Effect of different γ-subunit isoforms on the regulation of AMPK

Willows, Robin; Navaratnam, Naveenan; Lima, Ana; Read, Jon; Carling, David

doi:10.1042/bcj20170046

Cited by 49 publications

(36 citation statements)

References 46 publications

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“…This protein is also activated by the ADaM site ligand 991, and not by the ␤ 1 -specific A769662 (Fig. S2B), a very similar behavior to that of WT AMPK (16,27,35).…”

Section: A Cysteine-free Version Of Ampk Is Catalytically Active and mentioning

confidence: 80%

Conformational heterogeneity of the allosteric drug and metabolite (ADaM) site in AMP-activated protein kinase (AMPK)

Bridges

Yan

et al. 2018

Journal of Biological Chemistry

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AMP-activated protein kinase (AMPK) is a master regulator of energy homeostasis and a promising drug target for managing metabolic diseases such as type 2 diabetes. Many pharmacological AMPK activators, and possibly unidentified physiological metabolites, bind to the allosteric drug and metabolite (ADaM) site at the interface between the kinase domain (KD) in the α-subunit and the carbohydrate-binding module (CBM) in the β-subunit. Here, using double electron-electron resonance (DEER) spectroscopy, we demonstrate that the CBM-KD interaction is partially dissociated and the interface highly disordered in the absence of pharmacological ADaM site activators as inferred from a low depth of modulation and broad DEER distance distributions. ADaM site ligands such as 991, and to a lesser degree phosphorylation, stabilize the KD-CBM association and strikingly reduce conformational heterogeneity in the ADaM site. Our findings that the ADaM site, formed by the KD-CBM interaction, can be modulated by diverse ligands and by phosphorylation suggest that it may function as a hub for integrating regulatory signals.

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“…This protein is also activated by the ADaM site ligand 991, and not by the ␤ 1 -specific A769662 (Fig. S2B), a very similar behavior to that of WT AMPK (16,27,35).…”

Section: A Cysteine-free Version Of Ampk Is Catalytically Active and mentioning

confidence: 80%

Conformational heterogeneity of the allosteric drug and metabolite (ADaM) site in AMP-activated protein kinase (AMPK)

Bridges

Yan

et al. 2018

Journal of Biological Chemistry

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“…A recent study reported that in contrast to β1, Ser108 phosphorylation is not involved in binding of 991 to β2-containing AMPK complexes 54 . There is no high-resolution structural information available for AMPKβ2 complexes bound to ADaM site ligands, and so the molecular basis for the difference in requirement for Ser108 phosphorylation between the β-isoforms remains unknown.…”

Section: ) (Table 1)mentioning

confidence: 97%

“…In addition, variant forms of γ2 and γ3 transcripts have been identified that encode predicted proteins with truncated amino-terminal regions 50,51 . Recent studies demonstrate that the γ isoforms confer differential regulation of AMPK to both nucleotides and small molecule activators [52][53][54] . To date, the only crystal structures available for AMPK are for γ1-containing complexes, and further work is required to elucidate the precise roles of the amino-terminal regions of γ2 and γ3 on AMPK function.…”

Section: Commented [Ws1]: Au: First Sentence Of the Abstract Ok?mentioning

confidence: 99%

“…In this conformation, although the phosphate group of Thr172 is partially exposed to the solvent, it is not accessible to protein phosphatases. Allosteric activation following ligand binding at the ADaM site is dependent on the isoform composition of the AMPK complex 22,52,54 and involves the interaction between an α-helix immediately carboxy-terminal to the CBM (referred to as the C-interacting helix) and the αC helix of the kinase domain 22 . Mutation of Leu166 to Glu within the C-interacting helix, which is predicted to block interaction with the αC helix, significantly reduces allosteric activation of AMPK by 991 but has no effect on allosteric activation by AMP 22 .…”

Section: ) (Table 1)mentioning

confidence: 99%

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AMP-activated protein kinase: the current landscape for drug development

Steinberg

Carling

2019

Nat Rev Drug Discov

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466

405

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Since the discovery of AMP-activated protein kinase (AMPK) as a central regulator of energy homeostasis, many exciting insights into its structure, regulation and physiological roles have been revealed. While exercise, caloric restriction, metformin and many natural products increase AMPK activity and exert a multitude of health benefits, developing direct activators of AMPK to elucidate the role of AMPK in these beneficial effects has been challenging. However, in recent years, direct AMPK activators have been identified and tested in preclinical models, and a small number have entered clinical trials. Despite these advances, which disease(s) represent the best indications for therapeutic AMPK activation and the long-term safety of such approaches remain to be established. Commented [WS2]: Au: "The optimal consensus motif of AMPK substrates has been established (BOX 3)" OK?

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“…Although indirect AMPK activators, like metformin or AMP analogs like ZMP (the intracellular phosphorylation product of AICAR), activate both ␤ 1 -and ␤ 2 -containing AMPK isoforms, they are not specific for AMPK. In contrast, ADaM sitebinding pharmacological AMPK activators such as A769662 and especially 991 are specific but do not (A769662) (21) or relatively weakly (991) (22,23) activate ␤ 2 -containing complexes. Recently, both Merck (MK-9722) and Pfizer (PF-739) developed 991-derivative pan-AMPK activators (i.e.…”

mentioning

confidence: 93%

Structures of AMP-activated protein kinase bound to novel pharmacological activators in phosphorylated, non-phosphorylated, and nucleotide-free states

Yan

Zhou

Novick

et al. 2019

Journal of Biological Chemistry

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Edited by Wolfgang Peti AMP-activated protein kinase (AMPK) is an attractive therapeutic target for managing metabolic diseases. A class of pharmacological activators, including Merck 991, binds the AMPK ADaM site, which forms the interaction surface between the kinase domain (KD) of the ␣-subunit and the carbohydratebinding module (CBM) of the ␤-subunit. Here, we report the development of two new 991-derivative compounds, R734 and R739, which potently activate AMPK in a variety of cell types, including ␤ 2-specific skeletal muscle cells. Surprisingly, we found that they have only minor effects on direct kinase activity of the recombinant ␣ 1 ␤ 2 ␥ 1 isoform yet robustly enhance protection against activation loop dephosphorylation. This mode of activation is reminiscent of that of ADP, which activates AMPK by binding to the nucleotide-binding sites in the ␥-subunit, more than 60 Å away from the ADaM site. To understand the mechanisms of full and partial AMPK activation, we determined the crystal structures of fully active phosphorylated AMPK ␣ 1 ␤ 1 ␥ 1 bound to AMP and R734/R739 as well as partially active nonphosphorylated AMPK bound to R734 and AMP and phosphorylated AMPK bound to R734 in the absence of added nucleotides at <3-Å resolution. These structures and associated analyses identified a novel conformational state of the AMPK autoinhibitory domain associated with partial kinase activity and provide new insights into phosphorylation-dependent activation loop stabilization in AMPK. AMPK 2 is a three-subunit protein kinase that consists of two alternative kinase domain-containing ␣-subunits (␣ 1 and ␣ 2), two alternative carbohydrate-binding module-containing ␤-subunits (␤ 1 and ␤ 2), and three alternative AMP/ADP/ATPbinding ␥-subunits (␥ 1 , ␥ 2 , and ␥ 3) (1-3). In humans, the ␤ 1 isoform is only present in a fraction of AMPK complexes in liver but not immunologically detectable in skeletal muscle, whose predominant isoform is ␣ 2 ␤ 2 ␥ 1 (4-6). AMPK senses the energy state of cells by competitive binding of AMP, ADP, and ATP to three separate sites in its ␥-subunit. Upon energy stress, i.e. increases in the ratio of AMP and ADP to ATP, AMPK is activated ϳ100-fold by activation loop phosphorylation and an additional 2-10-fold by direct allosteric kinase activation (7). The activated enzyme phosphorylates key metabolic and regulatory proteins to stimulate ATP-generating catabolic pathways and inhibit ATP-consuming anabolic pathways and cellular programs (8-10). AMPK is an attractive potential target for the treatment of metabolic diseases, most prominently type 2 diabetes (9, 11, 12). Currently, metformin is the frontline drug for the treatment of type 2 diabetes. Metformin is taken up predominantly by the liver where it generates AMPK-activating energy stress by mildly inhibiting oxidative phosphorylation. In addition, it has been reported that at low pharmacological levels AMPK increases phosphorylation through LKB1 by inducing AMPK heterotrimer formation (13) and formation of an LKB1-This work was suppo...

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Effect of different γ-subunit isoforms on the regulation of AMPK

Cited by 49 publications

References 46 publications

Conformational heterogeneity of the allosteric drug and metabolite (ADaM) site in AMP-activated protein kinase (AMPK)

Conformational heterogeneity of the allosteric drug and metabolite (ADaM) site in AMP-activated protein kinase (AMPK)

AMP-activated protein kinase: the current landscape for drug development

Structures of AMP-activated protein kinase bound to novel pharmacological activators in phosphorylated, non-phosphorylated, and nucleotide-free states

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