A pleckstrin homology-related domain in SHIP1 mediates membrane localization during Fcγ receptor-induced phagocytosis.
SH2 domain-containing inositol-5'-phosphatase-1 (SHIP1) inhibits inflammation by hydrolyzing phosphoinositide-3'-kinase generated membrane phosphatidylinositol-3,4,5-trisphosphate (PIP(3)). 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 PIP(3). Wild-type PH-R domain bound PIP(3) 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.
Immunity and Infection Research Centre, Vancouver Coastal Health Research Institute, Vancouver, British Columbia, Canada.
Andrew
A
Ming-Lum
Shaheen
S
Shojania
Eva
E
So
Erin
E
McCarrell
Eileen
E
Shaw
David
D
Vu
Ida
I
Wang
Lawrence P
LP
McIntosh
Alice L-F
AL
Mui
D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D000072181:Inositol Polyphosphate 5-Phosphatases; D019906:Nuclear Magnetic Resonance, Biomolecular; D010587:Phagocytosis / Q000502:physiology*; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D017452:Receptors, IgG / Q000502:physiology*
D018129:Phosphatidylinositol Phosphates; D017452:Receptors, IgG; C060974:phosphatidylinositol 3,4,5-triphosphate; D010744:Phosphoric Monoester Hydrolases; D000072181:Inositol Polyphosphate 5-Phosphatases