Figure 1.
Expression levels of PTEN in various clones of U87MG cells.
U87MG human glioma cells, mock-treated or expressing WT-PTEN or two of its mutants (G129E-PTEN and C124S-PTEN) under the control of a tetracycline-inducible system, were incubated in full DMEM and treated with doxycycline as described in Materials and Methods. After 18 h, expression of PTEN was analyzed by Western blot in whole lysates using anti-PTEN and, as a loading control, anti-actin antibodies. Molecular weight markers are indicated on the right. PTEN and actin bands were quantified by densitometry and their ratios are shown in the histogram below. Results are means ± SD of three independent experiments. No significant differences were found among the cells that express WT-PTEN or its two mutants.
Figure 2.
Decreased levels of ubiquitinated proteins in U87MG cells that express PTEN.
The same U87MG cells from Figure 1 were incubated for 3 h in KH without (A) or with (B) amino acids and insulin. Where indicated, the cells were treated with the proteasome inhibitor MG132 (50 µM). Extracts (75 µg protein) of the cells were subjected to SDS-PAGE and analyzed by Western blot using two different antibodies that recognize ubiquitinated proteins (FK1 and FK2) and, as a loading control, an antibody that recognizes actin. The amount of ubiquitinated proteins (ubiq. prot.) was quantified by densitometry and normalized to the levels of actin. Results, which are means ± SD of three independent experiments, are shown below the representative Western blots. Differences from the corresponding values (with or without MG132) in mock-treated cells were found to be significant at **p<0.01 and ***p<0.005, respectively.
Figure 3.
Decreased chymotrypsin-like activity of proteasomes in U87MG cells that express PTEN.
The same cells from Figure 2 were cultured in KH without (A) or with (B) amino acids and insulin. The chymotrypsin-like peptidase activity of proteasomes was determined using the fluorogenic substrate N-Suc-LLVY-AMC as described in Materials and Methods. As a control, the specific proteasome inhibitor lactacystin (40 µM) was added and the activity of proteasomes was determined as the difference obtained in the absence and in the presence of lactacystin, which was about 12-15% of the activity measured without the inhibitor. Results are expressed in percentage relative to the fluorescence values of the mock-treated cells at 3.5 h and are means ± SD of five independent experiments, each of them run in duplicate.
Figure 4.
PTEN expression in U87MG cells increases the formation of autophagosomes.
A) U87MG cells expressing WT-PTEN, C124S-PTEN and G129E-PTEN or mock-treated were incubated in high (H) or low (L) proteolysis media (see Materials and Methods) for 2 h in the presence of lysosomal inhibitors (100 µM leupeptin and 20 mM NH4Cl). Extracts (75 µg protein) were analyzed by SDS-PAGE and immunoblot, with low and high exposure (exp.), using an antibody against LC3 and, as a loading control, an antibody that recognizes actin. A representative experiment is shown. The position of LC3-I and LC3-II bands are indicated on the left and molecular weight markers are indicated on the right. The histogram on the right shows the means ± SD of the densitometric analysis of the LC3-II/actin ratios from five different experiments. Stars indicate statistically significant differences from the corresponding (high or low proteolysis conditions) values in mock-treated cells at **p<0.01 and ***p<0.005. B) Representative fluorescent images of the indicated cell lines transitorily expressing EGFP-LC3 (at 48 h post-transfection) and incubated under high proteolysis conditions for 2 h. Quantification of autophagy by counting the number of EGFP-LC3 dots in the transfected cells (see Materials and Methods) is shown on the right. Stars indicate statistically significant differences from mock-treated cells at ***p<0.005. Bar: 20 µm.
Figure 5.
PTEN expression in U87MG cells increases the lysosomal mass.
A) Representative fluorescence images of cells expressing WT-PTEN, C124S-PTEN and G129E-PTEN and of the mock-treated U87MG cells (control) incubated under high proteolysis conditions for 2 h and with LysoTracker Red (75 nM) during the last 15 min. Fluorescence is higher in cells expressing WT-PTEN and G129E-PTEN. Bar: 20 μm. B) Representative electron micrographs of the cells incubated for 2 h under high and low proteolysis conditions. Cells that express WT-PTEN and the G129E-PTEN mutant show more autophagic vacuoles (arrows) compared to the mock-treated cells or the cells expressing the C124S-PTEN mutant. Bar: 0.6 μm. C) Cells were incubated as in A) but under high (KH), intermediate (KH plus insulin, Ins, or amino acids, EAA) and low (KH plus insulin and amino acids, Ins/EAA) proteolysis conditions (see Materials and Methods). LysoTracker Red fluorescence in the cells was analyzed by flow cytometry as described in Materials and Methods. Relative fluorescence units (RFU) are expressed in percentage of the values obtained in the mock-treated U87MG cells incubated under high proteolysis conditions (KH). Results are the mean ± SD from seven independent experiments with duplicate samples. Stars indicate statistically significant differences from the corresponding (high, intermediate and low proteolysis conditions) values in mock-treated cells at **p<0.01 and ***p<0.005.
Figure 6.
PTEN expression in U87MG cells increases the formation of autophagosomes under various proteolysis conditions.
U87MG cells expressing WT-PTEN, G129E-PTEN or C124S-PTEN (A) and mock-treated U87MG cells or expressing WT-PTEN or C124S-PTEN (B) were incubated under high (KH), intermediate (Ins or EAA)(A and B) and low (Ins/EAA)(only in A) proteolysis conditions for 2 h as in Figure 5C in the presence of lysosomal inhibitors (100 µM leupeptin and 20 mM NH4Cl). Extracts (75 µg protein) were analyzed by SDS-PAGE and immunoblot with antibodies that recognize LC3 and, as a loading control, actin. The position of LC3-I and LC3-II bands are indicated on the left and molecular weight markers are indicated on the right. The Western blots on the left show representative experiments and the histograms on the right show the means ± SD of the densitometric analysis of the LC3-II/ratios from five different experiments. Stars indicate statistically significant differences from the corresponding (high, intermediate or low proteolysis conditions) values in the cells expressing the C124S PTEN mutant (A) and mock-treated cells (B) at **p<0.01 and ***p<0.005.
Figure 7.
Levels of various components of the ubiquitin-proteasome system in U87MG cells expressing PTEN.
U87MG cells expressing WT-PTEN, C124S-PTEN or G129E-PTEN and mock-treated (control) were incubated under high (H) or low (L) proteolysis conditions (see Materials and Methods) for 2 h. Total cell extracts (75 µg protein in A and C and 50 µg protein in B) were separated in SDS-PAGE and the gels were immunoblotted with antibodies raised against the ubiquitin activating enzyme E1 (A), K63-linked polyubiquitin chains (B) and the following proteasome subunits as indicated: α7 and β4 (20S proteasome), β5i (immunoproteasome), S8 (19S regulatory particle) and PA28α (11S regulatory particle)(C). Actin was also detected as a loading control. The figure shows representative gels from three different experiments. Relative levels of the bands were calculated by densitometric analysis and are only shown for K63-linked polyubiquitin chains (B) and α7 (C). Stars indicate statistically significant differences from mock-treated cells at *p<0.05 (in B). No significant differences were found in the other analyses.
Figure 8.
PTEN expression in U87MG cells affects the PI3K class I/AKT/mTOR signaling pathway.
U87MG cells expressing WT-PTEN, C124S-PTEN or G129E-PTEN and mock-treated were incubated in high (H) and low (L) proteolysis media for 2 h. Total lysates were analyzed by Western blot using phospho-specific antibodies that recognize AKT-Thr308 (P-AKT-T) and AKT-Ser473 (P-AKT-S), p70S6K and 4-EBP1 and their respective pan-antibodies. The bands corresponding to the phosphorylated proteins, obtained from three different experiments, were densitometred and normalized to the corresponding total protein in the same sample. Results are shown on the histograms below the representative Western blots. Data are presented as percentage of the values in mock-treated (control) cells incubated in low proteolysis medium (L) and are means ± SD of three different experiments. Stars indicate statistically significant differences from the respective control values under high and low proteolysis conditions at *p<0.05 and ***p<0.005.
Figure 9.
Inhibition of the ubiquitin-proteasome pathway and activation of autophagy by PTEN in U87MG cells are independent of mTOR.
U87MG cells expressing WT-PTEN or C124S-PTEN (A) or WT-PTEN or G129E-PTEN and mock-treated (B) were incubated for 18 h with doxycycline and in the last 2 h the following inhibitors were added as indicated: rapamycin (RAP, 200 mM, mTOR inhibitor), KT5720 (25 µM, PKA inhibitor), PD98059 (10 µM, ERK1/2 inhibitor) and SB203580 (10 µM, p38 inhibitor). Before collecting the cells, proteasome (50 µM MG132, A) and lysosomal (100 µM leupeptin and 20 mM NH4Cl, B) inhibitors were also added for 1 h. Total lysates were analyzed by SDS-PAGE and Western blot with antibodies that recognize ubiquitinated proteins (FK1) (A), LC3 (B) and, as a loading control actin. Molecular weight markers are indicated on the right and in B the position of LC3-I, and LC3-II bands are also shown. The histograms on the right show means ± SD of the densitometric measurements from three different experiments and are expressed as amounts of ubiquitinated proteins (ubiq. prot.) normalized to the levels of actin (A) or as LC3II/actin ratios (B). Stars indicate statistically significant differences from the values without the corresponding inhibitor treatment at **p<0.01 and ***p<0.005.
Figure 10.
Lipid phosphatase-dependent and -independent roles of PTEN in the regulation of autophagy.
In a simplified scheme, insulin signalling to mTOR complex 1 (mTORC1) is mediated through phosphorylation of the insulin receptor 1 or 2 (IRS 1/2), subsequent activation of PI3K class I and protein kinase B/AKT, which either directly, or indirectly via inhibition of the tuberous sclerosis complex 1 and 2 (TSC1/TSC2) and activation of the small GTPase RHEB, activates mTORC1 (an inhibitor of autophagy). Adenosine 5-monophosphate-activated protein kinase (AMPK) can inhibit mTOR indirectly after activation of TSC1/TSC2 by phosphorylating residues different from those phosphorylated by AKT. PTEN, through its lipid phosphatase activity inhibits the generation of phosphatydilinositol (3,4,5)-trisphosphate (PIP3) from phosphatydilinositol (3,4)-bisphosphate (PIP2) caused by the above mentioned activation of class I PI3K. This leads to the inhibition of AKT/PKB signalling, which activates the TSC1/2 complex, restrains mTORC1 and consequently induces autophagy. By contrast, in U87MG cells, expression of PTEN under the control of a tetracycline-inducible system activates autophagy and inhibits the ubiquitin-proteasome system (UPS) through a lipid phosphatase-independent activity and in an mTOR-independent way. ERK1/2 and PKA may be involved in this signaling pathway. In contrast to autophagy, much less is known on the regulation of the ubiquitin-proteasome system. Both catabolic pathways are closely interrelated (indicated by the double headed arrow interconnecting them) and, therefore, it could be also possible that PTEN directly affects only one of them.