Abstract
AKT is a critical effector kinase downstream of the PI3K pathway that regulates a plethora of cellular processes including cell growth, death, differentiation, and migration. Mechanisms underlying activated phospho-AKT (pAKT) translocation to its action sites remain unclear. Here we show that NEDD4-1 is a novel E3 ligase that specifically regulates ubiquitin-dependent trafficking of pAKT in insulin-like growth factor (IGF)-1 signaling. NEDD4-1 physically interacts with AKT and promotes HECT domain-dependent ubiquitination of exogenous and endogenous AKT. NEDD4-1 catalyzes K63-type polyubiquitin chain formation on AKT in vitro. Plasma membrane binding is the key step for AKT ubiquitination by NEDD4-1 in vivo. Ubiquitinated pAKT translocates to perinuclear regions, where it is released into the cytoplasm, imported into the nucleus, or coupled with proteasomal degradation. IGF-1 signaling specifically stimulates NEDD4-1-mediated ubiquitination of pAKT, without altering total AKT ubiquitination. A cancer-derived plasma membrane-philic mutant AKT(E17K) is more effectively ubiquitinated by NEDD4-1 and more efficiently trafficked into the nucleus compared with wild type AKT. This study reveals a novel mechanism by which a specific E3 ligase is required for ubiquitin-dependent control of pAKT dynamics in a ligand-specific manner.
Highlights
After activation by phosphorylation, phospho-AKT is translocated to nucleus
NEDD4-1 Is an E3 Ligase for AKT—Nedd4-1Ϫ/Ϫ mouse embryonic fibroblasts (MEFs) are profoundly defective in AKT activation/phosphorylation by insulin and insulin-like growth factor (IGF)-1 stimulation, but not by EGF or serum (Fig. 1A and see Ref. 18)
We hypothesized that AKT is a direct substrate of NEDD4-1 E3 ligase activity and that AKT ubiquitination by NEDD4-1 is a necessary step for insulin- and insulin-like growth factor 1 (IGF-1)-induced AKT activation
Summary
Results: Ubiquitination of pAKT by NEDD4-1 is coupled to AKT activation at the plasma membrane by insulin-like growth factor (IGF)-1, which promotes pAKT nuclear trafficking. We show that NEDD4-1 is a novel E3 ligase that regulates ubiquitin-dependent trafficking of pAKT in insulin-like growth factor (IGF)-1 signaling. Upon ligand binding to receptor tyrosine kinases or G protein-coupled receptors, AKT activation is initiated by membrane recruitment, via interaction of its PH3 domain with. The C2-domain of NEDD4-1 interacts with the Src homology 2-domain of Grb10␣, whereas the BPS domain of Grb10␣ interacts with IGF1R upon ligand binding This Grb10-mediated interaction negatively regulates IGF1R signaling by promoting Nedd4-dependent IGF1R endocytosis [7]. Ubiquitination Regulates pAKT Nuclear Trafficking because PTEN protein levels are not altered in Nedd knockout mouse embryonic fibroblasts (MEFs) [17, 18]. We describe evidence demonstrating that AKT is a new substrate for NEDD4-1 E3 ligase, and AKT ubiquitination by NEDD4-1 positively regulating nuclear trafficking of the activated form of AKT
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