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 intracellular 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-α) and epidermal growth factor (EGF) share the same EGF receptor and previously indistinguishable intracellular signaling networks, only TGF-α stimulation causes exosome-mediated secretion. However, deduction of EGF-activated pathways from TGFα-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-α but not EGF signaling via threonine 308-phosphorylated Akt. PRAS40 knockdown (KD) or PRAS40 dominant-negative (DN) mutant overexpression blocks not only TGF-α- but also hypoxia- and H₂O₂-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 endoplasmic reticulum/Golgi pathway. Identification of PRAS40 as a linker protein paves the way for understanding how stress regulates exosome secretion under pathophysiological conditions.

KEYWORDS:

• stress
• growth factor
• signal transduction
• Akt
• HIF-1
• HSP
• exosome

ACKNOWLEDGMENTS

We have no commercial conflicts of interest to declare.

This work was supported by NIH grants GM066193 and GM067100 (to W.L.), AR46538 (to D.T.W.), AR33625 (M.C. and D.T.W.), and a VA Merit Award (to D.T.W.).

J.G. performed screening, cloning, and most of the key experiments. P.J. performed cloning of shRNAs into lentiviral vectors and rescue experiments by overexpressing PRAS40 and its mutants and produced recombinant Hsp90 proteins. J.D. helped J.G. through the early phase of the experiments. G.-B.J. and C.L. helped with confocal analysis. M.C. and D.T.W. designed the lentiviral constructs and virus production. P.W. and M.F. assisted with the isolation and analysis of exosomes. W.L. supervised the entire project.