PRAS40 Connects Microenvironmental Stress Signaling to Exosome-Mediated Secretion

Abstract

Secreted exosomes carrying lipids, proteins, and nucleic acids conduct cell-cell communications within the microenvironment of both physiological and pathological conditions. Exosome secretion is triggered by extracellular or intracellu- lar stress signals. Little is known, however, about the signal transduction between stress cues and exosome secretion. To identify the linker protein, we took advantage of a unique finding in human keratinocytes. In these cells, although transforming growth factor alpha (TGF-!) and epidermal growth factor (EGF) share the same EGF receptor and previously indistinguishable intracellular signaling networks, only TGF-! stimulation causes exosome-mediated secretion. However, deduction of EGF-activated pathways from TGF!-activated pathways in the same cells allowed us to identify the proline-rich Akt substrate of 40 kDa (PRAS40) as the unique downstream effector of TGF-! but not EGF signaling via threonine 308-phosphorylated Akt. PRAS40 knock- down (KD) or PRAS40 dominant-negative (DN) mutant overexpression blocks not only TGF-!- but also hypoxia- and H2 O2-induced exosome secretion in a variety of normal and tumor cells. Site-directed mutagenesis and gene rescue studies show that Akt-mediated activation of PRAS40 via threonine 246 phosphorylation is both necessary and sufficient to cause exosome secretion without affecting the endoplas- mic reticulum/Golgi pathway. Identification of PRAS40 as a linker protein paves the way for understanding how stress regulates exosome secretion under pathophysio- logical conditions. KEYWORDS