The Shc phosphotyrosine (pTyr) binding (PTB) domain interacts with the SH2-containing 5′ inositol phosphatase (SHIP) and the β common (βc) chain of the IL-3 receptor.
(A) Schematic representation of Shc domains expressed as glutathione-S-transferase (GST) fusion proteins. GST–PTB-M175 contains an arginine to methionine mutation in residue 175. (B) IC2 cells (4 × 107) were stimulated (+) with 200 ng/mL IL-3 for 5 minutes, lysed, and then incubated with GST alone or with different GST-Shc fusion proteins. Immunoprecipitation with anti-Shc and anti-βc chain antibodies in the same experiment is also shown. The bound proteins were analyzed with anti-pTyr antibodies. Arrows mark phosphorylated proteins in cells stimulated with IL-3. (C) IC2 cells were stimulated with 100 ng/mL IL-3 for 5 minutes, lysed, and then immunoprecipitated with either anti-βc or anti-SHIP antiserum. Western blot analysis was performed with anti-Shc antibodies. (D) GST precipitates from unstimulated or stimulated cells were analyzed with anti-SHIP and anti-βc immunoblotting. TCL indicates total cell lysate. Asterisks mark the position of the SHIP and βc chain isoforms.
