Role of the Brk SH3 domain in substrate recognition.
Breast tumor kinase (Brk) is a nonreceptor tyrosine kinase that is overexpressed in a high percentage of breast carcinomas. Brk contains SH3, SH2, and tyrosine kinase catalytic domains in a similar arrangement as Src family kinases. In this study, we explored the roles of the SH3 and SH2 domains in Brk regulation and substrate binding. We introduced a series of mutations into Brk that were predicted to disrupt the intramolecular interactions involving the SH3 and SH2 domains. These mutant forms of Brk displayed higher activity than wild-type Brk when expressed in human embryonic kidney HEK293 cells. These studies also allowed us to pinpoint the intramolecular binding site for the SH3 domain. To examine substrate binding, we compared binding and phosphorylation of Sam68, a physiological substrate of Brk. These experiments showed that the SH3 domain plays a particularly important role in substrate recognition. We confirmed this conclusion using a series of synthetic peptides in which a substrate sequence was coupled to an SH3 or SH2 ligand. The SH3-binding substrate had a significantly lower K(m) than a control, while no difference was observed between an SH2-binding substrate and a control. Taken together, our data suggest that SH3 interactions will govern phosphorylation of many substrates by Brk.
Qiu
Haoqun
H
Department of Physiology and Biophysics, Basic Science Tower, T-6, School of Medicine, State University of New York at Stony Brook, Stony Brook, NY 11794-8661, USA.
Miller
W Todd
WT
D048868:Adaptor Proteins, Signal Transducing; D016367:Baculoviridae / Q000235:genetics; D001665:Binding Sites; D020134:Catalytic Domain / Q000502:physiology; D002460:Cell Line; D004268:DNA-Binding Proteins; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000196:embryology; D007700:Kinetics; D008024:Ligands; D016297:Mutagenesis, Site-Directed; D009363:Neoplasm Proteins; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D011505:Protein-Tyrosine Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism; D013379:Substrate Specificity; D018909:src Homology Domains / Q000502:physiology*
D048868:Adaptor Proteins, Signal Transducing; D004268:DNA-Binding Proteins; C489559:KHDRBS1 protein, human; D008024:Ligands; D009363:Neoplasm Proteins; D010455:Peptides; D016601:RNA-Binding Proteins; D011505:Protein-Tyrosine Kinases; C088375:PTK6 protein, human