ampk α2β1γ1  (SignalChem)

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: 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