TRAF6-mediated ubiquitination of AKT1 in the nucleus occurs in a ß-arrestin2-dependent manner upon insulin stimulation.

AKT, also known as protein kinase B (PKB), serves as a crucial regulator of numerous biological functions, including cell growth, metabolism, and tumorigenesis. Increasing evidence suggests that the kinase activity of AKT is regulated via ubiquitination by various E3 ligase enzymes in response to different stimuli. However, the molecular mechanisms underlying insulin-induced AKT ubiquitination are not yet fully understood. Here, we show that activation of the insulin receptor (IR) leads to enhanced ubiquitination of AKT1 at K8 and K14 residues, facilitated by the cytosolic E3 ubiquitin ligase enzyme, TRAF6. Further investigation using AKT1 mutants with modified nucleocytoplasmic shuttling properties reveals that TRAF6-mediated AKT1 ubiquitination occurs within the nucleus in a ß-Arr2-dependent manner. The nuclear entry of TRAF6 depends on importin ß1, while ß-Arr2 regulates this process by facilitating the interaction between TRAF6 and importin ß1. Additionally, the ubiquitination of AKT1 is essential for its translocation to the activated IR on the plasma membrane, where it plays a functional role in recruiting Glut4 and facilitating glucose uptake. This study uncovers the cellular components and processes involved in insulin-induced ubiquitination and activation of AKT1, providing insights and detailed strategies for manipulating AKT1.

TRAF6-mediated ubiquitination of AKT in the nucleus is a critical event underlying the desensitization of G protein-coupled receptors.

BACKGROUND: Desensitization of G protein-coupled receptors (GPCRs) refers to the attenuation of receptor responsiveness by prolonged or intermittent exposure to agonists. The binding of ß-arrestin to the cytoplasmic cavity of the phosphorylated receptor, which competes with the G protein, has been widely accepted as an extensive model for explaining GPCRs desensitization. However, studies on various GPCRs, including dopamine D2-like receptors (D2R, D3R, D4R), have suggested the existence of other desensitization mechanisms. The present study employed D2R/D3R variants with different desensitization properties and utilized loss-of-function approaches to uncover the mechanisms underlying GPCRs homologous desensitization, focusing on the signaling cascade that regulates the ubiquitination of AKT. RESULTS: AKT undergoes K8/14 ubiquitination by TRAF6, which occurs in the nucleus and promotes its membrane recruitment, phosphorylation and activation under receptor desensitization conditions. The nuclear entry of TRAF6 relies on the presence of the importin complex. Src regulates the nuclear entry of TRAF6 by mediating the interaction between TRAF6 and importin ß1. Ubiquitinated AKT translocates to the plasma membrane where it associates with Mdm2 to phosphorylate it at the S166 and S186 residues. Thereafter, phosphorylated Mdm2 is recruited to the nucleus, resulting in the deubiquitination of ß-Arr2. The deubiquitinated ß-Arr2 then forms a complex with Gßγ, which serves as a biomarker for GPCRs desensitization. Like in D3R, ubiquitination of AKT is also involved in the desensitization of ß2 adrenoceptors. CONCLUSION: Our study proposed that the property of a receptor that causes a change in the subcellular localization of TRAF6 from the cytoplasm to the nucleus to mediate AKT ubiquitination could initiate the desensitization of GPCRs.