Stimulation of PI 3-kinase signaling via inhibition of the tumor suppressor phosphatase, PTEN

Class I phosphoinositide 3-kinases (PI 3-kinases) are growth factor and G-protein-coupled receptor activated enzymes that synthesize the ubiquitous lipid second messenger, phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3). PtdIns(3,4,5)P3 mediates downstream signaling through several effector proteins, such as the proto-oncogene product, protein kinase B/Akt, that selectively bind this lipid. The lipid phosphatase, PTEN (phosphatase and tensin homologue deleted on chromosome 10), is the product of a major tumor suppressor gene that antagonizes PI 3-kinase signaling by dephosphorylating the 3-position of the inositol ring of PtdIns(3,4,5)P3. PtdIns(3,4,5)P3 is also metabolized by removal of the 5-phosphate catalyzed by a distinct family of enzymes exemplarized by SHIP (SH2 domain containing inositol 5-phosphatase) and SHIP2. Mouse knockout studies, however, suggest that PTEN and SHIP2 have profoundly different biological functions where regulation of SHIP2 is geared towards stimulus dependent antagonism of PI 3-kinase signaling and regulation of PTEN is geared towards its inhibition or reduced cellular expression and hence functions to stimulate or enhance PI 3-kinase dependent signaling. Negative regulation of PTEN has been shown to occur via reactive oxygen species and serine/threonine phosphorylation. PTEN is a member of the protein tyrosine phosphatase family and shares a common catalytic motif involving a reactive cysteine residue. This can undergo reversible oxidation, via the formation of a disulphide bond with a neighboring cysteine, when cells are exposed to exogenous oxidants or in response to the growth factor-dependent production of endogenous reactive oxygen species. In order to examine systematically the importance of serine/threonine phosphorylation in the regulation of PTEN function we screened a large panel of protein kinases for their ability to phosphorylate PTEN. We also determined the sites phosphorylated in vitro and generated anti-phosphopeptide-specific antibodies to analyze their significance in vivo and to study their regulation. These studies suggest novel pathways that may contribute to negative regulation of PTEN activity and/or expression. Activation of class I PI 3-kinases and generation of the phospholipid second messenger PtdIns(3,4,5)P3, mediate downstream signaling through a variety of targets which include protein kinase B (PKB). PKB in turn phosphorylates protein targets that control gene transcription, glucose homeostasis, cell proliferation and survival. PtdIns(3,4,5)P3 levels are also increased through the inhibitory regulation of the tumor suppressor PTEN, either by reactive oxygen species or via phosphorylation of specific C-terminal serine and threonine residues that influence PTEN activity and/or localization. We have made use of a panel of protein kinases to identify novel PTEN regulatory kinases using an in vitro screen. The preliminary findings of this screen are described and their possible biological significance is discussed. © 2007 Elsevier Ltd. All rights reserved.