Phosphatidylinositol (3,4,5)P3 is essential but not sufficient for protein kinase B (PKB) activation; phosphatidylinositol (3,4)P2 is required for PKB phosphorylation at Ser-473: studies using cells from SH2-containing inositol-5-phosphatase knockout mice

• Published in: The Journal of biological chemistry 2002-03, Vol.277 (11), p.9027-9035
• Main Authors: Scheid, Michael P, Huber, Michael, Damen, Jacqueline E, Hughes, Michael, Kang, Veronica, Neilsen, Paul, Prestwich, Glenn D, Krystal, Gerald, Duronio, Vincent
• Format: Article
• Language: English
• Subjects: Animals; Enzyme Activation; Mice; Mice, Knockout; Phosphatidylinositol Phosphates - physiology; Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases; Phosphoric Monoester Hydrolases - physiology; Phosphorylation; Protein-Serine-Threonine Kinases; Proto-Oncogene Proteins - metabolism; Proto-Oncogene Proteins c-akt; Serine - metabolism; Stem Cell Factor - pharmacology
• ISSN: 0021-9258
• DOI: 10.1074/jbc.M106755200

Using bone marrow derived mast cells from SH2-containing inositol-5-phosphatase (SHIP) +/+ and minus sign/minus sign mice, we found that the loss of SHIP leads to a dramatic increase in Steel Factor (SF)-stimulated phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P(3)), a substantial reduction in PI(3,4)P(2), and no change in PI(4,5)P(2) levels. We also found that SF-induced activation of protein kinase B (PKB) is increased and prolonged in SHIP -/- cells, due in large part to more PKB associating with the plasma membrane in these cells. Pretreatment of SHIP -/- cells with 25 microm LY294002 resulted in complete inhibition of SF-induced PI(3,4)P(2), while still yielding PI(3,4,5)P(3) levels similar to those achieved in SHIP+/+ cells. This offered a unique opportunity to study the regulation of PKB by PI(3,4,5)P(3), in the absence of PI(3,4)P(2). Under these conditions, PKB activity was markedly reduced compared with that in SF-stimulated SHIP+/+ cells, even though more PKB localized to the plasma membrane. Although phosphoinositide-dependent kinase 1 mediated phosphorylation of PKB at Thr-308 was unaffected by LY294002, phosphorylation at Ser-473 was dramatically reduced. Moreover, intracellular delivery of PI(3,4)P(2) to LY294002-pretreated, SF-stimulated SHIP -/- cells increased phosphorylation of PKB at Ser-473 and increased PKB activity. These results are consistent with a model in which SHIP serves as a regulator of both activity and subcellular localization of PKB.