The PTEN tumour suppressor is a lipid and protein phosphatase that inhibits phosphoinositide 3-kinase (PI3K)-dependent signalling by dephosphorylating phosphatidylinositol 3,4,5-trisphosphate (PtdInsP 3 ). Here, we discuss the concept of PTEN as an 'interfacial enzyme', which exists in a high activity state when bound transiently at membrane surfaces containing its substrate and other acidic lipids, such as PtdIns(4,5)P 2 and phosphatidylserine (PtdSer). This mechanism ensures that PTEN functions in a spatially restricted manner, and may explain its involvement in forming the gradients of PtdInsP 3 , which are necessary for generating and/or sustaining cell polarity during motility, in developing neurons and in epithelial tissues. Coordinating PTEN activity with alternative mechanisms of PtdInsP 3 metabolism, by the tightly regulated SHIP 5-phoshatases, synthesizing the independent second messenger PtdIns(3,4)P 2 , may also be important for cellular polarization in some cell types. Superimposed on this interfacial mechanism are additional post-translational regulatory processes, which generally act to reduce PTEN activity. Oxidation of the active site cysteine residue by reactive oxygen species and phosphorylation of serine/threonine residues at sites in the C-terminus of the protein inhibit PTEN. These phosphorylation sites also appear to play a role in regulating both stability and localization of PTEN, as does ubiquitination of PTEN. Because genetic studies in mice show that the level of expression of PTEN in an organism profoundly influences tumour susceptibility, factors that regulate PTEN, localization, activity and turnover should be important in understanding its biological functions as a tumour suppressor.

Although PTEN (phosphatase and tensin homologue deleted on chromosome 10) is one of the most commonly mutated tumour suppressors in human cancers, loss of PTEN expression in the absence of mutation appears to occur in an even greater number of tumours. PTEN is phosphorylated in vitro on Thr366 and Ser370 by GSK3 (glycogen synthase kinase 3) and CK2 (casein kinase 2) respectively, and specific inhibitors of these kinases block these phosphorylation events in cultured cells. Although mutation of these phosphorylation sites did not alter the phosphatase activity of PTEN in vitro or in cells, blocking phosphorylation of Thr366 by either mutation or GSK3 inhibition in glioblastoma cell lines led to a stabilization of the PTEN protein. Our data support a model in which the phosphorylation of Thr366 plays a role in destabilizing the PTEN protein.

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.

Abstract:The tumour suppressor, Phosphatase and Tensin homolog deleted on chromosome ten (PTEN), has a well characterised and important lipid phosphatase activity and a poorly characterised protein phosphatase activity. We show that both activities are required together for the regulation of cellular invasion and most of its largest effects on gene expression. PTEN appears to dephosphorylate itself at Thr366 and mutation of this site makes lipid phosphatase activity sufficient for the regulation of invasion. We propose that the dominant role for PTEN's protein phosphatase activity is autodephosphorylation-mediated regulation of its lipid phosphatase activity. Since the regulation of invasion and these large gene expression changes do not correlate with total cellular levels of its PtdInsP 3 substrate and AKT activity, we speculatively propose a role for localised PtdInsP 3 signalling in the PTEN-mediated regulation of the former processes. Finally, in identifying a tumour-derived PTEN mutant selectively lacking protein phosphatase activity, we show that in some circumstances these processes, and not AKT, can correlate with PTEN-mediated tumour suppression.3

Gonadotropin-releasing hormone (GnRH) receptor agonists are extensively used in the treatment of sex hormone-dependent cancers via the desensitization of pituitary gonadotropes and consequent decrease in steroid sex hormone secretion. However, evidence now points to a direct inhibitory effect of GnRH analogs on cancer cells. These effects appear to be mediated via the G␣ i -type G protein, in contrast to the predominant G␣ q coupling in gonadotropes. Unlike G␣ q coupling, G␣ i coupling of the GnRH receptor can be activated by both agonists and antagonists. This unusual pharmacology suggested that the receptor involved in the cancer cells may not be the classical gonadotrope type I GnRH receptor. However, we have previously shown that a functional type II GnRH receptor is not present in man. In the present study, we show that GnRH agonists and selective GnRH antagonists exert potent antiproliferative effects on JEG-3 choriocarcinoma, benign prostate hyperplasia (BPH-1), and HEK293 cells stably expressing the type I GnRH receptor. This antiproliferative action occurs through a G␣ i -mediated activation of stress-activated protein kinase pathways, resulting in caspase activation and transmembrane transfer of phosphatidlyserine to the outer membrane envelope. Structurally related antagonistic GnRH analogs displayed divergent antiproliferative efficacies but demonstrated equal efficacies in inhibiting GnRH-induced G␣ q -based signaling. Therefore the ability of GnRH receptor antagonists to exert an antiproliferative effect on reproductive tumors may be dependent on ligand-selective activation of the G␣ i -coupled form of the type I GnRH receptor.