The tyrosine phosphorylation of ship is regulated by src kinases

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SHIP is tyrosine phosphorylated (Yphos) upon the addition of a wide variety of extracellular stimuli. However, little is known about the tyrosine kinase(s) involved. Using the Src-specific inhibitor, PP2, we found that SHIP's Yphos, following co-clustering of the B-cell receptor (BCR) with the FcγRIIB co-receptor in WEHI 231 cells, was dramatically reduced while the Syk inhibitor, piceatannol, had little or no effect. Comparable results were obtained following stimulation with interleukin-3 (IL-3) or Steel factor (SF) in Ba/F3 cells and bone marrow derived mast cells (BMMCs), respectively. To corroborate these results, BMMCs derived from Lyn −/− mice were examined. A time course of SF stimulation revealed that SHIP Yphos in Lyn −/− BMMCs was dramatically reduced though not completely abolished. SHIP Yphos was further reduced, however, by addition of PP2 but not piceatannol, suggesting that some or all of the residual SHIP Yphos was due to other Src family members. Binding studies using GST-beads containing the SH3 domain of different Src kinases and various truncated forms of SHIP's proline-rich C-terminus. Co-immunoprecipitation studies verified that SHIP and Lyn associate in WEHI 231 B-cells and that this interaction does not increase following stimulation. These results suggest that members of the Src family are key regulators of SHIP Yphos following activation of BCR and cytokine receptor systems.