A pleckstrin homology‐related domain in SHIP1 mediates membrane localization during Fcγ receptor‐induced phagocytosis

ABSTRACT SH2 domain‐containing inositol‐5′‐phosphatase‐1 (SHIP1) inhibits inflammation by hydrolyzing phosphoinositide‐3′‐kinase generated membrane phosphatidylinositol‐3,4,5‐trisphosphate (PIP3). Bioinformatic analysis of SHIP1 from multiple species revealed a pleckstrin homololgy‐related (PH‐R) domain, which we hypothesize mediates SHIP1′s association with the membrane, a requirement for its biological function. Recombinant murine SHIP1 PH‐R domain was subjected to biophysical and biochemical analysis. Residues K370 and K397 were found to be important for PH‐R domain association with membrane PIP3. Wild‐type PH‐R domain bound PIP3 with 1.9 ± 0.2 nM affinity, while the affinity of a K370A/K397A substituted mutant was too low to measure. Wild‐type (but not the K370A/K397A substituted) full‐length SHIP1 protein, reconstitutes normal inhibition of Fcγ receptor‐mediated phagocytosis when introduced into SHIP1−/− murine macrophages, reducing the number of phagocytic events by 2‐fold as compared to SHIP1−/− cells. In fact, the PH‐R‐mediated membrane interaction appears to be a major mechanism by which SHIP1 is recruited to the membrane, since the K370A/K397A substitution reduced the recruitment of both full‐length SHIP1 and the PH‐R domain by ≥2‐fold. We have previously shown that SHIP1 enzyme activity can be targeted for therapeutic purposes. The current studies suggest that molecules targeting the PH‐R domain can also modulate SHIP1 function.—Ming‐Lum, A., Shojania, S., So, E., McCarrell, E., Shaw, E., Vu, D., Wang, I., McIntosh, L. P., Mui, A. L. F. A pleckstrin homology‐related domain in SHIP1 mediates membrane localization during Fcγ receptor‐induced phagocytosis. FASEB J. 26, 3163–3177 (2012). www.fasebj.org