unctional Characterization of Brk and its Substrates in Cell Migration

Brk (Breast tumor kinase), a nonreceptor tyrosine kinase, was originally identified from a functional screen for identifying the tyrosine kinases overexpressed in metastatic breast cancers. Previous studies have shown that Brk is overexpressed in two-thirds of breast tumors and several other cancer types. However, the molecular mechanism in which this kinase participates with respect to tumorigenesis remains poorly characterized. In this thesis, we identified two substrates of Brk and raveled their signaling pathways to dissect Brk’s role in tumor progression. In the first part of study, we demonstrate that EGF stimulation activates the catalytic activity of Brk, which in turn phosphorylates paxillin at Y31 and Y118. These phosphorylation events provide the binding site for CrkII which recruits DOCK180-Elmo to promote the activation of small GTPase Rac1. Through this pathway, Brk possesses the ability to promote cell migration and invasion and functions as a mediator of EGF-induced migratory occurrence. In the second part of this thesis, we identified that p190RhoGAP is another substrate of Brk. Brk phosphorylates p190RhoGAP at the Y1105 residue, thereby promoting p190RhoGAP association with p120RasGAP. This complex formation between p190RhoGAP and p120RasGAP stimulates the ability of p190RhoGAP to downregulate RhoA, while inhibits p120RasGAP activity toward Ras inactivation. As a result, Brk oppositely regulates RhoA and Ras and mediates EGF-induced RhoA inactivation. Consistent with its inactivation of RhoA, Brk inhibits stress fiber formation. Furthermore, we demonstrated that p190RhoGAP is required for Brk-induced chemotactic migration towards EGF. Together, our findings reveal previously unknown function of Brk in regulating three small GTPases─Rho, Rac, Ras via phosphorylating paxillin and p190RhoGAP. The identification of these signaling pathways transduced by Brk would shed light on the role of Brk in tumor progression.