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

Abstract

Autophagy is 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 lysosomal protein with a unique structure of N-terminal PH (pleckstrin homology) domain and 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 lysosome enriched fraction after autophagy induction. Confocal microscopic analysis and BiFC analysis demonstrated that both Akt and Phafin2 accumulate in the lysosome after induction of autophagy. BiFC analysis using PtdIns (3)P interaction defective mutant of Phafin2 demonstrated that lysosomal accumulation of the Akt-Phafin2 complex and subsequent induction of autophagy were lysosomal PtdIns (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 lysosomal accumulation of Akt and Phafin2 is a critical step in the induction of autophagy via an interaction with PtdIns (3)P.

Phosphorylation‐dependent Akt–Inversin interaction at the basal body of primary cilia

“…Both ciliary length (Fig 5E and Appendix Fig S3E) and ciliogenesis (Appendix Fig S3D) were suppressed by Akt knockdown. Furthermore, reintroduction of siRNA‐resistant Akt (Matsuda‐Lennikov et al , 2014) rescued these effects on ciliary development (Fig 5E and Appendix Fig S3D). Consistent with these observations, primary cilia development was inhibited in Akt‐null MEFs that lacked Akt1, Akt2, and Akt1/2 (Appendix Fig S3F).…”

“…Statistical significance was analyzed by Student's t ‐test. Note that reintroduction of siRNA‐resistant Akt (Matsuda‐Lennikov et al , 2014) in Akt knockdown cells rescued the effect on ciliary length.…”

“…Statistical significance was analyzed by Student's t ‐test. Note that reintroduction of siRNA‐resistant Akt (Matsuda‐Lennikov et al , 2014) in Akt knockdown cells rescued the effect on ciliary length.Expression of 3A INVS (bottom panels) but not WT INVS (middle panels) inhibited the development of primary cilia as compared to control cells (top panels).Quantification of ciliary length. Results are mean ± SE of longitudinal length of acetylated tubulin (blue), a marker of primary cilia, in EGFP‐INVS (green) transfected in hTERT‐RPE1 cells ( n  = 58 for EGFP vector and for EGFP‐INVS WT, and n  = 52 for EGFP‐INVS 3A mutant).…”

Structural, Thermodynamic and Phosphatidylinositol 3-Phosphate Binding Properties of Phafin2

“…2 PtdIns(3)P is a hallmark of endosomes as it mediates the recruitment of effector proteins to these compartments; thus, both PtdIns(3)P-interacting domains can target Phafin2 to PtdIns(3)P-enriched membranes. Phafin2 regulates the function and structure of endosomes through a Rab5-dependent process 3 although no direct interaction of Phafin2 and Rab5 occurs.…”

“…Phafin2-mediated autophagy requires the presence of PtdIns(3)P at the lysosomal surface and the serine/threonine kinase Akt. 2 Binding of Phafin2 to Akt requires both the PH and FYVE domains. The interaction of Phafin2 with PtdIns(3)P allows localization of the Phafin2-Akt complex to the lysosomal surface.…”