Targeted disruption of SHIP leads to Steel factor-induced degranulation of mast cells.
To investigate the role of the src homology 2 (SH2)-containing inositol 5' phosphatase (SHIP) in growth factor-mediated signalling, we compared Steel factor (SF)-induced events in bone marrow-derived mast cells (BMMCs) from SHIP-/- and SHIP+/+ littermates. We found SF alone stimulated massive degranulation from SHIP-/- but none from SHIP+/+ BMMCs. This SF-induced degranulation, which was not due to higher c-kit levels in SHIP-/- cells, correlated with higher intracellular calcium than that in SHIP+/+ cells and was dependent on the influx of extracellular calcium. Both this influx and subsequent degranulation were completely inhibited by PI-3-kinase inhibitors, indicating that SF-induced activation of PI-3-kinase was upstream of extracellular calcium entry. A comparison of phosphatidylinositol-3,4,5-trisphosphate (PIP3) levels following SF stimulation of SHIP+/+ and SHIP-/- BMMCs suggested that SHIP restricted this entry by hydrolyzing PIP3. Although PI-3-kinase inhibitors blocked the release of intracellular calcium, implicating PIP3, and PLCgamma-2 was slightly more tyrosine phosphorylated in SHIP-/- cells, the increase in inositol-1,4,5-trisphosphate (IP3) and intracellular calcium levels were identical in SHIP-/- and SHIP+/+ BMMCs. These results suggest that SHIP prevents SF from triggering degranulation of normal BMMCs, and does so by hydrolyzing PIP3, which in turn limits extracellular calcium entry at a step after the release of intracellular calcium.
Huber
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Terry Fox Laboratory, BC Cancer Agency, 601 West 10th Avenue, Vancouver, BC, V5Z IL3, Canada.
Helgason
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Scheid
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Duronio
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Humphries
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Krystal
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D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D001854:Bone Marrow Cells / Q000378:metabolism*; D020013:Calcium Signaling; D015550:Cell Degranulation; D006868:Hydrolysis; D007377:Interleukin-3 / Q000494:pharmacology; D008407:Mast Cells / Q000378:metabolism*; D051379:Mice; D008817:Mice, Mutant Strains; D008954:Models, Biological; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D000072183:Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010744:Phosphoric Monoester Hydrolases / Q000172:deficiency* / Q000235:genetics; D019089:Stem Cell Factor / Q000494:pharmacology*
D007377:Interleukin-3; D018129:Phosphatidylinositol Phosphates; D000081082:Phosphoinositide-3 Kinase Inhibitors; D019089:Stem Cell Factor; C060974:phosphatidylinositol 3,4,5-triphosphate; D010744:Phosphoric Monoester Hydrolases; C098706:INPPL1 protein, human; D000072183:Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases