α2β1γ1  (Carna Inc)

Journal: iScience

Article Title: Activation of AMP-activated protein kinase (AMPK) through inhibiting interaction with prohibitins

doi: 10.1016/j.isci.2023.106293

Figure Lengend Snippet: PHBs are negative regulators of AMPK, related to Figures S3 and (A) HepG2 cells transfected with control (siCtrl) or PHB1 and PHB2 (siPHB1/2) siRNAs were subjected to immunoblot analysis with antibodies to the indicated proteins. The p-AMPKα/AMPKα, p-ACC/ACC, PHB1/β-actin, and PHB2/β-actin ratios were determined by densitometry and are expressed relative to the corresponding mean value for siCtrl-transfected cells. (B) HepG2 cells transfected with siCtrl or siPHB1/2 were incubated with [ 14 C]acetate and then assayed for fatty acid synthesis. (C) Immunoblot analysis of a whole cell extract (WCE) as well as of isolated mitochondrial (Mito) and cytosolic fractions of HepG2 cells with antibodies to AMPKα, to PHB1, to PHB2, to HSP90 (cytosolic marker), and to COX IV (mitochondrial marker). (D) Mitochondrial lysates prepared from HepG2 cells were subjected to immunoprecipitation (IP) with antibodies to AMPKα or with control immunoglobulin G (IgG), and the resulting precipitates as well as the original lysates (Input) were subjected to immunoblot analysis with antibodies to the indicated proteins. (E) Recombinant GST-PHB1 or GST alone, each bound to glutathione-agarose beads, was incubated with recombinant His 6 -tagged AMPK (α2β1γ1) in the absence or presence of RX-375 (2 μM), after which bead-bound proteins were subjected to immunoblot analysis with antibodies to the indicated proteins. (F) HepG2 cells expressing 3×Flag-tagged PHB1 and Myc epitope-tagged AMPKα2 were incubated with RX-375 (1 μM) for 3 h, after which cell lysates were subjected to immunoprecipitation with antibodies to Flag and the resulting precipitates were subjected to immunoblot analysis with antibodies to Myc and to Flag. All quantitative data are means ± s.e.m. from five (B) or six (A) independent experiments (n = 5 or 6, respectively). ∗p < 0.05 versus siCtrl, unpaired two-tailed Student’s t test (A and B).

Article Snippet: In brief, active recombinant human AMPK α1β1γ1 (02-113, Carna Biosciences, Kobe, Japan) or α2β1γ1 (02-114, Carna Biosciences) (0.3 nM) was incubated with indicated concentrations of RX-375 or Compound B, or 100 μM AMP in 10 μL of kinase buffer (50 mM HEPES-NaOH [pH 7.5], 1 mM EGTA, 10 mM MgCl 2 , 2 mM dithiothreitol, and 0.01% Tween 20) containing 50 nM ULight-SAMS peptide and 10 μM ATP in white OptiPlate 384-well microplates (6007290, PerkinElmer) at room temperature for 30 min. After the addition of 10 μL of detection mix (CR97–100, PerkinElmer) containing 40 mM EDTA and 2 nM Eu-conjugated antibodies to phospho-ACC (TRF0208, PerkinElmer) and incubation for an additional 1 h, phosphorylation of the peptide was determined by time-resolved fluorescence resonance energy transfer (excitation at 320 nm, emission at 615 and 665 nm).

Techniques: Transfection, Control, Western Blot, Incubation, Isolation, Marker, Immunoprecipitation, Recombinant, Expressing, Two Tailed Test

Journal: iScience

Article Title: Activation of AMP-activated protein kinase (AMPK) through inhibiting interaction with prohibitins

doi: 10.1016/j.isci.2023.106293

Figure Lengend Snippet:

Article Snippet: In brief, active recombinant human AMPK α1β1γ1 (02-113, Carna Biosciences, Kobe, Japan) or α2β1γ1 (02-114, Carna Biosciences) (0.3 nM) was incubated with indicated concentrations of RX-375 or Compound B, or 100 μM AMP in 10 μL of kinase buffer (50 mM HEPES-NaOH [pH 7.5], 1 mM EGTA, 10 mM MgCl 2 , 2 mM dithiothreitol, and 0.01% Tween 20) containing 50 nM ULight-SAMS peptide and 10 μM ATP in white OptiPlate 384-well microplates (6007290, PerkinElmer) at room temperature for 30 min. After the addition of 10 μL of detection mix (CR97–100, PerkinElmer) containing 40 mM EDTA and 2 nM Eu-conjugated antibodies to phospho-ACC (TRF0208, PerkinElmer) and incubation for an additional 1 h, phosphorylation of the peptide was determined by time-resolved fluorescence resonance energy transfer (excitation at 320 nm, emission at 615 and 665 nm).

Techniques: Produced, FLAG-tag, Control, Recombinant, Membrane, Transfection, Reverse Transcription, Silver Staining, Isolation, Cell Culture, Expressing, Plasmid Preparation, Luciferase, shRNA, Software

Journal: Biochemical Journal

Article Title: Structure-function analysis of the AMPK activator SC4 and identification of a potent pan AMPK activator

doi: 10.1042/BCJ20220067

Figure Lengend Snippet: GST-tagged AMPK from HEK293T/17 cells was immobilised on glutathione Sepharose and activities measured by SAMS assay in the presence of 0–30 µM AMPK activators. Dose-response curves for MSG011, MSG012, MK-8722 and PF-739 activation of ( A ) α1β1γ1, ( B ) α1β2γ1, ( C ) α2β1γ1 and ( D ) α2β2γ1. n = 3–6, data presented as mean fold AMPK activation relative to vehicle ± SEM. See .

Article Snippet: Recombinant full-length AMPK 6xHis α2β1γ1 was expressed in E. coli Rosetta 2 (DE3) (Merck Millipore) after double-transformation of pET-Duet-1 (α and γ subunits) and pCOLA (β subunit) vectors [ ].

Techniques: Activation Assay

Journal: Biochemical Journal

Article Title: Structure-function analysis of the AMPK activator SC4 and identification of a potent pan AMPK activator

doi: 10.1042/BCJ20220067

Figure Lengend Snippet: Parameters for compound activation of immobilised γ1 AMPK complexes

Article Snippet: Recombinant full-length AMPK 6xHis α2β1γ1 was expressed in E. coli Rosetta 2 (DE3) (Merck Millipore) after double-transformation of pET-Duet-1 (α and γ subunits) and pCOLA (β subunit) vectors [ ].

Techniques: Activation Assay

Journal: Biochemical Journal

Article Title: Structure-function analysis of the AMPK activator SC4 and identification of a potent pan AMPK activator

doi: 10.1042/BCJ20220067

Figure Lengend Snippet: ( A ) Cartoon representation of α2β1γ1 (α2, green; β1, cyan; γ1, magenta) in complex with MSG011 (yellow), staurosporine (orange), and two AMP molecules (white). Inset: zoomed view of MSG011 bound to the ADaM site shown as a surface representation. ( B ) Close up views of the ADaM site with critical polar residues that interact with MSG011 shown as sticks, α2 residues are in green and β1 residues are in cyan. For clarity the cartoon representation of α2β1γ1 is shown in grey (α2, dark grey; β1, light grey) with a transparent surface view. Left and right images are 90° rotations of the middle image.

Article Snippet: Recombinant full-length AMPK 6xHis α2β1γ1 was expressed in E. coli Rosetta 2 (DE3) (Merck Millipore) after double-transformation of pET-Duet-1 (α and γ subunits) and pCOLA (β subunit) vectors [ ].

Techniques:

Journal: bioRxiv

Article Title: Requirement of FLCN tumor suppressor gene for mTORC1-mediated inhibition of TFE3 transcriptional activity

doi: 10.1101/2020.07.08.193169

Figure Lengend Snippet: (A) TFE3 phosphorylation by mTORC1 in vitro . 32 P-phosphorylation of GST-Tfe3 and GST-S6K1 but not GST by the immunoprecipitated mTORC1 kinase complex (HA-Raptor/myc-mTOR). PP242, 200 nM. IP, immunoprecipitates. (B) TFE3 phosphorylation by AMPK in vitro . GST-TFE3, SAMS peptide and GST were incubated with recombinant AMPK (α2β1γ1) in the presence or absence of AMP (400 μM). (C) Fractionation of HEK293 cells transfected with wildtype or mutant TFE3 expression vectors. The ratios of nuclear to cytoplasmic TFE3 (Nuc/Cyto) are indicated. (D) GST pull-down experiment demonstrating the interaction between wildtype TFE3 (but not TFE3-S321A mutant) and 14-3-3 protein. (E) FLCN expression was required for TFE3 phosphorylation in cells under nutrient-rich conditions. Both mTORC1 activity and FLCN expression were required for TFE3 phosphorylation. UOK257 cells expressing either FLCN or lacZ were treated with (F) DMSO (Cont), AICAR (2 mM), PP242 (1 μM) and (G) chloroquine (100 μM). (H) Schematic diagram demonstrating a proposed mechanism for TFE3 regulation by the FLCN-AMPK-mTORC1 signaling network.

Article Snippet: 14-3-3 antibody and active recombinant AMPK (α2β1γ1) were from Millipore.

Techniques: In Vitro, Immunoprecipitation, Incubation, Recombinant, Fractionation, Transfection, Mutagenesis, Expressing, Activity Assay

Journal: Biochemical Journal

Article Title: Phosphorylation of AMPK by upstream kinases is required for activity in mammalian cells

doi: 10.1042/BCJ20170458

Figure Lengend Snippet: ( A ) AMPK α1β1γ1 and α2β1γ1 complexes were expressed in E. coli , purified and analysed by Western blotting using either an anti-pThr 172 antibody or an anti-β antibody. Recombinant complexes that had been phosphorylated with CaMKKβ [α1β1 γ1(P) and α2β1γ1(P)] are shown alongside the non-phosphorylated proteins. ( B ) AMPK activity of the non-phosphorylated complexes was measured using the SAMS peptide assay in the absence or presence of 991 (1 µM), AMP (10 µM) or both 991 and AMP. Results shown are plotted as nmol/min/mg and are the means ± SEM of three independent experiments. ( C ) Comparison of circular dichroism signal change measured at 306 nm for non-pThr172 (open symbols) and pThr172 (closed symbols) α1β1γ1 complexes as a function of 991/AMPK.

Article Snippet: We obtained the crystal structure of full-length human non-pThr 172 α2β1γ1 , in complex with 991, AMP and staurosporine at a resolution of 2.6 Å (crystallographic statistics are presented in ; co-ordinates deposited in the Protein Databank, PDB ID: 5ISO).

Techniques: Purification, Western Blot, Recombinant, Activity Assay, Comparison, Circular Dichroism

Journal: Biochemical Journal

Article Title: Phosphorylation of AMPK by upstream kinases is required for activity in mammalian cells

doi: 10.1042/BCJ20170458

Figure Lengend Snippet: Activity of non-pThr 172 AMPK complexes AMPK complexes were expressed in E. coli and activity of the purified complexes was measured using the SAMS peptide assay in the absence or presence of the allosteric activators, 991 (1 µM), AMP (10 µM) or both activators together. Fold-activation relative to activity measured in the absence of activator is also shown. In all cases, results shown are the mean (±SEM) determined from at least three independent experiments. In all cases, allosteric activation causes a statistically significant increase in AMPK activity compared with the control value ( P < 0.05).

Article Snippet: We obtained the crystal structure of full-length human non-pThr 172 α2β1γ1 , in complex with 991, AMP and staurosporine at a resolution of 2.6 Å (crystallographic statistics are presented in ; co-ordinates deposited in the Protein Databank, PDB ID: 5ISO).

Techniques: Activity Assay, Purification, Activation Assay, Control

Journal: Biochemical Journal

Article Title: Phosphorylation of AMPK by upstream kinases is required for activity in mammalian cells

doi: 10.1042/BCJ20170458

Figure Lengend Snippet: Equilibrium dissociation constants for 991 binding to AMPK complexes AMPK complexes were expressed in E. coli and dissociation constants ( K d ) for 991 were determined using circular dichroism (CD). The K d values are reported as the mean (±SEM) determined from at least three independent experiments. The values for the phosphorylated Thr 172 complexes (P) are taken from a previous study (Xiao et al. [ 9 ]).

Article Snippet: We obtained the crystal structure of full-length human non-pThr 172 α2β1γ1 , in complex with 991, AMP and staurosporine at a resolution of 2.6 Å (crystallographic statistics are presented in ; co-ordinates deposited in the Protein Databank, PDB ID: 5ISO).

Techniques: Binding Assay, Circular Dichroism

Journal: Biochemical Journal

Article Title: Phosphorylation of AMPK by upstream kinases is required for activity in mammalian cells

doi: 10.1042/BCJ20170458

Figure Lengend Snippet: ( A ) Bar diagram indicating the three subunits that make up the complex, showing the location of specific regions within the subunits (AID: autoinhibtory domain; CBM: carbohydrate-binding module; CBS: cystathionine-β-synthase domain), as well as the location of Thr 172 in the α subunit and Ser 108 in the β subunit. ( B ) Cartoon representation of full-length human α2β1γ1 in complex with 991. The domains of the three subunits are coloured according to ( A ). The orientation of the figure is similar to our earlier papers, so that the kinase domain is ‘upside-down’ with respect to the classical kinase orientation. 991, which binds at the interface of the kinase domain and CBM, is shown in stick representation, with its carbon atoms coloured magenta. The three AMP molecules bound in the γ1 subunit, and staurosporine bound to the kinase domain, are also shown. The α-hook (also known as αRIM2) region of α2 interacting at site 3 in the γ1 subunit is shown in blue. Thr 172 is shown in space-filling representation. ( C ) A detailed view of the α-hook interaction at site 3 with AMP bound. ( D ) Comparison of the AID region from the current structure (cyan) with the isolated AID of human α1 (PDB ID:4RED; grey). ( E ) 991-binding site at the interface of the CBM of β1 and the kinase domain, showing electrostatic interactions with pSer 108 . The co-ordinates of the non-pThr 172 AMPK structure have been deposited in the Protein Databank (PDB ID: 5ISO).

Article Snippet: We obtained the crystal structure of full-length human non-pThr 172 α2β1γ1 , in complex with 991, AMP and staurosporine at a resolution of 2.6 Å (crystallographic statistics are presented in ; co-ordinates deposited in the Protein Databank, PDB ID: 5ISO).

Techniques: Binding Assay, Comparison, Isolation

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Quantitative phosphoproteomic analysis reveals cAMP/vasopressin-dependent signaling pathways in native renal thick ascending limb cells

doi: 10.1073/pnas.1007424107

Figure Lengend Snippet: Quantification of in vitro NKCC2 phosphorylation by PKA and AMPK. (A) Representative MS1 time-course curves (Upper) quantifying phosphorylation of Ser126-containing peptide after incubation with purified PKA-α (blue) or AMPK-α2β1γ1 with (black) or without (green) 0.1 mM AMP. Immunoblotting confirmation (Lower) using pSer126-specific NKCC2 antibody. (B) Representative MS1 time-course curves (Upper) quantifying phosphorylation of S874-containing peptide after incubation with purified PKA. Immunoblotting confirmation (Lower) using pSer874-specific NKCC2 antibody.

Article Snippet: Both peptides (0.4 nmol) were incubated with purified active PKA-α ( PRKACA ) or purified active AMPK-α2β1γ1 (with or without 0.1 mM AMP) at a kinase:peptide molar ratio of 1:32 in kinase reaction buffer supplemented with 200 μM ATP for 1 h at 37 °C (all components from Cell Signaling).

Techniques: In Vitro, Incubation, Purification, Western Blot