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

Michael P Scheid,Michael Huber,Jacqueline E Damen,Michael Hughes,Veronica Kang,Paul Neilsen,Glenn D Prestwich,Gerald Krystal,Vincent Duronio

doi:10.1074/jbc.m106755200

Abstract

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.

Highlights

Introduction ofPI[3,4]P2 into bone marrow-derived mast cells (BMMCs)—SHIPϪ/Ϫ and ϩ/ϩ BMMCs were incubated for 14 h in cytokine-free RPMI plus 10% FCS
We found that Steel Factor (SF)-induced activation of protein kinase B (PKB) is increased and prolonged in SH2containing inositol-5-phosphatase (SHIP)؊/؊ cells, due in large part to more PKB associating with the plasma membrane in these cells
Even in unstimulated cells, not obvious because of the different scales employed in Fig. 1A, the level of PI[3,4,5]P3 in the SHIPϪ/Ϫ cells was consistently higher than in the SHIPϩ/ϩ cells (525 cpm compared with 195 cpm in the example shown), while PI[3,4]P2 levels were similar

Summary

Introduction

Introduction ofPI[3,4]P2 into BMMCs—SHIPϪ/Ϫ and ϩ/ϩ BMMCs were incubated for 14 h in cytokine-free RPMI plus 10% FCS. Di-C8 PI[3,4]P2 (20 ␮M) (Echelon Research Laboratories, Salt Lake City, UT) or a control inositol head group (IP6) (Echelon Research Laboratories) were complexed with histone (20 ␮M) as a shuttle to facilitate entry into cells [18] by sonicating in a water-bath sonicator for 2 min. Other experiments used di-C16-PI[3,4]P2, with di-C16-PI[3,5]P2 as a control. Reactions were initiated by pretreating 5 ϫ 106 cells with or without 25 ␮M LY294002 for 7 min. The lipid-shuttle complex, IP6-shuttle, or empty shuttle was added to cells and incubated for 3 min, followed by incubation with 100 ng/ml SF for 2 min. Reactions were terminated by quickly centrifuging the cells, aspirating off the supernatant, and quick freezing the pellets in methanol/dry ice

Methods

Results

Conclusion