Figure 2.
Interaction between p62dok and RasGAP. (a) Tagged p62dok was expressed in E. coli, purified, and incubated in a kinase reaction in the presence of Abl kinase (+) or buffer only (−). After the kinase reaction, p62dok was incubated in an in vitro-binding reaction with glutathione-Sepharose beads to which were bound GST(−), GST-GAP SH2-SH3-SH2 (232), GST-GAP N-SH2 (N), or GST-GAP C-SH2 (C). Proteins bound to GST fusion proteins were separated by SDS/PAGE, blotted to nitrocellulose, and detected by immunoblotting with antibodies to p62dok (α-p62dok) or phosphotyrosine (α-pTyr). (b) Mammalian-expressed p62dok associates with RasGAP in a phosphorylation-dependent manner. (Fig. 1b). C-terminally tagged Dok-1 cDNA was electroporated into 293 cell in the presence (+) or absence (−) of Bcr-Abl. After a 24-h incubation, cells were lysed, and lysates were utilized in an in vitro- binding reaction with glutathione-Sepharose beads to which were bound GST(−), GST-GAP SH2-SH3-SH2 (232), GST-GAP N-SH2 (N), or GST-GAP C-SH2 (C). Proteins bound to GST fusion proteins were separated by SDS/PAGE, blotted to nitrocellulose, and detected by immunoblotting with antibodies to the C-terminal tag (α-tag) or phosphotyrosine (α-pTyr).