Altered physical state of p210bcr-abl in tyrphostin AG957-treated K562 cells.

Abstract

AG957 is a tyrosine kinase antagonist which prior studies had shown inhibits p210bcr-abl tyrosine kinase activity and K562 chronic myelogenous leukemia cell growth. We report here the effects of AG957 on the physical state of p210bcr-abl and its signaling adapter molecules Shc and Grb2 in K562 cells. After exposure to AG957, the amount of tyrosine-phosphorylated native p210bcr-abl decreases, with the appearance of a high molecular weight (> 210 kDa) p210bcr-abl. This effect is seen after [32P]orthophosphate and [35S]methionine labeling. Material suggesting the involvement of p210bcr-abl in high molecular weight complexes also appears using anti-Shc, anti-Grb2 and anti-phosphotyrosine antibodies. AG957 also acts in vitro to shift native p210bcr-abl in anti-p210bcr-abl or anti-Grb2 immunoprecipitates to higher molecular weight forms under conditions where the drug can also act as an antagonist of p210bcr-abl autokinase activity. Incubation with dithiothreitol inhibits the appearance of > 210 kDa forms of p210bcr-abl in the in vitro reaction. These results leads to the hypothesis that AG957 does not act simply as a reversible tyrosine kinase antagonist, but can alter the normal amounts and physical associations of molecules important in tyrosine kinase signaling. These effects likely reflect covalent cross-links induced by the drug.