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.
Highlights
The transforming growth factor (TGF)- signaling cascade is well regulated and determined by post-translational modifications, protein localization, degradation, and inhibitory molecules [2, 3]
In this study we show that the negative regulator of TGF- signaling, Ski, is phosphorylated by Akt kinase on threonine 458
Smad7 and the decreased phosphorylation of Smads (Fig. 8, A–C). This finding is consistent with insulin-mediated inhibition of TGF- signaling through temporal regulation of Ski and DISCUSSION In this study we show that the negative regulator of TGF-
Summary
Cell Culture and Reagents—Human osteosarcoma U-2 OS cells were cultured in Dulbecco’s modified Eagle’s medium containing 15% fetal calf serum (Autogen Bioclear, Calne, UK). Goat anti-Ski (N-20), mouse monoclonal Sp-1 (1CG), rabbit p15, and Smad antibodies were obtained from Santa Cruz Biotechnology Inc. Nuclear and Cytosolic Fractions—The cells were washed twice with phosphate-buffered saline and lysed in 200 l of hypotonic buffer (20 mM Tris/HCl, pH 7.4, 20% glycerol, 10 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.1% Triton X-100, 25 mM NaF, 25 mM -glycerophosphate, 1 mM sodium orthovanadate, 1 mM dithiothreitol). G-Sepharose beads, and incubated for 30 min at 30 °C with human 100 ng of recombinant Akt (Cell Signaling Technology, Beverly, MA) in the presence of 2.5 mM -glycerophosphate, 1 mM EGTA, 0.4 mM EDTA, 5 mM MgCl2, 0.05 mM dithiothreitol, 25 M [␥-32P]ATP (PerkinElmer Life Science) in 5 mM MOPS pH 7.2 buffer. The primers for the amplification of the human Smad were as follows: forward, 5Ј-TCCAGATACCCGATGGATTTTC-3Ј, and reverse, 5Ј-GATTTTGCTCCGCACCTTCT-3Ј
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
