Figure 1.
Phafin2 associates with Akt in mammalian cells.
A. Flag-Phafin2 interacted only with HA-Akt, but not with HA-PDK1 or HA-PrKA. Similar levels of expression of HA-Akt, PDK1, PrKA, and Flag-Phafin2 were shown by immunoblot (HA = anti-HA antibody; F = anti-Flag antibody; C = Control antibody). B. Flag-tagged Phafin2 interacted with HA-tagged Akt1 (lane 1–3) and Akt2 (lane 4–6), but not with Akt3 (lane 7–9). Similar levels of three Akt isoforms and Phafin2 were expressed (lower panels). C. Endogenous Akt1 and Akt2 interacted with Phafin2 in HT1080 cells by co-immunoprecipitation assays. Expression of Akt isoforms and Phafin2 were shown (lower panels). D. The C-terminal Akt kinase domain is the binding domain for Phafin2 interaction in co-immunoprecipitation assays. Expression of the Flag-Phafin2 and HA-Akt subfragments were shown (lower panels). E. Structural features of Phafin2 used in this study are shown. Phafin2 consisted of N-terminal PH domain and C-terminal FYVE domain. Both PH domain and FYVE domain interacted with Akt in co-immunoprecipitation assays. Expression of Akt and Phafin2 subfragments were shown (lower panels). F. Interaction between recombinant Phafin2 and Akt was verified in GST pull-down assays. Recombinant active (a) or unactive (un) Akt was incubated with GST-Phafin2 beads and subsequently resolved onto SDS-PAGE and detected by immunoblotting using anti-Akt antibody (right panel). Levels of phosphorylation of active Akt (a) and unactive Akt (un) were shown (lower panels).
Figure 2.
Phafin2 displays accumulation at the lysosome with Akt after induction of autophagy.
A–B. HT1080 cells were cultured in DMEM supplemented with 10% FBS (A, non-treated) or HBSS (B), and sucrose gradient subcellular fractionation was conducted and resolved onto SDS-PAGE and immunoblotted by indicated antibodies. Acid phosphatase activity of each fraction was measured by optical absorption using Acid Phosphatase Assay kit (Sigma) and TEM examination of the crude fraction of lysosome before and after HBSS treatment to induce autophagy was also shown underneath. C–D. Cells were cultured in complete media (non-treated, left panels), treated with rapamycin (middle panels) or HBSS (right panels) to induce autophagy. HeLa cells were fixed and immunostained by anti-LAMP2 (green) along with anti-Phafin2 (red) antibodies (C). J774.1 cells were immunostained by anti-Akt (green) along with anti-Phafin2 (red) antibodies (D). Colocalized areas in the merged view were indicated by white arrow heads. The quantification of the colocalized area of Phafin2 with lysosome (C) or Akt with Phafin2 (D) per cell (%) was shown as a bar graph with statistical analysis by Mann Whitney U-test (right side). White scale bar represents 10 µm. The results were consistent between three independent experiments. E–K. Using BiFC, localization of Akt and Phafin2 with lysosome was examined (E). pCAGGS-VN-Phafin2 alone (F) or both pCAGGS-VN-Phafin2 and pCAGGS-VC-Akt2 (G–K) were transfected into HeLa cells and cultured in normal condition (G) or treated with rapamycin (H) and subsequent removal of rapamycin (I). For HBSS treated cells, the culture media was replaced with HBSS for 4 hours (J). HBSS were subsequently replaced with complete media (DMEM) with 10% FBS (K). The cells were fixed and immunostained with anti-LAMP2 antibody and visualized by confocal microscopy. Fluorescence intensities of BiFC (green) and AlexaFluor594 (red) along the line (a–b) were plotted underneath. White scale bar represents 10 µm.
Figure 3.
The interaction of the PtdIns(3)P with Phafin2 determines the lysosomal localization.
A. PIP strips were incubated with GST-Phafin2 WT, PH, or FYVE and the binding ability was examined by GST immunoblot. A comparable amount of recombinant wild type, N-term (PH domain), or C-term (FYVE domain) of Phafin2 was used (left panel). B. PtdIns(3)P interacting defective-Phafin2 (R53C, R171A, and R172A) failed to bind to PtdIns(3)P on PIP strip (right panel). C. BiFC analysis, 3-MA or wortmannin treatment abrogated the accumulation of the Akt-Phafin2 protein complexes at perinuclear lysosome after rapamycin treatment. D. Mutant Phafin2 displayed no accumulation of perinuclear lysosome after rapamycin treatment (right panels). E–F. Mutant Phafin2 failed to induce autophagy determined by decreased intensity of LC3-II band (E, lane 6). The percentage of autophagy inhibition was 29.3±7.50% out of three independent experiments. The observation was consistent, as determined by the absence of GFP-LC3 puncta using confocal microscopy (F, lower right side panels). Note that ectopic expression of wild type Phafin2 modestly enhanced autophagy induction determined by LC3-II blot (E, lane4 upper panel) and LC3 puncta (F. lower panels). White scale bar represents 10 µm.
Figure 4.
Presence of both Akt and Phafin2 are required for induction of autophagy.
A–D. Phafin2-siRNA transfected macrophages showed no inhibition on initial uptake of fluorescent bacteria (A–B, top panels). However, after HBSS treatment to induce autophagy, Phafin2-siRNAs transfected cells inhibited not only elimination of fluorescent bacteria (A–B, middle panels), but also induction of autophagy (A–B, bottom panels), which is reversible by re-introduction of human Phafin2 (C–D, bottom panels). Note that human Phafin2 is resistant for mouse Phafin2-siRNA. Quantification of LC3 puncta per cell with statistical analysis by Student's t-test is shown on the right side. White scale bar represents 10 µm. E–H. Using J774.1 murine macrophages, LPS-induced lysosomal accumulation of Akt was eliminated by Phafin2-siRNAs (E–F), which is associated with inhibition of autophagy (I–J, lower panels). The observations are reversible by re-introduction of human Phafin2 (G–H and K–L, lower panels). Quantification of the percent of colocalization area of Akt with LAMP2 with statistical analysis by Student's t-test is shown on the right side. I–L. Phafin2-siRNA transfected macrophages inhibited LPS-induced autophagy determined by LC3 pancta with Phafin2 expression shown in inset (bottom panels, compare I and J). Inhibition of autophagy by Phafin2-siRNAs can be reverted by re-introduction of human Phafin2, which is resistant for mouse Phafin2-siRNA (bottom panels, compare K and L). Quantification of LC3 puncta per cell with statistical analysis by Student's t-test is shown on the right side. M. LC3 immunoblot by the introduction of Phafin2-siRNA were shown. The percentage of autophagy inhibition out of three independent experiments was 53.3±21.4%. N–Q. Akt-siRNA (fig. S5) transfected macrophages retained LPS-induced lysosomal translocation/accumulation of Phafin2 (upper panels). Akt-siRNA, however, inhibited LPS-induced autophagy determined by LC3 pancta with Akt expression shown in inset (bottom panels, compare O and Q). Number of GFP-LC3 puncta per cells with statistical analysis by Student's t-test were shown as a bar graph. R–U. Akt-siRNA (fig. S5) transfected HeLa cells failed to induce autophagy determined by LC3 puncta on Ds-Red positive cells (inset). Further, inhibition of autophagy by Akt-siRNAs can be reversed by re-introduction of siRNA-resistant human Akt2 in HeLa cells (U). Number of GFP-LC3 puncta per cells with statistical analysis by Student's t-test were shown as a bar graph. V. LC3 immunoblot by the introduction of Akt siRNA were shown. The percentage of autophagy inhibition (shown in the bar graph) was 45.4±11.2% out of three independent experiments.
Figure 5.
Lysosomal interaction of Akt with Phafin2 is a critical step to induce autophagy.
PI3K-Akt pathway that mediates anti-apoptotic signal is suggested to play an important role in the regulation of autophagy in mammalian cells. However, molecular mechanisms by which Akt signal regulates autophagy are largely unknown. In this study, we demonstrated that the presence of both Akt and Phafin2 on the lysosome is critical in the induction of autophagy via interaction of lysosomal PtdIns(3)P in mammalian cells. Akt-Phafin2 functional interaction not only clarifies the molecular basis of the PI3K-Akt signaling pathway in the regulation of autophagy, but also shows how 3-MA (3-methyladenine), a widely used autophagy inhibitor, inhibits autophagy in mammalian cells at molecular levels. It has been suggested that Akt activation prevents induction of autophagy, however, the roles of Akt in the regulation of autophagy induction is not clear. Hence, the current studies add a new twist to the molecular regulation of autophagy via PI3K-Akt signaling pathways in mammalian cells.