The Src family kinase Hck interacts with Bcr-Abl by a kinase-independent mechanism and phosphorylates the Grb2-binding site of Bcr

Bcr-Abl, the oncogene causing chronic myeloid leukemia, encodes a fusion protein with constitutively active tyrosine kinase and transforming capacity in hematopoietic cells. Various intracellular signaling intermediates become activated and/or associate by/with Bcr-Abl, including the Src family kinase HCK. To elucidate some of the structural requirements and functional consequences of the association of Bcr-Abl with HCK, their interaction was investigated in transiently transfected COS7 cells. Neither the complex formation of HCK kinase with Bcr-Abl nor the activation of HCK by Bcr-Abl was dependent on the Abl kinase activity. Both inactivating point mutations of HCK and dephosphorylation of HCK enhanced its complex formation with Bcr-Abl, indicating that their physical interaction was negatively regulated by HCK (auto)phosphorylation. Finally, experiments with a series of kinase negative Bcr-Abl mutants showed that HCK phosphorylated Bcr-Abl and induced the binding of Grb2 to Tyr177 of Bcr-Abl. Taken together, our results suggest that Bcr-Abl preferentially binds inactive forms of HCK by an Abl kinase-independent mechanism. This physical interaction stimulates the HCK tyrosine kinase, which may then phosphorylate the Grb2-binding site in Bcr-Abl.