An inhibitory role of the G-protein regulator AGS3 in mTOR-dependent macroautophagy

Abstract

Macroautophagy is a cellular process whereby the cell sequesters and recycles cytosolic constituents in a lysosome-dependent manner. It has also been implicated in a number of disorders, including cancer and neurodegeneration. Although a previous report that AGS3 over-expression promotes macroautophagy suggests a stimulatory role of AGS3 in this process, we have found that knock-down of AGS3, unexpectedly, also induces macroautophagy, indicating an inhibitory function of endogenous AGS3 in macroautophagy. Interestingly, AGS3 phosphorylation is decreased upon induction of mammalian target of rapamycin (mTOR)-dependent macroautophagy. Moreover, unlike wild-type AGS3, over-expression of an AGS3 mutant lacking this modification fails to enhance macroautophagic activity. These observations imply that AGS3 phosphorylation may participate in the modulation of macroautophagy.

Figure 1. Both knock-down and over-expression of AGS3 lead to an increased LC3II level in HEK293 and HeLa cells.

Cells transfected with DNA (0.4 µg/ml) or siRNA (20 nM) were lysed 24 or 48 hrs after transfection, repectively, unless otherwise noted. β-actin was used as a loading control. (A) The effect of rapamycin (100 nM, 4 hrs) on the level of LC3II in HEK293 cells. (B) The impact of AGS3 over-expression on LC3II in HEK293 cells. Cells transfected with an empty vector (pcDNA3) were used as a negative control. (C) The influence of AGS3 knock-down on LC3II in HEK293 cells. Cells transfected with a non-targeting siRNA were used as a negative control. (D) The influence of AGS3 knock-down on LC3II in HeLa cells. Western blot LC3II band intensities are normalized to that of the control siRNA treated cells (arbitrarily set as 1X). (E) HEK293 cells were treated with either a non-targeting control siRNA or AGS3 siRNA1 and either starved (2 hrs) or not. (F) GFP-LC3 stable HEK293 cells were treated with either a non-targeting control siRNA or AGS3 siRNA1 and endogenous LC3 was assayed by western blot. Western blot band intensities are normalized to that of the control siRNA treated cells (arbitrarily set as 1X). (G) HEK293 cells stably expressing GFP-LC3 were treated with AGS3 siRNA1 (50 nM, 72 hrs) and either starved (2 hrs) or not; a higher AGS3 knock-down efficiency was obtained for the HEK293 GFP-LC3 stable cells when using a higher siRNA concentration and a longer incubation time. This could be due to differences in cell origin. The number of GFP positive puncta structures were quantified as described in the “Materials and Methods”. For the sake of clarity normalized GFP-LC3II counts/cell (n = 6, approximately 10,000 cells/condition) are shown. Two statistical tests (ANOVA and single-sample Student's t-test) performed on LOG transformed data revealed that the AGS3 effect was significant across all experimental conditions (p<0.05 for Starved, p<0.01 for all others). The images on the left side are representative full size acquired images, the inset to the right shows a close-up of one cell with indication of detected GPF puncta (red circles). The green dotted lines enclose the cell nuclei. Representative blots and images are shown (AGS3 was detected using a rabbit polyclonal antibody characterized below). Data presented in bar graphs is the average result from at least three independent experiments except in (G). Error bars: standard error of the mean. Asterisks - * = p<0.05, ** = p<0.01, paired Student's t-test.

Figure 2. The effect of AGS3 depletion on LC3II persists when the fusion between autophagosomes and lysosomes is inhibited in HEK293.

siRNA-mediated knock-down of AGS3 was performed as described in Figure 1. (A) The impact of AGS3 knock-down on the LC3II level in the presence of bafilomycin A1. Cells transfected with a non-targeting control siRNA or AGS3 siRNA1, were treated with either DMSO alone (as a negative control) or bafilomycin A1 (100 nM, 2 hrs). (B) The influence of AGS3 suppression on the number of GFP-LC3II puncta in HEK293 stable cells treated with bafilomycin A1 (100 nM, 2 hrs). Cells stably expressing GFP-LC3 were first transfected with a non-targeting control siRNA or AGS3 siRNA1 and the GFP-LC3II puncta was quantified and the statistical analysis performed as described in Figure 1; in both conditions the difference between control siRNA and AGS3 siRNA1 samples is statistically significant (p<0.01). Normalized GFP-LC3II counts/cell (n = 4, approximately 10,000 cells/condition) are shown. (C) The effect of AGS3 depletion on the mRNA level of LC3. HEK293 cells were treated with a non-targeting control siRNA or AGS3 siRNA1; total RNA was collected, reverse-transcribed and analyzed by real-time PCR. Results were normalized to four “house-keeping” gene products. (D) The LC3I levels in HEK293 cells treated with either a non-targeting control siRNA or AGS3 siRNA1 determined by western blot. Data presented in all bar graphs is the average result from three (A, C & D) or four (B) independent experiments. The values of untreated control and knock-down cells presented in (B) are provided for the sake of comparison and are the same as those that appear in Figure 1G. Error bars: standard error of the mean. Asterisks - * = p<0.05, ** = p<0.01, paired Student's t-test. Representative blots are shown. Similar results as those presented in 2A were obtained in HeLa cells (data not shown).

Figure 3. A rabbit polyclonal antibody detects an AGS3 post-translational modification in HEK293 and HeLa cells.

DNA and siRNA transfections were performed as described in Figure 1. (A) In HeLa cells, exogenously over-expressed untagged-AGS3 as well as endogenous AGS3 exhibited three differently migrating bands (indicated by asterisks). (B) Characterization of the putative AGS3 bands using lysates collected from HeLa cells transfected with AGS3 siRNAs. The presence of three differently migrating bands which are sensitive to the AGS3 siRNAs is also evident in HEK293 cells (Figures 1C, 1E & 2A). (C) Analysis of the band pattern of an HA-tagged AGS3 expressed in HeLa cells. (D) Comparison of the mobility between recombinant AGS3 expressed in E. coli and endogenous AGS3 from HeLa cell lysate.

Figure 4. Characterization of the slowest-migrating species of AGS3 and its sensitivity to mTOR-dependent autophagy.

DNA transfections were performed as described in Figure 1. (A) Western blot analysis of AGS3 immunoprecipitated from HeLa cell lysates in the presence or absence of Antarctic phophatase. (B) Western blot analysis of a truncated AGS3 construct (HA-AGS3TPR; a.a. 1–472) consisting of the N-terminal TPR domains immunoprecipitated from HeLa lysates. The lower light band likely represents a non-specific signal as it appears in both lanes. (C) Western blot analysis of the phosphatase treatment of a truncated AGS3 construct (HA-AGS3GPR; a.a. 461–650) consisting of the C-terminal GPR domains immunoprecipitated from HeLa lysates. (D) The influence of mTOR-dependent and –independent autophagic inductions on the triplet of AGS3. Autophagy was induced in HeLa cells by inhibiting mTOR activity, via either rapamycin (100nM, 4 hrs) or starvation (2 hrs), or by an mTOR-independent pathway via LiCl (10 mM, 24 hrs).

Figure 5. Over-expression of the AGS3 phospho-mutant does not induce autophagy in HEK293 HeLa or COS7 cells.

DNA transfections were performed as described in Figure 1. (A) Western blot analysis of the AGS3 GPR phospho-mutant (all Ser and Thr within last the C-terminal 183 a.a. mutated to Ala) expressed in HEK293 cells. (B) Co-immunoprecipitation of Gαi2/Gαi3 with either wild-type AGS3 or the AGS3 phospho-mutant from HEK293. (C) Assessment of the ability of AGS3 GPR phospho-mutant to induce autophagy in HEK293 cells. HEK293 cells transfected with the vector alone, AGS3 or AGS3 GPR phosphor-mutant were lysed and LC3II levels was assayed. (D) Assessment of the ability of AGS3 GPR phospho-mutant to induce autophagy in COS7 and HeLa cells. Representative blots are shown. Data presented in the bar graph in (B) are the average results from at least three independent experiments. Error bars: standard error of the mean. Asterisks = p<0.01, paired Student's t-test. Western blot band intensities in (D) are shown as normalized to that of vector (i.e. pcDNA3) transfected cells (arbitrarily set as 1X).