Journal:
Article Title: Insulin Enhances Post-translational Processing of Nascent SREBP-1c by
Promoting Its Phosphorylation and Association with COPII
Vesicles *
doi: 10.1074/jbc.M805746200
Figure Lengend Snippet: Effects of PKB on SREBP-1c phosphorylation and activation by insulin. A, hepatocytes infected with Ad-HisSREBP-1cFLAG were incubated with or without Akt inhibitor II (10 μm) or III (10 μm) for 30 min before adding insulin. Microsomal membranes (500 μg) were immunoprecipitated (IP) with anti-FLAG (full-length SREBP-1c) and immunoblotted for phosphoserine antibody or FLAG antibody (upper left panel). Hepatocytes infected with Ad-His-SREBP-1cFLAG were preincubated with or without Akt inhibitor II or III for 30 min and then treated with insulin for 1 h. Nuclear protein extracts (50 μg) were separated by SDS-PAGE and immunoblotted for anti-His (nSREBP-1c) or anti-histone H1 antibodies (upper right panel). Hepatocytes were treated with or without insulin (100 nm) in the presence and absence of Akt inhibitor II (10 μm) or III (10 μm), and cell extracts were prepared. An equal amount of protein (50 μg) from control and each treatment was analyzed by Western blotting for phosphorylated GSK-3β-specific antibodies. The blots were reprobed with anti-GSK-3β antibodies to assess the levels of total GSK-3β (lower panel). B, hepatocytes were incubated with or without insulin (100 nm) in the presence or absence of LY294002 (10 μm) or Akt inhibitor II (10 μm), and microsomes were prepared. Microsomal membrane proteins were immunoprecipitated (500 μg) with anti-SREBP-1c or preimmune IgG, fractionated by SDS-PAGE, and immunoblotted for Sec23 (left panel). Hepatocytes were incubated with or without insulin (100 nm) in the presence or absence of LY294002 (10 μm) or Akt inhibitor II (10 μm), and microsomes were prepared. Microsomal membrane proteins (500 μg) were immunoprecipitated with anti-Sec23 or preimmune IgG, fractionated by SDS-PAGE, and immunoreacted with anti-SREBP-1c antibodies (right panel). C, hepatocytes infected with Ad-HisSREBP-1cFLAG were incubated with or without insulin (100 nm) for 1 h, and cytosol and native membranes (control-treated) were prepared. Cytosol immunodepleted of PKB/Akt or treated with control IgG was incubated with urea-washed native membranes and assayed by GST-Sar1 pulldown assay as described under “Materials and Methods.” D, hepatocytes infected with Ad-HisSREBP-1cFLAG were incubated with or without insulin (100 nm) for 1 h, and cytosol and native membranes (control-treated) were prepared. Cytosol immunodepleted of PKB/Akt or treated with control IgG was incubated with urea-washed native membranes for 1 h at 37°C and an ATP-regenerating system containing 50 μCi of [γ-32P]ATP. Membranes were reisolated, solubilized, fractionated by SDS-PAGE, and subjected to autoradiography as described under “Materials and Methods.” E, recombinant SREBP-1c, recombinant active PKB, or recombinant SREBP-1c and active PKB were incubated in vitro in the presence of [γ-32P]ATP. Proteins were separated by SDS-PAGE, and autoradiographed. Alternatively, proteins were detected by immunoblotting using either SREBP-1 antibody or Akt antibody (left panel). Recombinant wild-type GSK-3β or its nonphosphorylatable mutant (S9A) and active PKB were incubated in the presence of [γ-32P]ATP. Proteins were separated by SDS-PAGE and autoradiographed or immunoblotted using GSK-3β or Akt antibody (right panel).
Article Snippet: Akt inhibitor II (SH-5) and Akt inhibitor III (SH-6) were procured from Invitrogen.
Techniques: Activation Assay, Infection, Incubation, Immunoprecipitation, SDS Page, Western Blot, Autoradiography, Recombinant, In Vitro, Mutagenesis
![Effects of PKB on SREBP-1c phosphorylation and activation by insulin. A, hepatocytes infected with Ad-HisSREBP-1cFLAG were incubated with or without <t>Akt</t> inhibitor II (10 μm) or <t>III</t> (10 μm) for 30 min before adding insulin. Microsomal membranes (500 μg) were immunoprecipitated (IP) with anti-FLAG (full-length SREBP-1c) and immunoblotted for phosphoserine antibody or FLAG antibody (upper left panel). Hepatocytes infected with Ad-His-SREBP-1cFLAG were preincubated with or without Akt inhibitor II or III for 30 min and then treated with insulin for 1 h. Nuclear protein extracts (50 μg) were separated by SDS-PAGE and immunoblotted for anti-His (nSREBP-1c) or anti-histone H1 antibodies (upper right panel). Hepatocytes were treated with or without insulin (100 nm) in the presence and absence of Akt inhibitor II (10 μm) or III (10 μm), and cell extracts were prepared. An equal amount of protein (50 μg) from control and each treatment was analyzed by Western blotting for phosphorylated GSK-3β-specific antibodies. The blots were reprobed with anti-GSK-3β antibodies to assess the levels of total GSK-3β (lower panel). B, hepatocytes were incubated with or without insulin (100 nm) in the presence or absence of LY294002 (10 μm) or Akt inhibitor II (10 μm), and microsomes were prepared. Microsomal membrane proteins were immunoprecipitated (500 μg) with anti-SREBP-1c or preimmune IgG, fractionated by SDS-PAGE, and immunoblotted for Sec23 (left panel). Hepatocytes were incubated with or without insulin (100 nm) in the presence or absence of LY294002 (10 μm) or Akt inhibitor II (10 μm), and microsomes were prepared. Microsomal membrane proteins (500 μg) were immunoprecipitated with anti-Sec23 or preimmune IgG, fractionated by SDS-PAGE, and immunoreacted with anti-SREBP-1c antibodies (right panel). C, hepatocytes infected with Ad-HisSREBP-1cFLAG were incubated with or without insulin (100 nm) for 1 h, and cytosol and native membranes (control-treated) were prepared. Cytosol immunodepleted of PKB/Akt or treated with control IgG was incubated with urea-washed native membranes and assayed by GST-Sar1 pulldown assay as described under “Materials and Methods.” D, hepatocytes infected with Ad-HisSREBP-1cFLAG were incubated with or without insulin (100 nm) for 1 h, and cytosol and native membranes (control-treated) were prepared. Cytosol immunodepleted of PKB/Akt or treated with control IgG was incubated with urea-washed native membranes for 1 h at 37°C and an ATP-regenerating system containing 50 μCi of [γ-32P]ATP. Membranes were reisolated, solubilized, fractionated by SDS-PAGE, and subjected to autoradiography as described under “Materials and Methods.” E, recombinant SREBP-1c, recombinant active PKB, or recombinant SREBP-1c and active PKB were incubated in vitro in the presence of [γ-32P]ATP. Proteins were separated by SDS-PAGE, and autoradiographed. Alternatively, proteins were detected by immunoblotting using either SREBP-1 antibody or Akt antibody (left panel). Recombinant wild-type GSK-3β or its nonphosphorylatable mutant (S9A) and active PKB were incubated in the presence of [γ-32P]ATP. Proteins were separated by SDS-PAGE and autoradiographed or immunoblotted using GSK-3β or Akt antibody (right panel).](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_8047/pmc02658047/pmc02658047__zbc0160970700011.jpg)