Altered physical state of p210bcr-abl in tyrphostin AG957-treated K562 cells.
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.
Kaur
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Laboratory of Biological Chemistry, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Sausville
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EA
D000818:Animals; D000911:Antibodies, Monoclonal; D001597:Benzylidene Compounds / Q000494:pharmacology*; D015153:Blotting, Western; D002455:Cell Division / Q000187:drug effects; D016044:Fusion Proteins, bcr-abl / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D007553:Isotope Labeling; D051379:Mice; D008970:Molecular Weight; D009570:Nitriles / Q000494:pharmacology*; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D011233:Precipitin Tests; D012697:Serine Endopeptidases; D015398:Signal Transduction / Q000187:drug effects; D013462:Sulfur Radioisotopes; D014407:Tumor Cells, Cultured; D020032:Tyrphostins
D000911:Antibodies, Monoclonal; D001597:Benzylidene Compounds; D009570:Nitriles; D010761:Phosphorus Radioisotopes; D013462:Sulfur Radioisotopes; D020032:Tyrphostins; C104965:tyrphostin AG957; D016044:Fusion Proteins, bcr-abl; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase