HAGBP disrupts Grb2-SoS complexes and reduces MAPK activity.

K562 cells were incubated as detailed in Materials and methods but with only 10 μM of the peptide indicated. This leads to a significant reduction of proliferation in the case of peptide P6 (HAGBP; Figure 1) but still allows some growth, which is essential to yield enough protein for the precipitation experiments. (A) Immunoprecipitation (IP) and Western blot (WB) were carried out with the antibodies indicated as described in Materials and methods. A total of 40 μg of total K562 lysate from untreated cells was also loaded as a control. Note that complexes of Grb2 are detectable with p210^Bcr-Abl, but not with the normal cellular p145^c-Abl protein. (B) Total lysates of K562 cells treated as in (A) were separated by SDS-PAGE, blotted, and probed with anti-MAPK or activation-specific phospho-MAPK antibodies, as described in Materials and methods. (C) A total of 250 μg of K562 total cell protein from cells treated with 10 μM of peptide, where indicated, was precipitated with anti-MAPK antibody in the presence of protease and phosphatase inhibitors to maintain the kinase activity state from the time point of cell lysis. After precipitating anti-MAPK with protein A–sepharose and removing unbound proteins by several wash steps, samples were analyzed by in vitro kinase assay with MBP as substrate. A representative experiment is shown. (D) Statistical analysis of 4 independent in vitro kinase experiments with lysates of cells treated with 10 μM of the indicated peptides. Incorporated radioactivity was determined by phosphoimager and quantified using the TINA 2.09d software.