Brk Activates Rac1 and Promotes Cell Migration and Invasion by Phosphorylating Paxillin

Abstract

Brk (for breast tumor kinase) is a nonreceptor tyrosine kinase containing SH3, SH2, and tyrosine kinase catalytic domains. Brk was originally identified from a human metastatic breast tumor, and its overexpression is frequently observed in breast cancer and several other cancer types. However, the molecular mechanism by which this kinase participates in tumorigenesis remains poorly characterized. In the present study, we not only identified paxillin as the binding partner and substrate of Brk but also discovered a novel signaling pathway by which Brk mediates epidermal growth factor (EGF)-induced paxillin phosphorylation. We show that EGF stimulation activates the catalytic activity of Brk, which in turn phosphorylates paxillin at Y31 and Y118. These phosphorylation events promote the activation of small GTPase Rac1 via the function of CrkII. Through this pathway, Brk is capable of promoting cell motility and invasion and functions as a mediator of EGF-induced migration and invasion. In accordance with these functional roles, Brk translocates to membrane ruffles, where it colocalizes with paxillin during cell migration. Together, our findings identify novel signaling and biological roles of Brk and indicate the first potential link between Brk and metastatic malignancy.Unraveling the signaling pathways responsible for the establishment of a metastatic phenotype in carcinoma cells is of crucial importance for the understanding of the pathology of cancer. The process of metastasis includes several components, such as the ability to invade through acquisition of cell motility, degradation of extracellular matrix and basement membrane, cell proliferation, and survival signaling. Aberrant tyrosine kinase signaling via stimulation of growth factor receptors or intracellular tyrosine kinases has been shown to contribute to various steps of tumor development and progression, including metastasis (6). Brk is an intracellular tyrosine kinase that was identified in a study for screening kinases expressed in human metastatic breast tumors (36). In addition to a typical tyrosine kinase domain, Brk possesses both SH3 and SH2 domains and thus is related to Src family kinases (36). However, unlike Src family kinases, Brk lacks an N-terminal consensus sequence for myristoylation and membrane association (36). Its genomic structure is also distinct from that of Src family kinases, suggesting that Brk has diverged significantly from Src kinases in evolution (37). The expression pattern and subcellular localization of Brk have suggested its role in tumorigenesis. In normal tissues, the expression of Brk or its mouse ortholog Sik is restricted to differentiating epithelial cells of the skin and gastrointestinal tract (27). However, it is highly expressed in many breast carcinoma cell lines and a significant portion of breast tumor tissues but not in human mammary epithelial cells (3, 34, 36) and mouse mammary glands at various developmental stages (27). Elevated expression of Brk has also been detected in metastatic melanoma cell li...

TIMP-2 upregulates RECK expression via dephosphorylation of paxillin tyrosine residues 31 and 118

“…On the contrary, the complex formation of paxillin with Crk and C3G, an activator of Rap1, is not reduced, rather slightly but reproducibly increased by TIMP-2. This result is consistent with our previous finding (Oh et al, 2004) and also with the recent reports that phosphorylation of paxillin at Tyr-31/118 promotes its association with Crk and DOCK180 and the activation of Rac1 (Chen et al, 2004;Valles et al, 2004). The potential involvement of p130 CAS , another major target protein of FAK/Src kinase, in TIMP-2 signaling was examined by immunoprecipitation followed by immunoblotting.…”

SH2 Domains Recognize Contextual Peptide Sequence Information to Determine Selectivity

“…The presence of non-permissive residues for Brk and permissive residues for Crk within YXXP motifs in Cbl and p130Cas helps explain why Crk is a highly connected node within this particular network whereas Brk is poorly connected. As an aside, the localization and interaction data suggest that paxillin (Pxn) must function as a hub for interaction with both Crk and Brk connecting them to the signaling machinery involved in cell spreading (59,60). Recruitment of Brk is an important step for the phosphorylation of paxillin.…”

Identification of STAT3 as a specific substrate of breast tumor kinase

“…To eliminate the possibility that STAT3 is phosphorylated in response to autocrine interleukin-6 (IL-6), we tested the effect of inhibition of the JAK-STAT3 pathway stimulated by IL-6. The reason for testing this pathway is that one of the few direct substrates identified for Brk is paxillin, and phosphorylated paxillin activates Rac1 (Chen et al, 2004). Our previous study established the ability of activated Rac1 to induce the production and autocrine action of IL-6 leading to the phosphorylation of STAT3.…”