Suppression of cellular proliferation and invasion by the concerted lipid and protein phosphatase activities of PTEN

Abstract

PTEN is a tumour suppressor with phosphatase activity in vitro against both lipids and proteins and other potential non-enzymatic mechanisms of action. Although the importance of PTEN’s lipid phosphatase activity in regulating the PI3K signalling pathway is recognised, the significance of PTEN’s other mechanisms of action is currently unclear. Here, we describe the systematic identification of a PTEN mutant, PTEN Y138L, with activity against lipid, but not soluble substrates. Using this mutant we provide evidence for the interfacial activation of PTEN against lipid substrates. We also show that when re-expressed at physiological levels in PTEN null U87MG glioblastoma cells the protein phosphatase activity of PTEN is not required to regulate cellular PtdInsP3 levels or the downstream protein kinase Akt/PKB. Finally, in 3D Matrigel cultures of U87MG cells similarly re-expressing PTEN mutants, both the protein and lipid phosphatase activities were required to inhibit invasion, but either activity alone significantly inhibited proliferation, albeit only weakly for the protein phosphatase activity. Our data provides a novel tool to address the significance of PTEN’s separable lipid and protein phosphatase activities and suggest that both activities act to suppress proliferation and act together to suppress invasion.

Disruption of epithelial architecture caused by loss of PTEN or by oncogenic mutant p110α/PIK3CA but not by HER2 or mutant AKT1

“…To further investigate the mechanisms by which PTEN affects epithelial morphology, we used a series of well characterised functionally selective PTEN mutants that we have developed in our laboratory. PTEN C124S lacks all phosphatase activity and two further mutants selectively lack either lipid (PTEN G129E) or protein (PTEN Y138L) phosphatase activity [37,38]. PTEN C124S and PTEN G129E do not affect the PtdInsP 3 -dependent AKT kinases, whereas PTEN Y138L appears to suppress the phosphorylation and activation of AKT as efficiently as wild-type PTEN [37][38][39].…”

“…PTEN C124S lacks all phosphatase activity and two further mutants selectively lack either lipid (PTEN G129E) or protein (PTEN Y138L) phosphatase activity [37,38]. PTEN C124S and PTEN G129E do not affect the PtdInsP 3 -dependent AKT kinases, whereas PTEN Y138L appears to suppress the phosphorylation and activation of AKT as efficiently as wild-type PTEN [37][38][39]. In order to test the function of different PTEN mutants in NMuMG cells we made silent mutations in the PTEN cDNA making it resistant to our PTEN-targeting shRNA mediated knockdown.…”

“…All cells were grown at 37°C, 5%CO 2 . HEK293T cells were cultured and lentiviral particles prepared as previously described [37]. NMuMG, MDCK and MDA-MB-468 cells were transduced with lentiviruses in media supplemented with 16 g/ml of polybrene (hexadimethrine bromide, Sigma).…”

“…Cell colonies with one distinct central hollow lumen were scored blind using a phase contrast microscope to allow rapid focusing through each colony. Proliferation of matrigel cultures was performed using a Lactate Dehydrogenase (LDH) assay kit purchased from Roche as previously described [37].…”

PTEN Protein Phosphatase Activity Correlates with Control of Gene Expression and Invasion, a Tumor-Suppressing Phenotype, But Not with AKT Activity

“…We used this mutant to show that both lipid and protein phosphatase activities are required together for PTEN to inhibit glioma cell invasion (21). Here we provide a mechanistic basis for the concerted actions of PTEN's two activities and show that they are also required together to mediate many of the effects of PTEN on gene expression.…”