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

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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

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