The PTEN ( phosphatase and tensin homologue deleted on chromosome 10) tumor suppressor is a phosphatidylinositol 3,4,5-trisphosphate (PtdInsP(3)) 3-phosphatase that plays a crucial role in regulating many cellular processes by antagonizing the phosphoinositide 3-kinase signaling pathway. Although able to metabolize soluble inositol phosphates in vitro, the question of their significance as physiological substrates is unresolved. We show that inositol phosphates are not regulated by wild type PTEN, but that a synthetic mutant, PTEN M-CBR3, previously thought to be inactive toward inositides, can selectively regulate inositol 1,3,4,5,6-pentakisphosphate (Ins(1,3,4,5,6) P-5). Transfection of U87-MG cells with PTEN M-CBR3 lowered Ins(1,3,4,5,6) P-5 levels by 60% without detectable effect on PtdInsP(3). Although PTEN M-CBR3 is a 3-phosphatase, levels of myo-inositol 1,4,5,6-tetrakisphosphate were not increased, whereas myo-inositol 1,3,4,6-tetrakisphospate levels increased by 80%. We have used PTEN M-CBR3 to study the physiological function of Ins(1,3,4,5,6) P-5 and have found that Ins(1,3,4,5,6) P-5 does not modulate PKB phosphorylation, nor does it regulate clathrin-mediated epidermal growth factor receptor internalization. By contrast, PTEN M-CBR3 expression, and the subsequent lowering of Ins(1,3,4,5,6) P-5, are associated with reduced anchorage-independent colony formation and anchorage-dependent proliferation in U87-MG cells. Our results, together with previously published data, suggest that Ins(1,3,4,5,6) P-5 has a role in proliferation.

The tumour suppressor PTEN is a PtdIns(3,4,5)P-3 phosphatase that regulates many cellular processes through direct antagonism of PI 3-kinase signalling. Here we show that oxidative stress activates PI 3-kinase-dependent signalling via the inactivation of PTEN. We use two assay systems to show that cellular PTEN phosphatase activity is inhibited by oxidative stress induced by 1 mM hydrogen peroxide. PTEN inactivation by oxidative stress also causes an increase in cellular PtdIns(3,4,5)P-3 levels and activation of the downstream PtdIns(3,4,5)P-3 target, PKB/Akt, that does not occur in cells lacking PTEN. We then show that endogenous oxidant production in RAW264.7 macrophages inactivates a fraction of the cellular PTEN, and that this is associated with an oxidant-dependent activation of downstream signalling. These results show that oxidants, including those produced by cells, can activate downstream signalling via the inactivation of PTEN. This demonstrates a novel mechanism of regulation of the activity of this important tumour suppressor and the signalling pathways it regulates. These results may have significant implications for the many cellular processes in which PtdIns(3,4,5)P-3 and oxidants are produced concurrently.

The tumour suppressor protein, PTEN (phosphatase and tensin homologue deleted on chromosome 10) is a member of the mixed function, serine/threonine/tyrosine phosphatase subfamily of protein phosphatases. Its physiological substrates, however, are primarily 3-phosphorylated inositol phospholipids, which are products of phosphoinositide 3-kinases. PTEN thus antagonizes PI 3-kinase-dependent signalling pathways, which explains to a large extent its tumour suppressor status. We have examined the kinetic behaviour, substrate specificity and regulation of PTEN both in vitro and in a variety of cellular models. Although PTEN can utilize both phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P(3)] and its water-soluble headgroup, inositol 1,3,4,5-tetrakisphosphate, as substrates, it displays classical features of interfacial catalysis, which greatly favour the lipid substrate (by as much as 1000-fold as judged by K(cat)/K(m) values). Expression of PTEN in U87 cells (which lack endogenous PTEN) and measuring the levels of all known 3-phosphorylated lipids suggests that phosphatidylinositol 3,4-bisphosphate and PtdIns(3,4,5)P(3) are both substrates, but that phosphatidylinositol 3-phosphate and phosphatidylinositol 3,5-bisphosphate are not. PTEN binds to several PDZ-domain-containing proteins via a consensus sequence at its extreme C-terminus. Disruption of targeting to PDZ-domain proteins selectively blocks some PTEN functions, but not others, suggesting the existence of spatially localized, functionally dedicated pools of signalling lipids. We have also shown recently that PTEN expression is controlled at the transcriptional level and is profoundly upregulated by peroxisome proliferator activated receptor gamma agonists, thereby providing possible implications for these drugs in diabetes, inflammation and cancer.

The tumour suppressor protein PTEN (phosphatase and tensin homolog deleted on chromosome 10) is a lipid phosphatase which can antagonize the phosphoinositide 3-kinase (PI 3-kinase) signalling pathway, promoting apoptosis and inhibiting cell-cycle progression and cell motility. We show that very little cellular PTEN is associated with the plasma membrane, but that artificial membrane-targeting of PTEN enhances its inhibition of signalling to protein kinase B (PKB). Evidence for potential targeting of PTEN to the membrane through PDZ domain-mediated protein-protein interactions led us to use a PTEN enzyme with a deletion of the C-terminal PDZ-binding sequence, that retains full phosphatase activity against soluble substrates, and to analyse the efficiency of this mutant in different cellular assays. The extreme C-terminal PDZ-binding sequence was dispensable for the efficient down-regulation of cellular PtdIns(3,4,5)P-3 levels and a number of PI 3-kinase-dependent signalling activities, including PKB and p70S6K. However, the PDZ-binding sequence was required for the efficient inhibition of cell spreading. The data show that a PTEN mutation, similar to those found in some tumours, affects some functions of the protein but not others, and implicate the deregulation of PTEN-dependent processes other than PKB activation in the development of some tumours. Significantly, this hypothesis is supported by data showing low levels of PKB phosphorylation in a glioblastoma sample carrying a mutation in the extreme C-terminus of PTEN compared with tumours carrying phosphatase-inactivating mutations of the enzyme. Our data show that deregulation of PKB is not a universal feature of tumours carrying PTEN mutations and implicate other processes that may be deregulated in these tumours.