Analysis of the cellular functions of PTEN using catalytic domain and C-terminal mutations: differential effects of C-terminal deletion on signallingpathways downstream of phosphoinositide 3-kinase

The tumour suppressor protein, PTEN (phosphatase and tensin homolog deleted on chromosome in), is a phosphatase that can dephosphorylate tyrosine-cont aining peptides, She, focal adhesion kinase and phosphoinositide substrates . In cellular assays, PTEN has been shown to antagonize the PI-3K-dependent activation of protein kinase B (PE;B) and to inhibit cell spreading and mo tility. It is currently unclear, however, whether PTEN accomplishes these e ffects through its lipid- or protein-phosphatase activity, although strong evidence has demonstrated the importance of the latter for tumour suppressi on by PTEN. By using a PTEN G129E (Gly(120) --> Glu) mutant that has lost i ts lipid phosphatase activity, while retaining protein phosphatase activity , we demonstrated a requirement for the lipid phosphatase activity of PTEN in the regulation of PKB activity, cell viability and membrane ruffling. We also made a small C-terminal deletion of PTEN, removing a putative PDZ (PS D95, Dig and ZO1)-binding motif, with no detectable effect on the phosphata se activity of the protein expressed in HEK393 cells (human embryonic kidne y 293 cells) assayed in vitro. Surprisingly, expression of this mutant reve aled differential requirements for the C-terminus in the different function al assays. Wild-type and C-terminally deleted PTEN appeared to be equally a ctive in down-regulating PE;B activity, but this mutant enzyme had no effec t on platelet-derived growth factor (PDGF)-induced membrane ruffling and wa s only partially active in a cell viability assay. These results stress the importance of the lipid phosphatase activity of PTEN in the regulation of several signalling pathways. They also identify a mutation, similar to muta tions that occur in some human tumours, which removes the effect of PTEN on membrane ruffling but not that on PKB.