Lysosomal Interaction of Akt with Phafin2: A Critical Step in the Induction of Autophagy

Autophagyis an evolutionarily conserved mechanism for the gross disposal of intracellular proteins in mammalian cells and dysfunction in this pathway has been associated with human disease. Although the serine threonine kinase Akt is suggested to play a role in this process, little is known about the molecular mechanisms by which Akt induces autophagy. Using a yeast two-hybrid screen, Phafin2 (EAPF or PLEKHF2), a lysosomalprotein with a unique structure of N-terminal PH ( pleckstrin homology) domainand C-terminal FYVE (Fab 1, YOTB, Vac 1, and EEA1) domain was found to interact with Akt. A sucrose gradient fractionation experiment revealed that both Akt and Phafin2 co-existed in the same lysosomeenriched fraction after autophagyinduction. Confocal microscopic analysis and BiFC analysis demonstrated that both Akt and Phafin2 accumulate in the lysosomeafter induction of autophagy. BiFC analysis using PtdIns (3)P interaction defective mutant of Phafin2 demonstrated that lysosomalaccumulation of the Akt-Phafin2 complex and subsequent induction of autophagywere lysosomalPtdIns (3)P dependent events. Furthermore, in murine macrophages, both Akt and Phafin2 were required for digestion of fluorescent bacteria and/or LPS-induced autophagy. Taken together, these findings establish that lysosomalaccumulation of Akt and Phafin2 is a critical step in the induction of autophagyvia an interaction with PtdIns (3)P.