Targeted disruption of SHIP leads to Steel factor-induced degranulation ofmast 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 S HIP-/- and SHIP+/+ littermates, We found SF alone stimulated massive degran ulation from SHIP-/- but none from SHIP+/+ BMMCs, This SF-induced degranula tion, which was not due to higher c-kit levels in SHIP-/- cells, correlated with higher intracellular calcium than that in SHIP+/+ cells and was depen dent on the influx of extracellular calcium, Both this influx and subsequen t degranulation were completely inhibited by PI-3-kinase inhibitors, indica ting that SF-induced activation of PI-3-kinase was upstream of extracellula r calcium entry, A comparison of phosphatidylinositol-3,4,5-trisphosphate ( PIP3) levels following SF stimulation of SHIP+/+ and SHIP-/- BMMCs suggeste d that SHIP restricted this entry by hydrolyzing PIP3, Although PI-3-kinase inhibitors blocked the release of intracellular calcium, implicating PIP3, and PLC gamma-2 was slightly more tyrosine phosphorylated in SHIP-/- cells , the increase in inositol-1,4,5-trisphosphate (IP3) and intracellular calc ium levels were identical in SHIP-/- and SHIP+/+ BMMCs, These results sugge st that SHIP prevents SF from triggering degranulation of normal BMMCs, and does so by hydrolyzing PIP3, which in turn limits extracellular calcium en try at a step after the release of intracellular calcium.