Src family kinases phosphorylate the Bcr-Abl SH3-SH2 region and modulate Bcr-Abl transforming activity.
Bcr-Abl is the oncogenic protein-tyrosine kinase responsible for chronic myelogenous leukemia. Recently, we observed that inhibition of myeloid Src family kinase activity (e.g. Hck, Lyn, and Fyn) induces growth arrest and apoptosis in Bcr-Abl-transformed cells, suggesting that cell transformation by Bcr-Abl involves Src family kinases (Wilson, M. B., Schreiner, S. J., Choi, H. J., Kamens, J., and Smithgall, T. E. (2002) Oncogene 21, 8075-8088). Here, we report the unexpected observation that Hck, Lyn, and Fyn strongly phosphorylate the SH3-SH2 region of Bcr-Abl. Seven phosphorylation sites were identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry: Tyr89 and Tyr134 in the Abl-derived SH3 domain; Tyr147 in the SH3-SH2 connector; and Tyr158, Tyr191, Tyr204, and Tyr234 in the SH2 domain. SH3 domain Tyr89, the most prominent phosphorylation site in vitro, was strongly phosphorylated in chronic myelogenous leukemia cells in a Src family kinase-dependent manner. Substitution of the SH3-SH2 tyrosine phosphorylation sites with phenylalanine substantially reduced Bcr-Abl-mediated transformation of TF-1 myeloid cells to cytokine independence. The positions of these tyrosines in the crystal structure of the c-Abl core and the transformation defect of the corresponding Bcr-Abl mutants together suggest that phosphorylation of the SH3-SH2 region by Src family kinases impacts Bcr-Abl protein conformation and signaling.
Meyn
Malcolm A
MA
Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261, USA.
Wilson
Matthew B
MB
Abdi
Fadi A
FA
Fahey
Nathalie
N
Schiavone
Anthony P
AP
Wu
Jiong
J
Hochrein
James M
JM
Engen
John R
JR
Smithgall
Thomas E
TE
D001665:Binding Sites; D002471:Cell Transformation, Neoplastic; D016044:Fusion Proteins, bcr-abl / Q000737:chemistry*; D006801:Humans; D009154:Mutation; D010766:Phosphorylation; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D015398:Signal Transduction; D014443:Tyrosine / Q000737:chemistry; D018909:src Homology Domains; D019061:src-Family Kinases / Q000737:chemistry / Q000502:physiology*
D014443:Tyrosine; D016044:Fusion Proteins, bcr-abl; D019061:src-Family Kinases