Tyrosine phosphorylation in the SH3 domain disrupts negative regulatory interactions within the c-Abl kinase core.
Recent studies have shown that trans-phosphorylation of the Abl SH3 domain at Tyr89 by Src-family kinases is required for the full transforming activity of Bcr-Abl. Tyr89 localizes to a binding surface of the SH3 domain that engages the SH2-kinase linker in the crystal structure of the c-Abl core. Displacement of SH3 from the linker is likely to influence efficient downregulation of c-Abl. Hydrogen-deuterium exchange (HX) and mass spectrometry (MS) were used to investigate whether Tyr89 phosphorylation affects the ability of the SH3 domain to interact intramolecularly with the SH2-kinase linker in cis as well as other peptide ligands in trans. HX MS analysis of SH3 binding showed that when various Abl constructs were phosphorylated at Tyr89 by the Src-family kinase Hck, SH3 was unable to engage a high-affinity ligand in trans and that interaction with the linker in cis was reduced dramatically in a construct containing the SH3 and SH2 domains plus the linker. Phosphorylation of the Abl SH3 domain on Tyr89 also interfered with binding to the negative regulatory protein Abi-1 in trans. Site-directed mutagenesis of Tyr89 and Tyr245, another tyrosine phosphorylation site located in the linker that may also influence SH3 binding, implicated Tyr89 as the key residue necessary for disrupting regulation after phosphorylation. These results imply that phosphorylation at Tyr89 by Src-family kinases prevents engagement of the Abl SH3 domain with its intramolecular binding partner leading to enhanced Abl kinase activity and cellular signaling.
Chen
Shugui
S
Chemistry & Chemical Biology and The Barnett Institute of Chemical & Biological Analysis, Northeastern University, Boston, MA 02115, USA.
O'Reilly
Linda P
LP
Smithgall
Thomas E
TE
Engen
John R
JR
D000595:Amino Acid Sequence; D001665:Binding Sites; D016044:Fusion Proteins, bcr-abl / Q000737:chemistry / Q000378:metabolism; D016313:Genes, abl; D006801:Humans; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011487:Protein Conformation; D016315:Proto-Oncogene Proteins c-abl / Q000737:chemistry* / Q000378:metabolism*; D015398:Signal Transduction; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism*; D018909:src Homology Domains / Q000235:genetics
D014443:Tyrosine; D016044:Fusion Proteins, bcr-abl; D016315:Proto-Oncogene Proteins c-abl
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