Fig 1: NBR2 promotes AMPK kinase activity(a, b) Protein lysates were prepared from HEK293T (a) or UMRC2 cells (b) with overexpression of EV or NBR2 expression vectors, and analyzed by Western blotting. (c) UMRC2 cells stably expressing EV or NBR2 expression vectors were cultured in 25 mM glucose-containing medium for different days as indicated, and then subjected to cell proliferation analysis (Mean ± s.d., n=3 biologically independent extracts, two-tailed paired Student’s t-test). (d) Left panel: Schematic diagram showing different truncation mutants of NBR2 #1 and the summary of their binding capabilities to AMPK α. Right panel: In vitro-synthesized biotinylated sense (S), antisense (AS), or different truncation (T) mutants of NBR2 #1 were incubated with protein lysates from 786-O cells which had been cultured in glucose free medium for 24 hours. Precipitation reactions were conducted using streptavidin beads and then subjected to Western blotting. (e) Protein lysates were prepared from HEK293T or UMRC2 cells with overexpression of EV, NBR2 #1 full length (FL), or T1 mutant expression vectors, and analyzed by Western blotting. (f) UMRC2 cells stably expressing EV, NBR2 #1 FL, or T1 mutant expression vectors were cultured in 25 mM glucose-containing medium for different days as indicated, and then subjected to cell proliferation analysis (Mean ± s.d., n=3 biologically independent extracts, two-tailed paired Student’s t-test). (g) AMPK complex precipitated from HEK293T cells was subjected to the kinase assay in the presence of ATP, in vitro synthesized RNAs and GST-ACC 1–130 aa fusion proteins as indicated. The kinase activity of AMPK was measured by phosphorylation of ACC at S79 site. (h) In vitro purified active human AMPK complex was subjected to in vitro kinase assays in the presence of ATP, SAMS peptide and in vitro synthesized biotinylated sense (S)/antisense (AS)/T1 mutant (T1) NBR2 #1 or several chemical compounds (Compound C, A769662, AMP) as indicated (see Materials & Methods for details). The Kinase activity was measured by the luminescence with a plate-reading illuminometer (Mean ± s.d., n=3 biologically independent extracts, two-tailed paired Student’s t-test). Source data for c, f, h can be found in Supplementary Table 1. Unprocessed original scans of blots are shown in Supplemental Fig. 8.
Fig 2: AMPK directly phosphorylates Skp2 at S256 to promote Skp2 SCF complex formation. a HEK293 cells transfected with Skp2 ΔN, 1–200 and the indicated HA-AMPKα (CA) constructs were subjected to immunoprecipitation, followed by immunoblotting. b The sequence of optimal AMPK motif, Skp2 and known AMPK substrates are listed. The Serine (S) residues that phosphorylated by AMPK are highlighted. c HEK293 cells transfected with Skp2 WT, S256, and the indicated HA-AMPKα (CA) constructs were subjected to immunoprecipitation, followed by immunoblotting. d Constitutively active (CA) and kinase dead (KD) HA-AMPKα1 proteins purified from 293T cells and recombinant GST-Skp2 WT purified from bacteria were subjected to in vitro kinase assay, followed by Immunoblotting. e Constitutively active (CA) HA-AMPKα1 purified from 293T cells and recombinant GST-Skp2 WT, S256A purified from bacteria were subjected to in vitro kinase assay, followed by Immunoblotting. f In vitro kinase assay of recombinant GST-Skp2 and active AMPKα1β1γ1 complex purified from sf9 cells with or without Compound C (5 μM) for 30 min followed by Immunoblotting. g Immunoblotting of control (shLuc) and AMPKα1 knockdown (#1) MDA-MB-231 cells treated with EGF, hypoxia (1% O2), glucose deprivation and H2O2 using Skp2 S256 phosphor-specific antibody. h In vivo ubiquitination assay in 293T cells transfected with the indicated plasmids was performed, followed by immunoblotting. i 293T cells transfected with Flag-Skp2 WT, S256A/D, and the indicated HA-AMPKα (CA) constructs were subjected to immunoprecipitation with Flag antibody, followed by immunoblotting. j MDA-MB-231 cells treated with or without glucose free medium and compound C were subjected to immunoprecipitation with Skp2 antibody, followed by immunoblotting. k The potential binding pocket on the interface of Skp2-Cullin1. Skp2 is shown in green and Cullin1 is displayed in yellow. l 293T cells transfected with Flag-Cullin1 WT and K221R were subjected to immunoprecipitation with Flag antibody, followed by immunoblotting
Fig 3: AMPK-Skp2-Akt axis is critical for survival under stress and glucose deprivation induced VEGF secretion and EGF-induced glycolysis and migration. a, c Cell survival analysis of MDA-MB-231 cells with control (shLuc), AMPKa1 knockdown, AMPKa1 knockdown along with Myr-Akt, Skp2 WT, or Skp2 S256A or Skp2 S256D restoration under hypoxia (1% O2) for 72 h. b, d Cell survival analysis of MDA-MB-231 cells with control (shLuc), AMPKa1 knockdown, AMPKa1 knockdown along with Myr-Akt, Skp2 WT, or Skp2 S256A or Skp2 S256D restoration under glucose deprivation for 16 h. e MDA-MB-231 cells with control (shLuc), AMPKa1 knockdown and AMPKa1 knockdown with vector control, Skp2 WT and S256A or Skp2 S256D restoration were treated with a glucose-free medium for 8 h, and supernatant was collected for HUVEC tube formation assay. Each value represents the mean ± SEM in three independent experiments. **P < 0.01. f MDA-MB-231 cells with control (shLuc), AMPKa1 knockdown (#1) and Myr-Akt restoration in AMPKa1 knockdown were starved in DMEM glucose-free medium for 4 h and added with 2-NBDG for 30 min. Cells were then subjected to FACS analysis. Each value represents the mean ± SEM (n = 4 per group) in three independent experiments. **P < 0.01. g Immunoblotting of control (shLuc) and AMPKa knockdown (#1) MDA-MB-231 cells serum starved and treated with EGF for 15 and 30 min. h Immunoblotting for MDA-MB-231 cells with control (shLuc), AMPKa1 knockdown and AMPKa1 knockdown along with Myr-Akt restoration. i MDA-MB-231 AMPKa1 knockdown cells with Skp2 WT, S256A or S256D restoration were starved in DMEM glucose-free medium for 4 h and added with 2-NBDG for 30 min. Cells were then subjected to FACS analysis. Each value represents the mean ± SEM (n = 4 per group) in three independent experiments. **P < 0.01. j Cell migration assay of MDA-MB-231 cells with control (shLuc), AMPKa1 knockdown and AMPKa1 knockdown along with Myr-Akt restoration. k MDA-MB-231 AMPKa1 knockdown cells with Skp2 WT, S256A or S256D restoration were subject to cell migration assay with or without EGF
Fig 4: The functional effects of NBR2 are partially mediated by AMPK(A and B) UMRC2 cells stably expressing EV or NBR2 expression vectors were transfected with AMPK siRNA (AMPK si1 or si2) or control siRNA (Ctrl si). Protein lysates were prepared and analyzed by Western blotting (a), or cells were cultured in 25 mM glucose-containing medium for different days as indicated, and then subjected to cell proliferation analysis (b) (Mean ± s.d., n=3 biologically independent extracts, two-tailed paired Student’s t-test). (c–g) MDA-MB-231 cells with stable expression of control shRNA (Ctrl sh) or NBR2 shRNA (NBR2 sh) were infected with empty vector (EV) or constitutively active AMPK (AMPK CA). These cells were cultured in 25 or 0 mM glucose-containing medium for 24 hours, and protein lysates were prepared and analyzed by Western blotting (c); The cells were cultured in 0 mM glucose-containing medium for different days as indicated, and then subjected to crystal violet staining to measure cell number (d) (Mean ± s.d., n=3 biologically independent extracts, two-tailed paired Student’s t-test); The cells were cultured in 25 or 0 mM glucose-containing medium for 24 hours, then subjected to Annexin V/PI staining followed by FACS analysis to measure the percentages of Annexin V positive/PI negative cells cells (e) (Mean ± s.d., n=5 fields per group, each field was assessed from an independent experiment, two-tailed paired Student’s t-test), or to Western blotting analysis to measure PARP cleavage (f); The cells were seeded in soft agar containing high or low concentrations of glucose as indicated. Bar graph showing the mean colony numbers from the soft agar assay (g) (Mean ± s.d., n=5 fields per group, each field was assessed from an independent experiment, two-tailed paired Student’s t-test). (h) Relative tumor volumes of MDA-MB-231 xenograft tumors of different genotypes at different weeks (Mean ± s.d., n = 5 xenograft tumors, *: P < 0.05, **: P < 0.01, two-tailed paired Student’s t-test). Source data for b, d, e can be found in Supplementary Table 1. Unprocessed original scans of blots are shown in Supplemental Fig. 8.
Fig 5: AMPK is required for Akt phosphorylation and activation under various cellular stresses. a Screening of kinases in the hypoxia-induced Akt phosphorylation using kinase inhibitor library. Immunoblotting of HEK293 cells pretreated with indicated inhibitors at 10 µ? for 2 h and challenged with 1% O2 for another 4 h. b Immunoblotting of control (shLuc) and AMPKa1 knockdown MDA-MB-231 cells (#1, #2) treated with 1% O2 for 4 h. c–e Immunoblotting of WT and AMPKa1/a2 double knockout (AMPKa-/-) restored with vector, WT or kinase dead AMPKa under hypoxia (1% O2), 100 µ? H2O2 or glucose deprivation for indicated time. In each restoration experiment, 5 µg of the plasmid was transfected into indicated cell lines. f, g Immunoblotting of with control (shLuc) and AMPKa1 knockdown MDA-MB-231 cells under H2O2 (100 µ?) or glucose deprivation for indicated time
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