PTEN protein phosphatase activity correlates with control of gene expression and invasion, a tumor-suppressing phenotype, but not with AKT activity.

The tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN) has a well-characterized lipid phosphatase activity and a poorly characterized protein phosphatase activity. We show that both activities are required for PTEN to inhibit cellular invasion and to mediate most of its largest effects on gene expression. PTEN appears to dephosphorylate itself at threonine 366, and mutation of this site makes lipid phosphatase activity sufficient for PTEN to inhibit invasion. We propose that the dominant role for PTEN's protein phosphatase activity is autodephosphorylation-mediated regulation of its lipid phosphatase activity. Because PTEN's regulation of invasion and these changes in gene expression required lipid phosphatase activity, but did not correlate with the total cellular abundance of its phosphatidylinositol 3,4,5-trisphosphate (PIP3) lipid substrate or AKT activity, we propose that localized PIP3 signaling may play a role in those PTEN-mediated processes that depend on both its protein and lipid phosphatase activities. Finally, we identified a tumor-derived PTEN mutant selectively lacking protein phosphatase activity, indicating that in some circumstances the regulation of invasion and not that of AKT can correlate with PTEN-mediated tumor suppression.

Ubiquitination of PTEN (phosphatase and tensin homolog) inhibits phosphatase activity and is enhanced by membrane targeting and hyperosmotic stress.

The PTEN (phosphatase and tensin homolog) tumor suppressor is a phosphatase that inhibits phosphoinositide 3-kinase-dependent signaling by metabolizing the phosphoinositide lipid phosphatidylinositol 3,4,5-trisphosphate (PtdInsP(3)) at the plasma membrane. PTEN can be mono- or polyubiquitinated, and this appears to control its nuclear localization and stability, respectively. Although PTEN phosphorylation at a cluster of C-terminal serine and threonine residues has been shown to stabilize the protein and inhibit polyubiquitination and plasma membrane localization, details of the regulation of ubiquitination are unclear. Here, we show that plasma membrane targeting of PTEN greatly enhances PTEN ubiquitination and that phosphorylation of PTEN in vitro does not affect subsequent ubiquitination. These data suggest that C-terminal phosphorylation indirectly regulates ubiquitination by controlling membrane localization. We also show that either mono- or polyubiquitination in vitro greatly reduces PTEN phosphatase activity. Finally, we show that hyperosmotic stress increases both PTEN ubiquitination and cellular PtdInsP(3) levels well before a reduction in PTEN protein levels is observed. Both PTEN ubiquitination and elevated PtdInsP(3) levels were reduced within 10 min after removal of the hyperosmotic stress. Our data indicate that ubiquitination may represent a regulated mechanism of direct reversible control over the PTEN enzyme.

PTEN is destabilized by phosphorylation on Thr366.

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.

A novel leptin signalling pathway via PTEN inhibition in hypothalamic cell lines and pancreatic beta-cells.

In obesity and diabetes, the ability of hypothalamic neurons to sense and transduce changes in leptin and insulin levels is compromised. The effects of both hormones require intracellular signalling via the PI3-kinase pathway, which is inhibited by the phosphatase PTEN. We show that leptin-stimulated F-actin depolymerization in mouse hypothalamic cells is inhibited by PTEN, a process involving independent effects of both its lipid and protein phosphatase activities. Potentially mediating this F-actin depolymerization, leptin, but not insulin, stimulated the phosphorylation of PTEN in a CK2 dependent manner, and inhibited its phosphatase activity. Similarly, hyperpolarization of mouse pancreatic beta-cells by leptin also requires coincident PtdIns(3,4,5)P3 generation and actin depolymerization, and could be inhibited by mechanisms requiring both the lipid and protein phosphatase activities of PTEN. These results demonstrate a critical role for PTEN in leptin signalling and indicate a mechanism by which leptin and insulin can produce PI3K dependent differential cellular outputs.

The tumour-suppressor function of PTEN requires an N-terminal lipid-binding motif.

The PTEN (phosphatase and tensin homologue deleted on chromosome 10) tumour-suppressor protein is a phosphoinositide 3-phosphatase which antagonizes phosphoinositide 3-kinase-dependent signalling by dephosphorylating PtdIns(3,4,5)P3. Most tumour-derived point mutations of PTEN induce a loss of function, which correlates with profoundly reduced catalytic activity. However, here we characterize a point mutation at the N-terminus of PTEN, K13E from a human glioblastoma, which displayed wild-type activity when assayed in vitro. This mutation occurs within a conserved polybasic motif, a putative PtdIns(4,5)P2-binding site that may participate in membrane targeting of PTEN. We found that catalytic activity against lipid substrates and vesicle binding of wild-type PTEN, but not of PTEN K13E, were greatly stimulated by anionic lipids, especially PtdIns(4,5)P2. The K13E mutation also greatly reduces the efficiency with which anionic lipids inhibit PTEN activity against soluble substrates, supporting the hypothesis that non-catalytic membrane binding orientates the active site to favour lipid substrates. Significantly, in contrast to the wild-type enzyme, PTEN K13E failed either to prevent protein kinase B/Akt phosphorylation, or inhibit cell proliferation when expressed in PTEN-null U87MG cells. The cellular functioning of K13E PTEN was recovered by targeting to the plasma membrane through inclusion of a myristoylation site. Our results establish a requirement for the conserved N-terminal motif of PTEN for correct membrane orientation, cellular activity and tumour-suppressor function.

Hypo-osmotic stress activates Plc1p-dependent phosphatidylinositol 4,5-bisphosphate hydrolysis and inositol Hexakisphosphate accumulation in yeast.

Polyphosphoinositide-specific phospholipases (PICs) of the delta-subfamily are ubiquitous in eukaryotes, but an inability to control these enzymes physiologically has been a major obstacle to understanding their cellular function(s). Plc1p is similar to metazoan delta-PICs and is the only PIC in Saccharomyces cerevisiae. Genetic studies have implicated Plc1p in several cell functions, both nuclear and cytoplasmic. Here we show that a brief hypo-osmotic episode provokes rapid Plc1p-catalyzed hydrolysis of PtdIns(4,5)P2 in intact yeast by a mechanism independent of extracellular Ca2+. Much of this PtdIns(4,5)P2 hydrolysis occurs at the plasma membrane. The hydrolyzed PtdIns(4,5)P2 is mainly derived from PtdIns4P made by the PtdIns 4-kinase Stt4p. PtdIns(4,5)P2 hydrolysis occurs normally in mutants lacking Arg82p or Ipk1p, but they accumulate no InsP6, showing that these enzymes normally convert the liberated Ins(1,4,5)P3 rapidly and quantitatively to InsP6. We conclude that hypo-osmotic stress activates Plc1p-catalyzed PtdIns(4,5)P2 at the yeast plasma membrane and the liberated Ins(1,4,5)P3 is speedily converted to InsP6. This ability routinely to activate Plc1p-catalyzed PtdIns(4,5)P2 hydrolysis in vivo opens up new opportunities for molecular and genetic scrutiny of the regulation and functions of phosphoinositidases C of the delta-subfamily.