FIGURE 1.
NEDD4-1 is a novel AKT E3 ligase involved in IGF-1 and insulin signaling. A, Western blot analysis (WB) of cell lysates from WT and Nedd4-1−/− MEFs after the indicated treatments (C, control; Stv, starvation; Ins, insulin; Ser, serum). B, in vivo analysis of AKT ubiquitination in MCF7 cells transfected with His-ubiquitin (His-Ub), AKT or NEDD4-1 (N4–1). After His-tag pulldown (His-pdn), ubiquitinated AKT was detected by anti-AKT immunoblotting (IB). Protein input was monitored by direct Western blot (WB). C, in vivo analysis of endogenous AKT ubiquitination by NEDD4-1 overexpression in MCF7 cells. D, in vivo ubiquitination of endogenous AKT in Nedd4-1+/+ and Nedd4-1−/− MEFs. E, coimmunoprecipitation of endogenous AKT with transfected NEDD4-1 (HA-N4–1) in MCF7 cells. F, in vitro ubiquitination of AKT by recombinant NEDD4-1 (rN4-1). pUb-AKT, polyubiquitinated AKT; Ub-AKT, ubiquitinated AKT.