Abstract
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.
Highlights
Frequently lost tumor suppressors in human cancer [3]
Several binding partners and regulators of the PTEN protein appear to act as oncogenes and tumor suppressors principally through their ability to control PTEN [7,8,9,10,11,12]
NEDD4-1 has been identified as an efficient E3 ubiquitin ligase for PTEN in vitro that is capable of controlling PTEN ubiquitination and expression in cells [11]
Summary
Cell Culture and Transfection—U87MG glioblastoma cells and HEK293T cells were obtained from the European Collection of Animal Cell Cultures and maintained in the recommended medium (10% fetal bovine serum). Transfection of U87MG cells was performed using TransITLT1 reagent (Mirus) when U87MG cells were ϳ50% confluent. Plasmids encoding PTEN, the PTEN A3 mutant (S380A/ T382A/T383A), or the myristoylated PTEN construct and/or FLAG-tagged ubiquitin were used in transfection as indicated. Cells were treated with or without inhibitor(s) for the indicated times. Cells were washed with phosphate-buffered saline and lysed. The following agents were purchased from Calbiochem: proteasome inhibitor I (PSI), calpeptin, and the CK2 inhibitors DMAT and 4,5,6,7-tetrabromo-.
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