Abstract
Two major mechanisms of intracellular protein degradation, autophagy and the ubiquitin-proteasome pathway, operate in mammalian cells. PTEN, which is frequently mutated in glioblastomas, is a tumor suppressor gene that encodes a dual specificity phosphatase that antagonizes the phosphatidylinositol 3-kinase class I/AKT/mTOR pathway, which is a key regulator of autophagy. Here, we investigated in U87MG human glioma cells the role of PTEN in the regulation of autophagy and the ubiquitin-proteasome pathway, because both are functionally linked and are relevant in cancer progression. Since U87MG glioma cells lack a functional PTEN, we used stable clones that express, under the control of a tetracycline-inducible system (Tet-on), wild-type PTEN and two of its mutants, G129E-PTEN and C124S-PTEN, which, respectively, lack the lipid phosphatase activity only and both the lipid and the protein phosphatase activities of this protein. Expression of PTEN in U87MG glioma cells decreased proteasome activity and also reduced protein ubiquitination. On the contrary, expression of PTEN increased the autophagic flux and the lysosomal mass. Interestingly, and although PTEN negatively regulates the phosphatidylinositol 3-kinase class I/AKT/mTOR signaling pathway by its lipid phosphatase activity, both effects in U87MG cells were independent of this activity. These results suggest a new mTOR-independent signaling pathway by which PTEN can regulate in opposite directions the main mechanisms of intracellular protein degradation.
Highlights
Macroautophagy, hereafter called autophagy, and the ubiquitin-proteasome system (UPS) are the main pathways of intracellular protein degradation in mammalian cells [1]
We have examined here the possible regulation of autophagy and the UPS by the two purported phosphatase activities of PTEN under high and low proteolysis conditions in U87MG human glioma cells that lack a functional PTEN. We report that both intracellular protein degradation systems become affected by the conditional expression of PTEN in opposite directions and that, surprisingly, in U87MG human glioma cells these effects, including the activation of autophagy, are mainly independent of the lipid phosphatase activity of PTEN
PTEN is not expressed in U87MG glioma cells and ectopic expression of wild type PTEN (WT-PTEN) in these cells results in cell death, as it has been described in other cancer cell lines [23]
Summary
Macroautophagy, hereafter called autophagy, and the ubiquitin-proteasome system (UPS) are the main pathways of intracellular protein degradation in mammalian cells [1]. Autophagy is essentially responsible for the degradation under starvation conditions of long-lived proteins, cytoplasmic organelles and other cell components. The initial step is the formation of a double membrane structure, called the phagophore, which surrounds cytoplasmic material and closes to form a double membrane autophagosome. In contrast to autophagy, which is less selective and more adapted to cell survival under starvation, the UPS is highly selective and mainly involved in the prompt degradation of abnormal, improperly assembled and other short-lived proteins, controlling both protein quantity and quality and playing a crucial role in the survival of eukaryotic cells under basal conditions [4]. Various ubiquitin molecules are attached to a protein substrate through specific ligases (E3) in concert with other enzymes (E1 and E2) and, the polyubiquitinated protein is deubiquitinated to release reusable ubiquitin molecules and the remaining protein is degraded by the 26S/30S proteasome [5,6]
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