Figure 5

Effect of sumoylation on the activity of CoCoA C-terminal AD. (A) In vitro-translated CoCoA(1–190) wild type, CoCoA(1–190, K29R), SUMO1-CoCoA(1–190) fusion protein, or SUMO1 were incubated with GST or GST-CoCoA(470–691) fusion protein bound to glutathione-Sepharose. Bound proteins were eluted and analyzed by autoradiography. (B) CV1 cells were transfected in 24-well plates with GK1-Luc reporter plasmid (150 ng) and plasmids encoding Gal4 DBD or Gal4 DBD-CoCoA(470–691) (50 ng), and HA-CoCoA(1–190) wild type or K29R mutant or SUMO1-CoCoA(1–190) (100 or 150 ng), as indicated. Cell extracts were assayed for luciferase activity 48 h after transfection. Luciferase activity results shown are from a single experiment which is representative of four independent experiments. (C) 293T cells were transfected in 24-well plates with GK1-Luc reporter plasmid (150 ng) and plasmids encoding Gal4 DBD-CoCoA(470–691) (50 ng), p300 (25 ng), and pSG5.HA-CoCoA(1–190) wild type or K29R mutant or SUMO1-CoCoA(1–190) (50, 100 or 150 ng) as indicated. Cell extracts were assayed for luciferase activity 48 h after transfection. Results shown are representative of two independent experiments. (D) CV-1 cells were transfected in 12-well plates with pGL3OT reporter plasmid (200 ng), pSG5.HA-LEF1 (10 ng), pSG5.HA-β-catenin (50 ng), and a pSG5.HA vector encoding CoCoA or SUMO1-fused CoCoA, as indicated. Results shown are representative of three independent experiments. (E) CV-1 cells were transfected in 12-well plates with MMTV(ERE)-LUC reporter plasmid (200 ng), pHE0 (2 ng), pSG5.HA-GRIP1 (50 ng), and a pSG5.HA vector encoding CoCoA or SUMO1-fused CoCoA, as indicated. Results shown are representative of four independent experiments. For immunoblots CV-1 cells do not produce enough protein, so COS-7 cells were transfected with 200 or 400 ng of CoCoA expression plasmids. Cell extracts were subjected to immunoblot analysis with anti-HA antibody.