Coexistence of phosphotyrosine-dependent and -independent interactions between Cbl and Bcr-Abl.
Cbl is one of the major tyrosine-phosphorylated proteins in Bcr-Abl-expressing cells. A direct association between the SH2 domain of Bcr-Abl and tyrosine-phosphorylated Cbl has been demonstrated. The purpose of this study was to determine if and how unphosphorylated Cbl and Bcr-Abl may associate. Interactions between Cbl and Bcr-Abl were investigated in yeast two- and three-hybrid systems, gel overlay assays, and immunoprecipitates from mammalian cells expressing wild-type and the Y177F mutant of Bcr-Abl. No direct interaction between Bcr-Abl and unphosphorylated Cbl was observed. Bcr-Abl did, however, associate with Grb2, an adaptor protein that binds tyrosine 177 of Bcr-Abl. Additionally, Grb2 interacted with Cbl. In a yeast three-hybrid assay, Grb2 mediated an interaction between Cbl and Bcr-Abl that was dependent on a functional Grb2 binding site. This interaction was confirmed in vitro using purified proteins. In cells expressing Bcr-Abl with a mutation in the Grb2 binding site, binding of Cbl to Bcr-Abl was significantly reduced, but Cbl tyrosine phosphorylation was maintained. Imatinib treatment of these cells further reduced but did not abrogate Cbl binding, reflecting residual kinase activity. Multiple phosphotyrosine-dependent and -independent interactions stabilize the interaction between Cbl and Abl. Grb2 or another, yet unidentified, protein may mediate an initial interaction between Cbl and Bcr-Abl that is independent of Cbl tyrosine phosphorylation. Following this initial interaction, Cbl can then become tyrosine phosphorylated and interact with the SH2 domain of Bcr-Abl, further stabilizing the complex.
Gaston
Isabelle
I
Department of Molecular Microbiology and Immunology, Oregon Health and Science University, Portland, Ore. 97239, USA.
Johnson
Kara J
KJ
Oda
Tsukasa
T
Bhat
Arun
A
Reis
Margaret
M
Langdon
Wallace
W
Shen
Lei
L
Deininger
Michael W
MW
Druker
Brian J
BJ
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D001549:Benzamides; D016044:Fusion Proteins, bcr-abl / Q000378:metabolism*; D051380:GRB2 Adaptor Protein; D006801:Humans; D000068877:Imatinib Mesylate; D051379:Mice; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism*; D010879:Piperazines / Q000494:pharmacology; D011506:Proteins / Q000737:chemistry / Q000502:physiology; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D050721:Proto-Oncogene Proteins c-cbl; D011743:Pyrimidines / Q000494:pharmacology; D020798:Two-Hybrid System Techniques; D014443:Tyrosine / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases; D018909:src Homology Domains
D048868:Adaptor Proteins, Signal Transducing; D001549:Benzamides; D051380:GRB2 Adaptor Protein; C495948:GRB2 protein, human; C495949:Grb2 protein, mouse; D010879:Piperazines; D011506:Proteins; D011518:Proto-Oncogene Proteins; D011743:Pyrimidines; D019000:Phosphotyrosine; D014443:Tyrosine; D000068877:Imatinib Mesylate; D050721:Proto-Oncogene Proteins c-cbl; D044767:Ubiquitin-Protein Ligases; D016044:Fusion Proteins, bcr-abl; C067419:CBL protein, human; C493849:Cbl protein, mouse