Targeted disruption of SHIP leads to Steel factor-induced degranulation of mast cells

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Abstract
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 PLCγ‐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.