Figure 2.

Receptor-interacting protein 1 induces activation of Akt and downregulates p27^Kip1 through a phosphatidylinositol 3-kinase-dependent pathway. (A) Western blot showing that LY294002 (LY; 10 μM) prevents RIP1-mediated downregulation of p27^Kip1 in RIP1^−/− MEFs. The lower panel shows RIP1 expression and shows that RIP1 expression is similar in LY294002-treated cells compared with DMSO-treated cells. Cells were cultured in DMEM with 10% FBS in experiments B–G. (B) Western blot showing that introduction of RIP1 adenovirus into an RIP1^−/− immortalized MEF cell line for 24 h induces phosphorylation of Akt (S473), whereas introduction of GFP or p27^Kip1 adenovirus does not. (C) Introduction of RIP1 into RIP1^−/− cells also results in phosphorylation of Akt on Thr 308 (T308). (D) Akt kinase assay using Akt immunoprecipitated from RIP1^−/− immortalized MEFs with or without RIP1 reconstitution. Akt was immunoprecipitated 48 h after RIP1 infection. (E) The kinase domain of RIP1 is not required for activation of pAkt or downregulation of p27^Kip1. Wild-type RIP1 (WT) or a kinase domain deletion mutant (DKD) was introduced into RIP1^−/− cells followed by western blot. The middle panel shows expression of wild-type RIP1 and the DKD mutant. Densitometry values show relative signal intensity normalized for ERK2 (loading) signal. (F) RIP1-induced phosphorylation of Akt blocked by LY294002 (10 μM). (G) RIP1 expression induces phosphorylation of GSK3. (H) RIP1 expression induces phosphorylation of p70S6K (Thr 389). Three independent experiments were performed and a representative experiment is shown. ERK2, extracellular signal-regulated kinase 2; GFP, green fluorescent protein; GSK3, glycogen synthase kinase-3; MEF, mouse embryonic fibroblast; pAkt, phosphorylated Akt; RIP1, receptor-interacting protein 1.