

“…Transfection and lysis of cells were performed as reported previously (44). To activate AMPK by TAK1 in HeLa cells, 24 h post-transfection cells were serum-starved for 4 h prior to treatment with 1 mM H 2 O 2 for 15 min and subsequent lysis (36).…”

Section: Mutagenesis-kinase-deficient (K63w) Kinase Domain ␣Gmentioning

confidence: 99%

“…AMPK activity was determined by Western blot analysis of AMPK␣ Thr-172 phosphorylation and activity assay using the artificial peptide substrate with the sequence HMRSAMSGLHLVKRR as reported previously (53). Activity of immunoprecipitated AMPK from HeLa cell lysates was assayed as described (44).…”

Section: Mutagenesis-kinase-deficient (K63w) Kinase Domain ␣Gmentioning

confidence: 99%

“…Usually, 2 liters of autoinduction medium (52) were inoculated, and bacteria were grown at 37°C for 3 h before cultures were shifted to room temperature with continuous shaking at 220 rpm for 22 h. Cells were harvested and proteins were extracted as previously described (46). Proteins were purified by application of a two-dimensional purification protocol as reported earlier (44). Briefly, the soluble protein fraction was subjected to batch affinity purification for 60 -90 min at room temperature using 1 ml of nickel-Sepharose HP (GE Healthcare).…”

Section: Mutagenesis-kinase-deficient (K63w) Kinase Domain ␣Gmentioning

confidence: 99%

“…Direct phosphorylation of Thr-172 in the activation segment of the AMPK kinase domain by Ca 2ϩ /calmodulin-dependent protein kinase kinase-2 (CamKK2) (41) or the tumor suppressor kinase complex LKB1-MO25-STRAD (42) is the crucial step in AMPK activation, which then leads to autophosphorylation of multiple sites on its ␣-and ␤-subunits (43). Recently, we described a distal recognition site for the upstream kinases LKB1 and CamKK2 that includes residues of the amphipathic kinase domain ␣G-helix of AMPK, namely Val-219 and Phe-223 (44).…”

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confidence: 99%

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

Scholz

Sidler

Thali

et al. 2010

Journal of Biological Chemistry

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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...

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Section: Methodsmentioning

confidence: 99%

Section: Mutagenesis-kinase-deficient (K63w) Kinase Domain ␣Gmentioning

confidence: 99%

Section: Mutagenesis-kinase-deficient (K63w) Kinase Domain ␣Gmentioning

confidence: 99%

Section: Mutagenesis-kinase-deficient (K63w) Kinase Domain ␣Gmentioning

confidence: 99%

mentioning

confidence: 99%

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

Scholz

Sidler

Thali

et al. 2010

Journal of Biological Chemistry

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Long‐range molecular dynamics show that inactive forms of Protein Kinase A are more dynamic than active forms

et al. 2018

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Many protein kinases are characterized by at least two structural forms corresponding to the highest level of activity (active) and low or no activity, (inactive). Further, protein dynamics is an important consideration in understanding the molecular and mechanistic basis of enzyme function. In this work, we use protein kinase A (PKA) as the model system and perform microsecond range molecular dynamics (MD) simulations on six variants which differ from one another in terms of active and inactive form, with or without bound ligands, C‐terminal tail and phosphorylation at the activation loop. We find that the root mean square fluctuations in the MD simulations are generally higher for the inactive forms than the active forms. This difference is statistically significant. The higher dynamics of inactive states has significant contributions from ATP binding loop, catalytic loop, and αG helix. Simulations with and without C‐terminal tail show this differential dynamics as well, with lower dynamics both in the active and inactive forms if C‐terminal tail is present. Similarly, the dynamics associated with the inactive form is higher irrespective of the phosphorylation status of Thr 197. A relatively stable stature of active kinases may be better suited for binding of substrates and detachment of the product. Also, phosphoryl group transfer from ATP to the phosphosite on the substrate requires precise transient coordination of chemical entities from three different molecules, which may be facilitated by the higher stability of the active state.

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Plant SnRK1 Kinases: Structure, Regulation, and Function

Margalha

Valerio

Baena-González

2016

Experientia Supplementum

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SnRK1 is an evolutionarily conserved protein kinase complex that regulates energy homeostasis in plants. In doing so, it promotes tolerance to adverse environmental conditions and influences a large array of growth and developmental processes. SnRK1 shares clear structural and functional similarities with its orthologs, yeast SNF1 and mammalian AMPK, but has evolved unique features that presumably provide a better adaptation to an autotrophic lifestyle. In this chapter, we review current knowledge on SnRK1, an atypical member of the SNF1/AMPK family, providing insight into its structure, regulation, and functions.

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Homo-oligomerization and Activation of AMP-activated Protein Kinase Are Mediated by the Kinase Domain αG-Helix

Cited by 25 publications

References 89 publications

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

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

Long‐range molecular dynamics show that inactive forms of Protein Kinase A are more dynamic than active forms

Plant SnRK1 Kinases: Structure, Regulation, and Function

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