Functional Role of mTORC2 versus Integrin-Linked Kinase in Mediating Ser473-Akt Phosphorylation in PTEN-Negative Prostate and Breast Cancer Cell Lines
Figure 5
Evidence that ILK binds and phosphorylates Akt in PC-3 cells.
(A) Co-immunoprecipitation analysis reveals the association of ILK with Akt in PC-3 cells, which can be attenuated by T315. PC-3 cells were treated with 2.5 µM T315 or DMSO control for 24 h. Equal amounts of cell lysates were immunoblotted (WB) with antibodies against ILK, p-Ser473-Akt, or Akt (Input; lower panel), or were immunoprecipitated (IP) with anti-Akt antibody and protein A/G agarose followed by immunoblotting with antibodies against ILK, p-Ser473-Akt, or Akt (upper panel). (B) Effect of T315 on the kinase activity of ILK. Equal amounts of immunocomplexes obtained by immunoprecipitation of ILK from PC-3 cell lysates were incubated with bacterially expressed GST-Akt and 200 mM of ATP in the presence of DMSO or T315 at indicated concentrations for 20 min, and were then subjected to Western blotting with antibodies against p-Ser-473-Akt, Akt, or ILK. The immunoprecipitation procedure using IgG was performed as control. Left panel, Western blot analysis of the dose-dependent effect of T315 on the kinase activity of ILK. Right panel, relative expression levels of phosphorylated Ser-473-Akt after normalization to total Akt and subtraction from control (time zero). Amounts of immunoblotted proteins from three independent experiments were quantitated by densitometry. The abundance of phosphorylated Ser-473-Akt in T315-treated cells was expressed as a percentage of that in the DMSO control cells (0 µM). Values, means; bars, S.D. (n = 3). All immunoblots are representative of three independent experiments. *P<0.05, compared to DMSO control (0 µM).