| The SH2-domain containing 5′-inositol phosphatase SHIP1, an enzyme that hydrolyzes the signaling lipid phosphatidylinositol-(3,4,5)-trisphosphate [PtdIns(3,4,5)P3], is an important regulator of B cell signaling. Ligation of the B cell antigen receptor leads to the production of PtdIns(3,4,5)P3, resulting in calcium mobilization and the activation of Akt and ERK. These events are inhibited when the antigen receptor is co-ligated with Fc γRIIB1. SHIP1-deficient B cell lines and B cells from SHIP1-deficient mice are unable to inhibit these signaling cascades, indicating that SHIP1 is a critical downstream mediator of FcγRIIB1.; The N-terminal SH2 domain of SHIP1 binds to phosphorylated FcγRIIB1, recruiting SHIP 1 to the plasma membrane where it can hydrolyze PtdIns(3,4,5)P 3. Surprisingly, a mutant SHIP1 that lacked only the non-catalytic C-terminus failed to mediate FcγRIIB1 inhibition of calcium flux. To identify characteristics of the SHIP1 C-terminus that are required for function in the FcγRIIB1 system, two naturally occurring SHIP1 splice isoforms were used to reconstitute SHIP1-deficient DT40 B cells. When compared to full length SHIP1α, SHIP1β contains a small deletion in the C-terminus while SHIP1δ lacks most of the C-terminus. Expression of SHIP1β restored FcγRIIB1-mediated inhibition of calcium flux, Akt activation, and ERK activation, while expression of SHIP1δ failed to restore inhibition of calcium flux. This suggested that proteins that bind to SHIP1α and SHIP1β, but not to SHIP1δ, are required for their involvement in inhibitory signaling.; Proteins capable of binding to SHIP1α, but not to SHIP1δ, were examined by comparing immunoprecipitations from cells expressing either isoform. CIN85 was identified as a protein that, via its SH3 domains, interacts with the C-termini of SHIP1α and SHIP1β but not SHIP1δ in 293T cells. To test the requirement for the CIN85: SHIP1 interaction in F cγRIIB1-mediated inhibition of calcium flux, putative dominant negative CIN85 mutants were expressed in SHIP1-expressing cells. This approach did not demonstrate a role for the SHIP1:CIN85 interaction in FcγRIIB1-mediated inhibition of calcium flux. The above data confirm and extend the critical role played by the C-terminus of SHIP1. Although our studies failed to demonstrate a role for the SHIP1:CIN85 interaction, this interaction might prove important in future studies on FcγRIIB1-mediated inhibition of BCR signaling. |
