Inhibition of Protein Synthesis Alters Protein Degradation through Activation of Protein Kinase B (AKT)

Chun-Ling Dai,Jianhua Shi,Yanxing Chen,Khalid Iqbal,Fei Liu,Cheng-Xin Gong

doi:10.1074/jbc.m112.445148

Abstract

The homeostasis of protein metabolism is maintained and regulated by the rates of protein biosynthesis and degradation in living systems. Alterations of protein degradation may regulate protein biosynthesis through a feedback mechanism. Whether a change in protein biosynthesis modulates protein degradation has not been reported. In this study, we found that inhibition of protein biosynthesis induced phosphorylation/activation of AKT and led to phosphorylation of AKT target substrates, including FoxO1, GSK3α/β, p70S6K, AS160, and the E3 ubiquitin ligase MDM2. Phosphorylation of ribosomal protein S6 was also modulated by inhibition of protein biosynthesis. The AKT phosphorylation/activation was mediated mainly through the PI3K pathway because it was blocked by the PI3K inhibitor LY294002. The activated AKT phosphorylated MDM2 at Ser(166) and promoted degradation of the tumor suppressor p53. These findings suggest that inhibition of protein biosynthesis can alter degradation of some proteins through activation of AKT. This study reveals a novel regulation of protein degradation and calls for caution in blocking protein biosynthesis to study the half-life of proteins.

Highlights

Whether a change in protein biosynthesis modulates protein degradation is unknown
We found that inhibition of protein biosynthesis induced phosphorylation/activation of AKT and led to phosphorylation of AKT target substrates, including FoxO1, GSK3␣/␤, p70S6K, AS160, and the E3 ubiquitin ligase MDM2
Inhibition of Protein Synthesis Promotes Phosphorylation and Alters Degradation of AKT—When we tried to determine the half-lives of HA-tagged WT and mutant (T308A/S473A, T308A, and S473A) AKT by measuring the level of HA-AKT with anti-HA antibody at various time points after treatment with the protein synthesis inhibitor cycloheximide at the concentration (100 ␮M) that is used in many studies [16, 17], we found that AKT with a single mutation (T308A or S473A) was more stable than WT AKT, whereas the turnover of the T308A/ S473A mutant was more rapid compared with WT AKT (Fig. 1, a–e)

Summary

Background

Whether a change in protein biosynthesis modulates protein degradation is unknown. Results: Inhibition of protein biosynthesis induces activation of AKT (protein kinase B), leading to activation of E3 ubiquitin ligase and degradation of its substrate proteins. The activated AKT phosphorylated MDM2 at Ser166 and promoted degradation of the tumor suppressor p53 These findings suggest that inhibition of protein biosynthesis can alter degradation of some proteins through activation of AKT. The functions of AKT3 are not well known, AKT3-deficient mice exhibit a reduction in brain weight resulting from decreases in both cell size and cell number but maintain normal glucose homeostasis and body weight [14, 15]. During investigation of the regulation and turnover of AKT, we observed that inhibition of protein biosynthesis by cycloheximide induced phosphorylation/activation of AKT and altered its degradation. Cycloheximide Alters Protein Degradation biosynthesis was inhibited and that this phenomenon involved AKT activation

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION