THE SH2-CONTAINING ADAPTER PROTEIN GRB10 INTERACTS WITH BCR-ABL

Bcr-Abl is an oncogenic tyrosine kinase expressed in tumor cells of CM L and a subset of ALL which in its unregulated and activated state is thought to cause cell transformation and leukemia. Bcr-Abl contains se veral autophosphorylation sites which serve as potential docking sites for SH2-containing signaling molecules; Mutational analysis has indic ated that these autophosphorylation sites play a critical role in the transforming capability of Bcr-Abl, It has been shown that the SH2-con taining adapter protein Grb(2) binds to the autophosphorylation site T yr(p)177 whereby it couples Bcr-Abl to the Ras pathway. The biological consequences of this interaction, however, are presently unclear, A T yr177-mutated Bcr-Abl which lacks the ability to interact with the Grb 2-SH2 domain still transforms myeloid cells and generates tumors in nu de mice. We performed a yeast two-hybrid screen to identify signaling proteins which bind to distinct Bcr-Abl autophosphorylation sites. Aut ophosphorylation of Bcr-Abl in yeast was accomplished by using the DNA binding protein LexA which permits dimerization and crossphosphorylat ion of the fused bait. Using a LexA-Bcr-Abl full length fusion protein as bait, we identified several SH2-containing proteins. Among them we confirmed molecules already shown by others to interact with Bcr-Abl, irt vivo, including Grb2, PI-3-kinase and Crk indicating that dimeriz ation in yeast leads to autophosphorylation of tyrosine residues cruci al for Bcr-Abl signaling in vivo. More importantly, we identified the SH2-containing protein Grb10 as a new binding partner for Bcr-Abl. Thi s binding occurs in a phosphotyrosine-dependent manner at Bcr sites of Bcr-Abl. Both Abl and Bcr alone, as well as a kinase-defective Bcr-Ab l, failed to interact with Grb10 in yeast. Mutational analysis uncover ed a new SH2 binding site in Bcr-Abl located between Bcr aa242-446, wh ich is different from the Grb2 binding site. Binding could be demonstr ated in vitro and also in vivo as shown by co-immunoprecipitation anal ysis in CML cells. Using a temperature sensitive Bcr-Abl stably overex pressed in hematopoetic cells, we demonstrated that complex formation of Grb10 with Bcr-Abl was kinase activation-dependent in vivo. Notably , a Bcr-Abl mutant protein (Bcr/1-242-Abl) which lacks the ability to interact with Grb10 partially alleviated IL-3 dependence of Ba/F3 cell s, indicating that the Grb10/Bcr-Abl interaction is important for Bcr- Abl-induced IL-3 independence of Ba/F3 cells. In addition, the Bcr/1-2 42-Abl mutant has a reduced capacity to induce focus formation in fibr oblasts.