The SH2-containing adapter protein GRB10 interacts with BCR-ABL.
Bcr-Abl is an oncogenic tyrosine kinase expressed in tumor cells of CML and a subset of ALL which in its unregulated and activated state is thought to cause cell transformation and leukemia. Bcr-Abl contains several autophosphorylation sites which serve as potential docking sites for SH2-containing signaling molecules. Mutational analysis has indicated that these autophosphorylation sites play a critical role in the transforming capability of Bcr-Abl. It has been shown that the SH2-containing adapter protein Grb2 binds to the autophosphorylation site Tyr(p)177 whereby it couples Bcr-Abl to the Ras pathway. The biological consequences of this interaction, however, are presently unclear. A Tyr177-mutated Bcr-Abl which lacks the ability to interact with the Grb2-SH2 domain still transforms myeloid cells and generates tumors in nude mice. We performed a yeast two-hybrid screen to identify signaling proteins which bind to distinct Bcr-Abl autophosphorylation sites. Autophosphorylation of Bcr-Abl in yeast was accomplished by using the DNA binding protein LexA which permits dimerization and crossphosphorylation 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, in vivo, including Grb2, PI-3-kinase and Crk indicating that dimerization in yeast leads to autophosphorylation of tyrosine residues crucial for Bcr-Abl signaling in vivo. More importantly, we identified the SH2-containing protein Grb10 as a new binding partner for Bcr-Abl. This 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-Abl, failed to interact with Grb10 in yeast. Mutational analysis uncovered a new SH2 binding site in Bcr-Abl located between Bcr aa242-446, which is different from the Grb2 binding site. Binding could be demonstrated in vitro and also in vivo as shown by co-immunoprecipitation analysis in CML cells. Using a temperature sensitive Bcr-Abl stably overexpressed 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 cells, indicating that the Grb10/Bcr-Abl interaction is important for Bcr-Abl-induced IL-3 independence of Ba/F3 cells. In addition, the Bcr/1-242-Abl mutant has a reduced capacity to induce focus formation in fibroblasts.
Bai
R Y
RY
Department of Internal Medicine III, Technical University of Munich, Germany.
Jahn
T
T
Schrem
S
S
Munzert
G
G
Weidner
K M
KM
Wang
J Y
JY
Duyster
J
J
D000042:Absorption; D002460:Cell Line; D002471:Cell Transformation, Neoplastic / Q000235:genetics; D004789:Enzyme Activation; D066246:ErbB Receptors / Q000378:metabolism; D016044:Fusion Proteins, bcr-abl / Q000235:genetics / Q000378:metabolism*; D051383:GRB10 Adaptor Protein; D005822:Genetic Vectors / Q000378:metabolism; D006801:Humans; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000378:metabolism; D009154:Mutation; D011485:Protein Binding; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011506:Proteins / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics; D014162:Transfection; D014407:Tumor Cells, Cultured; D018909:src Homology Domains
D011506:Proteins; D051383:GRB10 Adaptor Protein; D066246:ErbB Receptors; D011505:Protein-Tyrosine Kinases; D016044:Fusion Proteins, bcr-abl