SHIP's C-terminus is essential for its hydrolysis of PIP3 and inhibition of mast cell degranulation.
The SH2-containing inositol-5'-phosphatase, SHIP, restrains bone marrow-derived mast cell (BMMC) degranulation, at least in part, by hydrolyzing phosphatidylinositol (PI)-3-kinase generated PI-3,4,5-P(3) (PIP3) to PI-3,4-P(2). To determine which domains within SHIP influence its ability to hydrolyze PIP3, bone marrow from SHIP(-/-) mice was retrovirally infected with various SHIP constructs. Introduction of wild-type SHIP into SHIP(-/-) BMMCs reverted the Steel factor (SF)-induced increases in PIP3, calcium entry, and degranulation to those observed in SHIP(+/+) BMMCs. A 5'-phosphatase dead SHIP, however, could not revert the SHIP(-/-) response, whereas a SHIP mutant in which the 2 NPXY motifs were converted to NPXFs (2NPXF) could partially revert the SHIP(-/-) response. SF stimulation of BMMCs expressing the 2NPXF, which could not bind Shc, led to the same level of mitogen-activated protein kinase (MAPK) phosphorylation as that seen in BMMCs expressing the other constructs. Surprisingly, C-terminally truncated forms of SHIP, lacking different amounts of the proline rich C-terminus, could not revert the SHIP(-/-) response at all. These results suggest that the C-terminus plays a critical role in enabling SHIP to hydrolyze PIP(3) and inhibit BMMC degranulation.
Damen
J E
JE
Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, British Columbia, Canada.
Ware
M D
MD
Kalesnikoff
J
J
Hughes
M R
MR
Krystal
G
G
D020816:Amino Acid Motifs; D000818:Animals; D015153:Blotting, Western; D001854:Bone Marrow Cells / Q000201:enzymology / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D015550:Cell Degranulation / Q000187:drug effects*; D005822:Genetic Vectors; D006868:Hydrolysis / Q000187:drug effects; D008407:Mast Cells / Q000502:physiology*; D051379:Mice; D018345:Mice, Knockout; D016297:Mutagenesis, Site-Directed; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism*; D000072183:Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D019089:Stem Cell Factor / Q000494:pharmacology; D014161:Transduction, Genetic
D018129:Phosphatidylinositol Phosphates; D019089:Stem Cell Factor; D010744:Phosphoric Monoester Hydrolases; C098706:INPPL1 protein, human; D000072183:Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases; D002118:Calcium