SHIP-1–mediated inositol 5′-phosphatase activity associates with shc and CD16 ζ chain after receptor engagement.

(A) Cell lysates from 3 × 10⁷ unstimulated cultured NK cells were immunoprecipitated with anti-SHIP or control mAb; lysates from 10 × 10⁷ unstimulated or anti-CD16–stimulated NK cells were immunoprecipitated with anti-ζ or anti-shc mAbs. Immunoprecipitates were assayed for 5′-inositol phosphatase activity toward ³²P-PI3,4,5P₃, and the reaction products were subjected to TLC followed by autoradiography. The positions of PI3,4P₂ and PI3,4,5P₃ are indicated on the right. (B) The 10 × 10⁶ cell equivalent of the immunoprecipitated samples showed in panel A were analyzed by immunoblot with the indicated antibodies. The numbers to the left of the blots indicate molecular weight. (C) The percentage of substrate conversion (± SD) obtained by densitometric analysis of 4 different experiments is shown. The catalytic activity associated with SHIP-1 immunoprecipitates has been arbitrarily fixed at 100%. The percentage of conversion has been calculated on the basis of equivalent cell number within the single experiment.