The phosphatidylinositol 3-kinase/Akt pathway regulates transforming growth factor-{beta} signaling by destabilizing ski and inducing Smad7

Abstract

Ski is an oncoprotein that negatively regulates transforming growth factor (TGF)-beta signaling. It acts as a transcriptional co-repressor by binding to TGF-beta signaling molecules, Smads. Efficient TGF-beta signaling is facilitated by rapid proteasome-mediated degradation of Ski by TGF-beta. Here we report that Ski is phosphorylated by Akt/PKB kinase. Akt phosphorylates Ski on a highly conserved Akt motif at threonine 458 both in vitro and in vivo. The phosphorylation of Ski at threonine 458 is induced by Akt pathway activators including insulin, insulin-like growth factor-1, and hepatocyte growth factor. The phosphorylation of Ski causes its destabilization and reduces Ski-mediated inhibition of expression of another negative regulator of TGF-beta, Smad7. Induction of Smad7 levels leads to inactivation of TGF-beta receptors and TGF-beta signaling cascade, as indicated by reduced induction of TGF-beta target p15. Therefore, Akt modulates TGF-beta signaling by temporarily adjusting the levels of two TGF-beta pathway negative regulators, Ski and Smad7. These novel findings demonstrate that Akt pathway activation directly impacts TGF-beta pathway.

FIGURE 1.

Ski interacts with Akt. A, Ski interacts with Akt1. Cos 7 cells were transiently transfected with Ski-HA and Akt1-HA expression plasmids for 24 h. The cells were lysed, and the lysates were immunoprecipitated (IP) with control IgG or anti-Ski or anti-Akt antibody followed by immunoblotting (WB) with anti-HA antibody. Input lysate is shown to the left. B, Ski interacts with Akt1 and Akt2 but not with Akt3. 293T cells were transiently transfected with Ski-HA and either Akt1-HA, Akt2-HA, or Akt3-HA expression plasmids. The cell lysates were immunoprecipitated with anti-Ski antibody and immunoblotted with anti-HA antibody. C, Ski interacts with both active and inactive forms of Akt1. Cos 7 cells were transiently transfected with Ski-HA and either with Akt1-HA, kinase-dead (KD) Akt1-HA, or constitutively active myristylated Akt1-HA (Akt1myr). The cells were then treated with LY294002 (25 μm) overnight to inactivate Akt as indicated. The cell lysates were immunoprecipitated with anti-Ski antibody and were immunoblotted with anti-HA antibody. The cellular lysates were analyzed as control. The data shown are representative of two to three similar experiments.

FIGURE 2.

Ski is phosphorylated by Akt. A, conservation of a putative Ski Akt phosphorylation site across species. B, Akt phosphorylates Ski at threonine 458. Ski wild-type (wt), Ski threonine 458 alanine (A), or aspartic acid (D) mutants were co-transfected with Akt1. The lysates were immunoprecipitated (IP) with anti-Ski antibody (Ski) and probed with anti-Akt substrate and anti-HA antibody as indicated (WB). C, in vitro Akt assay. 293T cells were transfected with PCIneo vector, Ski wt, or Ski A and were additionally treated with LY294002 as indicated. The lysates were immunoprecipitated with anti-HA antibody, and in vitro Akt assay with recombinant Akt (rAkt) was performed as described under “Experimental Procedures.” The bands were quantified using ImageJ software. D, in vitro Akt assay in the presence of [γ-³²P]ATP (³²P). The data shown in B and C are representative of two similar experiments. WB, Western blotting.

FIGURE 3.

Insulin and other growth factors induce phosphorylation of Ski. A, basal phosphorylation of Ski is abrogated by a PI3K inhibitor, LY294002, and an Akt inhibitor, tricibine. U-2OS cells were transiently transfected with Ski for 24 h and were treated with LY294002 (25 μm) or tricibine (5 μm) overnight. The lysates were immunoprecipitated (IP) and then Western blotted (WB) with either Akt substrate or anti-HA antibodies as shown. The cellular lysates were analyzed as control. B, time course of the phosphorylation of Ski after insulin treatment. U-2 OS cells were transiently transfected with Ski for 24 h, serum-starved overnight, and then stimulated with insulin (100 nm) for the times indicated. The lysates were immunoprecipitated with anti-Ski antibody and probed with anti-Akt substrate, anti-HA, and phospho-Akt antibodies (P308Akt and P473Akt) as indicated. C, insulin increases phosphorylation of endogenous Ski. MDA-MB 435 cells were serum-starved overnight and treated with LY294002 (25 μm) for 6 h prior to adding insulin (100 nm) for 1 h. Ski was immunoprecipitated and probed with anti-Akt substrate antibody, anti-Ski antibody, and anti-phospho-Akt antibody. D, U-2 OS cells were transfected with Ski, starved and treated with insulin (100 nm), IGF-I (10 ng/ml), HGF (50 ng/ml), and epidermal growth factor (100 ng/ml) for 1 h. Ski, Akt, and their phospho-forms were analyzed as in A. The cellular lysates were analyzed as control. The data in C and D are representative of two similar experiments.

FIGURE 4.

Phosphorylated Ski is present both in the nucleus and in the cytoplasm. U2-OS cells were transfected with Ski expression vector. The cells were starved overnight and stimulated with insulin (100 nm) for 1 h. The cells were then separated into cytosolic (C) and nuclear (N) fractions followed by immunoprecipitation (IP) as indicated. Total Ski and phosphorylated Ski were detected with anti-HA and anti-Akt substrate antibodies by immunoblotting (WB). β-Tubulin and Sp1 were used as cytosolic and nuclear markers, respectively. The data shown are representative of three similar experiments.

FIGURE 5.

Smad3 competes with Akt for the binding of Ski. 293T cells were co-transfected with Ski-HA and Akt1-HA and an increasing amount of Smad3-Myc expression vector. Ski was immunoprecipitated (IP) and blotted (WB) with anti-HA and anti-Myc antibodies. Cellular lysates were analyzed as control. The data shown are representative of four similar experiments.

FIGURE 6.

Phosphorylation at threonine 458 destabilizes Ski. A, U-2 OS stably expressing wt Ski or Ski threonine 458 alanine mutant (Ski A) were treated either with CHX (50 μg/ml), MG132 (MG, 20 μm), and 5 μm LY294002 overnight and with insulin (100 nm) for one h. The cells were lysed, and the levels of Ski wt and A mutant were measured by immunoblotting (WB) using anti-HA antibody. The bands were quantified using ImageJ software and normalized against a nonspecific band. B, degradation of Ski wt and Ski A. The cells were treated with insulin (100 nm) for 30 min, after which CHX (50 μg/ml) was added as indicated. The proteins were analyzed by Western blotting, and the signals were quantified. The protein bands were quantified using FluorChem^TM densitometry. The data in B are representative of two similar experiments.

FIGURE 7.

Insulin does not alter the degradation rate of endogenous Ski by TGF-β. A, MDA-MB 453 cells were treated with or without insulin (100 nm) for 30 min, after which TGF-β (3 ng/ml) was added for the indicated times. The cell lysates were blotted (WB) with the indicated antibodies. An asterisk indicates a nonspecific (ns) band. The bands were quantified using ImageJ software and normalized against a nonspecific band. B, Ski levels in A were quantified from two independent experiments.

FIGURE 8.

Insulin-mediated destabilization of Ski induces expression of Smad7 and reduces TGF-β signaling. A, insulin destabilizes Ski and increases Smad7 expression. MDA-MB 435 cells were treated with insulin (100 nm) as indicated, and cell lysates were immunoblotted (WB) with anti-Ski, anti-Smad7, anti-P473Akt, anti-P308Akt, and anti-Akt antibody. B, insulin increases Smad7 expression temporarily. MDA-MB 435 cells were serum-starved overnight and stimulated with insulin (100 nm) as indicated. Smad7 mRNA was analyzed using RT-PCR. The bands were quantified using FluorChem^TM densitometry and were normalized against GADPH. Ski and Smad7 levels were simultaneously analyzed by Western blotting. C, TGF-β signaling is inactivated by insulin. MDA-MB 435 cells were first stimulated with insulin after which TGF-β (3 ng/ml) was added for the last 30 min. The cells were lysed and immunoblotted with anti-phospho-Smad2/3 (PSmad2/3), anti-Smad2/3 or anti-TGF-β receptor I (TGF-β RI) antibodies. D, insulin treatment prevents the induction of p15 by TGF-β. MDA-MB 435 cells were serum-starved overnight, stimulated with insulin followed by the addition of TGF-β (3 ng/ml) for the last 2 h or TGF-β alone (lane 2). E, model of parallel regulation of TGF-β and Akt pathways of TGF-β downstream regulators. The data shown are representative of at least two similar experiments.