Supplementary Figure 4 from Protection from Rapamycin-Induced Apoptosis by Insulin-Like Growth Factor-I Is Partially Dependent on Protein Kinase C Signaling
ARTICLE ABSTRACT
Rapamycin-induced apoptosis in sarcoma cells is inhibited by insulin-like growth factor-I (IGF-I) through a signaling pathway independent of Ras-extracellular signal-regulated kinase 1/2 and Akt. IGF-I induces Bad phosphorylation (Ser112, Ser136, and Ser155) in a pathway involving phosphoinositide 3′ kinase (PI3K) and protein kinase C (PKC; μ, ϵ, or θ) resulting in sequestering Bad from mitochondria and subsequently interacting with 14-3-3γ in the cytosol. Gene knockdown of Bad, Bid, Akt1, Akt2, PKC-μ, PKC-ϵ, or PKC-θ was achieved by transient transfection using small interfering RNAs. Results indicate that IGF-I signaling to Bad requires activation of PI3K and PKC (μ, θ, ϵ) but not mTOR, Ras-extracellular signal-regulated kinase 1/2, protein kinase A, or p90RSK. Wortmannin blocked the phosphorylation of PKC-μ (Ser744/Ser748), suggesting that PI3K is required for the activation of PKCs. PKCs phosphorylate Bad under in vitro conditions, and the association of phosphorylated Bad with PKC-μ or PKC-ϵ, as shown by immunoprecipitation, indicated direct involvement of PKCs in Bad phosphorylation. To confirm these results, cells overexpressing pEGFP-N1, wt-Bad, or Bad with a single site mutated (Ser112Ala; Ser136Ala; Ser155Ala), two sites mutated (Ser112/136Ala; Ser112/155Ala; Ser136/155Ala), or the triple mutant were tested. IGF-I protected completely against rapamycin-induced apoptosis in cells overexpressing wt-Bad and mutants having either one or two sites of phosphorylation mutated. Knockdown of Bid using small interfering RNA showed that Bid is not required for rapamycin-induced cell death. Collectively, these data suggest that IGF-I–induced phosphorylation of Bad at multiple sites via a pathway involving PI3K and PKCs is important for protecting sarcoma cells from rapamycin-induced apoptosis. Cancer Res; 70(5); 2000–9
