Endosomal H(2)O(2) Molecules Act as Signaling Mediators in Akt/PKB Activation.

Receptor-mediated endocytosis (RME) is a commonly recognized receptor internalization process of receptor degradation or recycling. However, recent studies have supported that RME is closely related to signal propagation and amplification from the plasma membrane to the cytosol. Few studies have elucidated the role of H(2)O(2), a mild oxidant among reactive oxygen species (ROS) in RME and second messenger of signal propagation. In the present study, we investigated the regulatory function of H(2)O(2) in early endosomes during signaling throughout receptor-mediated endocytosis. In mammalian cells with a physiological amount of H(2)O(2) generated during epidermal growth factor (EGF) activation, fluorescence imaging showed that the levels of two activating phosphorylations on Ser(473) and Thr(308) of Akt were transiently increased in the plasma membrane, but the predominant p-Akt on Ser(473) appeared in early endosomes. To examine the role of endosomal H(2)O(2) molecules as signaling mediators of Akt activation in endosomes, we modulated endosomal H(2)O(2) through the ectopic expression of an endosomal-targeting catalase (Cat-Endo). The forced removal of endosomal H(2)O(2) inhibited the Akt phosphorylation on Ser(473) but not on Thr(308). The levels of mSIN and rictor, two components of mTORC2 that work as a kinase in Akt phosphorylation on Ser(473), were also selectively diminished in the early endosomes of Cat-Endo-expressing cells. We also observed a decrease in the endosomal level of the adaptor protein containing the PH domain, the PTB domain, and the Leucine zipper motif 1 (APPL1) protein, which is an effector of Rab5 and key player in the assembly of signaling complexes regulating the Akt pathway in Cat-Endo-expressing cells compared with those in normal cells. Therefore, the H(2)O(2)-dependent recruitment of the APPL1 adaptor protein into endosomes was required for full Akt activation. We proposed that endosomal H(2)O(2) is a promoter of Akt signaling.