Figure 2
Figure 2. The PI3K/AKT1 signaling pathway is implicated in TGF-β–induced degradation of TAL1. (A) Wortmannin reverses TGF-β-induced degradation of TAL1. HeLa cells were transfected with 1 μg of pSGF-TAL1 and 10 μM wortmannin was added (lane 3) or not (lanes 1 and 2) to the culture medium 30 minutes before treatment with 10 ng/mL TGF-β1 for 9 hours (lanes 2 and 3). Cellular extracts were analyzed by immunoblot using antibodies to TAL1 (top panel) or to β-actin (bottom panel). (B) AKT1 increases TAL1 polyubiquitylation. HeLa cells were transfected with 1 μg of pSGF-TAL1 and 0.5 μg of pSG-HA-Ub, as well as 1 μg of vectors expressing HA-tagged AKT1, either wild-type (WT) or mutated in the kinase site,18 as indicated. Cell lysates were immunoprecipitated with an antibody to FLAG and immunoblot analysis was done using antibodies to HA (top panel) and to FLAG (bottom panel). (C) TAL1 T90A mutant is resistant to TGF-β–induced degradation. HeLa cells were transfected with 1 μg of pSGF-TAL1 either wild type (lanes 1 and 2) or including a change of threonine 90 to alanine (lanes 3 and 4). Cells were treated or not by TGF-β and analyzed as described for panel A. (D) TAL1 phosphorylation favors interaction with CHIP. HeLa cells were transfected with 1 μg of pRK1M/Myc-CHIP19 and HA-TAL1, either WT or including mutations T90A and T90D13 as indicated. Cell lysates were immunoprecipitated with an antibody to HA. Immunoblot analysis was done using antibodies to HA (top panel) and to Myc (bottom panel). (E) Presence of E47 prevents TAL1 interaction with CHIP. HeLa cells were transfected with 1 μg of vectors expressing pSGF-TAL1, Myc-E47,20 and Myc-CHIP as indicated. Cell lysates were immunoprecipitated with an antibody to FLAG and immunoblot analysis was done using antibodies to Myc (top panel) and to FLAG (bottom panel). (F) Schematic representation of the effect of the TGF-β/PI3K/AKT1 signaling pathway on TAL1 degradation. According to the model, TGF-β induces a PI3K/AKT1 dependent phosporylation of TAL1, which favors its interaction with CHIP, increasing TAL1 ubiquitylation and further proteolysis by the proteasome. E47, by associating with TAL1, hinders CHIP interaction.

The PI3K/AKT1 signaling pathway is implicated in TGF-β–induced degradation of TAL1. (A) Wortmannin reverses TGF-β-induced degradation of TAL1. HeLa cells were transfected with 1 μg of pSGF-TAL1 and 10 μM wortmannin was added (lane 3) or not (lanes 1 and 2) to the culture medium 30 minutes before treatment with 10 ng/mL TGF-β1 for 9 hours (lanes 2 and 3). Cellular extracts were analyzed by immunoblot using antibodies to TAL1 (top panel) or to β-actin (bottom panel). (B) AKT1 increases TAL1 polyubiquitylation. HeLa cells were transfected with 1 μg of pSGF-TAL1 and 0.5 μg of pSG-HA-Ub, as well as 1 μg of vectors expressing HA-tagged AKT1, either wild-type (WT) or mutated in the kinase site,18  as indicated. Cell lysates were immunoprecipitated with an antibody to FLAG and immunoblot analysis was done using antibodies to HA (top panel) and to FLAG (bottom panel). (C) TAL1 T90A mutant is resistant to TGF-β–induced degradation. HeLa cells were transfected with 1 μg of pSGF-TAL1 either wild type (lanes 1 and 2) or including a change of threonine 90 to alanine (lanes 3 and 4). Cells were treated or not by TGF-β and analyzed as described for panel A. (D) TAL1 phosphorylation favors interaction with CHIP. HeLa cells were transfected with 1 μg of pRK1M/Myc-CHIP19  and HA-TAL1, either WT or including mutations T90A and T90D13  as indicated. Cell lysates were immunoprecipitated with an antibody to HA. Immunoblot analysis was done using antibodies to HA (top panel) and to Myc (bottom panel). (E) Presence of E47 prevents TAL1 interaction with CHIP. HeLa cells were transfected with 1 μg of vectors expressing pSGF-TAL1, Myc-E47,20  and Myc-CHIP as indicated. Cell lysates were immunoprecipitated with an antibody to FLAG and immunoblot analysis was done using antibodies to Myc (top panel) and to FLAG (bottom panel). (F) Schematic representation of the effect of the TGF-β/PI3K/AKT1 signaling pathway on TAL1 degradation. According to the model, TGF-β induces a PI3K/AKT1 dependent phosporylation of TAL1, which favors its interaction with CHIP, increasing TAL1 ubiquitylation and further proteolysis by the proteasome. E47, by associating with TAL1, hinders CHIP interaction.

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