Brk activates rac1 and promotes cell migration and invasion by phosphorylating paxillin

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.     

Regulation of the nonreceptor tyrosine kinase Brk by autophosphorylation and by autoinhibition

Brk (breast tumor kinase) is a nonreceptor tyrosine kinase that is most closely related to the Frk family of kinases, and more distantly to Src family kinases. Brk was originally identified in a screen for tyrosine kinases that are overexpressed in human metastatic breast tumors. To shed light on the activity and regulation of Brk and related tyrosine kinases, we expressed and purified Brk using the Sf9/baculovirus system. We characterized the substrate specificity of Brk using synthetic peptides, and we show that the kinetic parameters K(m) and k(cat) both play a role in specificity. We carried out mass spectrometry experiments to show that Brk autophosphorylates within the predicted kinase activation loop and at additional sites in the N terminus. Autophosphorylation increases enzyme activity of wild-type Brk but not of a Y342A mutant form of Brk. We also carried out experiments to address the possible involvement of the Src homology (SH) 2 and SH3 domains of Brk in enzyme regulation. Mutation of a C-terminal tyrosine (Tyr-447) increases enzyme activity and SH2 domain accessibility, consistent with a role for this residue in autoinhibition. A proline-rich peptide activates Brk, suggesting that the SH3 domain is also involved in maintaining an inactive form of Brk. These biochemical results for Brk may aid in the understanding of other tyrosine kinases in the Frk family.     

HER2 regulates Brk/PTK6 stability via upregulating calpastatin,an inhibitor of calpain

Breast tumor kinase (Brk), also known as protein kinase-6 (PTK6), is a nonreceptor protein-tyrosine kinase that has a close functional relationship with the human epidermal growth factor receptor 2 (HER2). High levels of Brk were found in HER2-positive tumor specimens from patients with invasive ductal breast cancer; however, the underlying mechanism of the co-overexpression of Brk and HER2 remains elusive. In the current study, we explored the mechanism of HER2 and Brk co-overexpression in breast cancer cells by investigating the effect of overexpression and knockdown of HER2 on the level of Brk in breast cancer cells. We found that Brk was more stable in HER2-elevated cells than in control vector-transfected cells and was less stable in HER2 siRNA-treated cells than in control siRNA-treated cells, suggesting that HER2 regulates Brk protein stability. Further studies indicated that degradation of Brk involved a calpain-1-mediated proteolytic pathway and indicated an inverse relationship between the level of HER2 expression and calpain-1 activity. We found that HER2 inhibited calpain-1 activity through upregulating calpastatin, an endogenous calpain inhibitor. Silencing of HER2 downregulated calpastatin, and the downregulation could be rescued by overexpression of constitutively active MEK. Together, these data offer novel mechanistic insights into the functional relationship between Brk and HER2.     

Inhibitors of farnesyl protein transferase and MEK1,2 induce apoptosis in fibroblasts transformed with farnesylated but not geranylgeranylated H-Ras

Farnesyl protein transferase inhibitors (FTIs) reverse the transformed phenotype of fibroblasts expressing activated H-Ras and block anchorage-independent growth and tumorigenesis of tumor cell lines independent of their Ras mutational status. FTIs induce significant tumor regression accompanied by apoptosis in several transgenic mouse tumor models. FTI treatment of tumor cells in vitro is proapoptotic under certain cell culture conditions. Induction of apoptosis by FTIs in vitro generally requires a second death-promoting signal. To better understand FTI-induced apoptosis we analyzed the effect of SCH 66336, a tricyclic FTI, on apoptosis of Ras-transformed Rat2 fibroblasts. Treatment of H-Ras-CVLS-transformed fibroblasts with MEK1,2 inhibitors provides a pharmacological second signal to enhance FTI-induced apoptosis. Simultaneous treatment of these cells with a MEK1,2 inhibitor markedly enhanced caspase-3 activity and the apoptotic response to SCH 66336. The combination treatment resulted in a more complete and sustained inhibition of MAPK pathway activity than observed with either drug alone. Surprisingly, after treatment with either agent alone or in combination, no apoptotic response was observed in Rat2 cells transformed with a geranylgeranylated form of H-Ras (H-Ras-CVLL). Differences were also observed when SCH 66336 treatment was combined with forced suspension growth or serum withdrawal, in that an increase in drug-induced apoptosis was observed in H-Ras-CVLS-transformed Rat2 cells but not H-Ras-CVLL-transformed Rat2 cells. The lack of apoptotic effect of SCH 66336 and MEK inhibitor, alone or in combination, in H-Ras-CVLL-transformed cells suggests a difference in the reliance of cells transformed with farnesylated and geranylgeranylated forms of H-Ras on the MAPK signal transduction cascade for survival. K-Ras-transformed cells underwent apoptosis upon MEK1,2 inhibition but not in response to SCH 66336 treatment. The apoptotic response induced by MEK1,2 inhibitors is much greater in magnitude in H-Ras-transformed cells than in K-Ras-transformed cells, also pointing to differences in pathway utilization and/or dependence for these two Ras isoforms.     

Brk/Protein Tyrosine Kinase 6 Phosphorylates p27KIP1, Regulating the Activity of Cyclin D–Cyclin-Dependent Kinase 4

Cyclin D and cyclin-dependent kinase 4 (cdk4) are overexpressed in a variety of tumors, but their levels are not accurate indicators of oncogenic activity because an accessory factor such as p27^Kip1 is required to assemble this unstable dimer. Additionally, tyrosine (Y) phosphorylation of p27 (pY88) is required to activate cdk4, acting as an “on/off switch.” We identified two SH3 recruitment domains within p27 that modulate pY88, thereby modulating cdk4 activity. Via an SH3-PXXP interaction screen, we identified Brk (breast tumor-related kinase) as a high-affinity p27 kinase. Modulation of Brk in breast cancer cells modulates pY88 and increases resistance to the cdk4 inhibitor PD 0332991. An alternatively spliced form of Brk (Alt Brk) which contains its SH3 domain blocks pY88 and acts as an endogenous cdk4 inhibitor, identifying a potentially targetable regulatory region within p27. Brk is overexpressed in 60% of breast carcinomas, suggesting that this facilitates cell cycle progression by modulating cdk4 through p27 Y phosphorylation. p27 has been considered a tumor suppressor, but our data strengthen the idea that it should also be considered an oncoprotein, responsible for cyclin D-cdk4 activity.     

BRCA1 facilitates stress-induced apoptosis in breast and ovarian cancer cell lines

The BRCA1 tumor suppressor gene has previously been implicated in induction of high levels of apoptosis in osteocarcinoma cell lines. Overexpression of BRCA1 was shown to induce an apoptotic signaling pathway involving the c-Jun N-terminal kinase (JNK), but the signaling steps upstream and downstream of JNK were not delineated. To better understand the role of BRCA1 in apoptosis, we examined the effect of wild-type and C-terminal-truncated dominant negative BRCA1 on breast and ovarian cancer cell lines subjected to a number of different pro-apoptotic stimuli, including growth factor withdrawal, substratum detachment, ionizing radiation, and treatment with anticancer agents. All of these treatments were found to induce substantial levels of apoptosis in the presence of wild-type BRCA1, whereas dominant negative BRCA1 truncation mutants diminished the apoptotic response. Subsequent mapping of the apoptotic pathway induced by growth factor withdrawal demonstrated that BRCA1 enhanced signaling through a pathway that sequentially involved H-Ras, MEKK4, JNK, Fas ligand/Fas interactions, and caspase-9 activation. In addition, the pathway functioned independently of the p53 tumor suppressor. These data suggest that BRCA1 is an important modulator of the response to cellular stress and that loss of this apoptotic potential due to BRCA1 mutations may contribute to tumor development.     

ROR1 is expressed in human breast cancer and associated with enhanced tumor-cell growth

Receptor-tyrosine-kinase-like orphan receptor 1 (ROR1) is expressed during embryogenesis and by certain leukemias, but not by normal adult tissues. Here we show that the neoplastic cells of many human breast cancers express the ROR1 protein and high-level expression of ROR1 in breast adenocarcinoma was associated with aggressive disease. Silencing expression of ROR1 in human breast cancer cell lines found to express this protein impaired their growth in vitro and also in immune-deficient mice. We found that ROR1 could interact with casein kinase 1 epsilon (CK1ε) to activate phosphoinositide 3-kinase-mediated AKT phosphorylation and cAMP-response-element-binding protein (CREB), which was associated with enhanced tumor-cell growth. Wnt5a, a ligand of ROR1, could induce ROR1-dependent signaling and enhance cell growth. This study demonstrates that ROR1 is expressed in human breast cancers and has biological and clinical significance, indicating that it may be a potential target for breast cancer therapy.     

Repression of dpp targets by binding of brinker to mad sites

Signaling by decapentaplegic (Dpp), a Drosophila member of the transforming growth factor (TGF) beta superfamily of growth factors, has recently been shown to activate targets such as vestigial (vg) indirectly through negative regulation of brinker (brk). Here we show that the Brk protein functions as a repressor by binding to Dpp response elements. The Brk DNA binding activity was localized to an amino-terminal region containing a putative homeodomain. Brk bound to a Dpp response element of the Ultrabithorax (Ubx) midgut enhancer at a sequence that overlaps a binding site for the Smad protein, Mothers Against Dpp (Mad). Furthermore, Brk was able to compete with Mad for occupancy of this binding site. This recognition of overlapping binding sites provides a potential explanation for why the G/C-rich Mad binding site consensus differs the Smad3/Smad4 binding site consensus. We also found that the Dpp response element from Ubx was more sensitive than the vg quadrant enhancer to repression by Brk. This difference correlates with short-range activation of Ubx by Dpp in the visceral mesoderm, whereas vg exhibits a long-range response to Dpp in the wing imaginal disc, indicating that Brk binding sites may play a critical role in limiting thresholds for activation by Dpp. Finally, we provide evidence that Brk is capable of functioning as an active repressor. Thus, whereas Brk and Mad compete for regulation of Ubx and vg, Brk may regulate other Dpp targets without direct involvement of Mad.     

Differential regulation of the clusterin gene by Ha-ras and c-myc oncogenes and during apoptosis

Clusterin (ApoJ) is an extracellular glycoprotein expressed during processes of tissue differentiation and regression that involve programmed cell death (apoptosis). Increased clusterin expression has also been found in tumors, however, the mechanism underlying this induction is not known. Apoptotic processes in tumors could be responsible for clusterin gene activation. Alternatively, oncogenic mutations could modulate signal transduction, thereby inducing the gene. We examined the response of the rat clusterin gene to two oncogenes, Ha-ras and c-myc, in transfected Rat1 fibroblasts. While c-myc overexpression did not modify clusterin gene activity, the Ha-ras oncogene produced a seven to tenfold repression of clusterin mRNA; this down-regulation was also observed in the presence of c-myc. Since no induction of the clusterin gene was observed by the two oncogenes, we tested the alternative mechanism involving apoptosis. Growth factor withdrawal induced apoptosis, as shown by DNA degradation and micronuclei formation in the floating cells. Concomittantly we observed a three to tenfold increase in the amount of clusterin mRNA in the adhering cells of Rat1 and the c-myc transformed cell lines, and a weaker induction in the Ha-ras transformed cell line. On the basis of our results, we suggest that clusterin gene induction in the vital cells is produced by signaling molecules that are generated by the apoptotic cells. We conclude that apoptotic processes, not oncogenic mutations, are responsible for increased clusterin expression in tumors.     

PTK6 Inhibits Down-regulation of EGF Receptor through Phosphorylation of ARAP1

PTK6 (also known as Brk) is a non-receptor-tyrosine kinase containing SH3, SH2, and catalytic domains, that is expressed in more than 60% of breast carcinomas but not in normal mammary tissues. To analyze PTK6-interacting proteins, we have expressed Flag-tagged PTK6 in HEK293 cells and performed co-immunoprecipitation assays with Flag antibody-conjugated agarose. A 164-kDa protein in the precipitated fraction was identified as ARAP1 (also known as centaurin δ-2) by MALDI-TOF mass analysis. ARAP1 associated with PTK6 in an EGF/EGF receptor (EGFR)-dependent manner. In addition, the SH2 domain of PTK6, particularly the Arg¹⁰⁵ residue that contacts the phosphate group of the tyrosine residue, was essential for the association. Moreover, PTK6 phosphorylated residue Tyr²³¹ in the N-terminal domain of ARAP1. Expression of ARAP1, but not of the Y231F mutant, inhibited the down-regulation of EGFR in HEK293 cells expressing PTK6. Silencing of endogenous PTK6 expression in breast carcinoma cells decreased EGFR levels. These results demonstrate that PTK6 enhances EGFR signaling by inhibition of EGFR down-regulation through phosphorylation of ARAP1 in breast cancer cells.     

Three Brick genes have distinct functions in a common pathway promoting polarized cell division and cell morphogenesis in the maize leaf epidermis

We have taken a genetic approach to investigating cytoskeleton-dependent mechanisms governing cell morphogenesis in the maize leaf epidermis. Previously, we showed that the Brick1 (Brk1) gene is required for the formation of epidermal cell lobes as well as for properly polarized divisions of stomatal subsidiary mother cells, and encodes an 8 kDa protein highly conserved in plants and animals. Here, we show that two additional Brick genes, Brk2 and Brk3, are involved in the same aspects of epidermal cell morphogenesis and division. As shown previously for Brk1, analysis of the cytoskeleton shows that Brk2 and Brk3 are required for the formation of local F-actin enrichments associated with lobe outgrowth in wild-type cells. Analysis of brk1;brk2, brk1;brk3 and brk2;brk3 double mutants shows that their phenotypes are the same as those of brk single mutants. Mosaic analysis shows that Brk1 acts non cell-autonomously over a short distance. By contrast, Brk2 and Brk3 act cell-autonomously to promote pavement cell lobe formation, but Brk3 acts non cell-autonomously, and Brk2 partially non cell-autonomously, to promote polarized subsidiary mother cell divisions. Together, these observations indicate that all three Brk genes act in a common pathway in which each Brk gene has a distinct function. Recent work demonstrating a function for the mammalian homolog of BRK1 (HSPC300) in activation of Arp2/3-dependent actin polymerization implicates the Brk pathway in local regulation of actin polymerization in plant cells.     

Ras-dependent apoptosis correlates with persistent activation of stress-activated protein kinases and induction of isoform(s) of Bcl-x

Ras proteins are signal transducers for many cellular responses. However, it is not well established whether Ras-signaling also contributes to apoptosis. We have constructed H-RasR12-transformed Rat1 fibroblasts using tetracycline operator/repressor (TetO/TetR)-based conditional vectors. Rat1/TetO-RasR12 (Rat1-Ras) cells produced high levels of H-RasR12 protein and exhibited oncogenic transformation. Treatment of Rat1-Ras cells with 0.1% serum triggered massive apoptosis. Rat1-Ras cells expressed increased basal activities of extracellular response kinase (ERK) and p46/p54 stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK). Interestingly, Ras-dependent apoptosis correlated with further persistent activation of both p46 and p54 SAPK/JNK and concurrent inhibition of ERK. Differential modulation of SAPK/JNK and ERK was not detected in tetracycline-treated cells that did not commit apoptosis. Furthermore, two Bcl-x related proteins of 15 kDa and 18 kDa were highly induced in apoptotic Rat1-Ras cells. Our results establish a direct role for Ras in apoptosis, and suggest a functional relationship between H-Ras, SAPK/JNK, ERK and Bcl-x in regulating apoptosis.     

Hpr6.6 protein mediates cell death from oxidative damage in MCF-7 human breast cancer cells

Reactive oxygen species (ROS) cause cell death and are associated with a variety of maladies, from trauma and infection to organ degeneration and cancer. Cells mount a complex response to oxidative damage that includes signaling from transmembrane receptors and intracellular kinases. We have analyzed the response to oxidative damage in human breast cancer cells expressing the Hpr6.6 (human membrane progesterone receptor) protein. Although Hpr6.6 is related to a putative progesterone-binding protein, Hpr6.6 is widely expressed in epithelial tissues and shares close homology with a budding yeast damage response protein called Dap1p (damage response protein related to membrane progesterone receptor). We report here that the Hpr6.6 protein regulates the response to oxidative damage in breast cancer cells. Expression of Hpr6.6 in MCF-7 cells sensitized the cells to death following long-term/low dose or short-term/high dose treatment with hydrogen peroxide. Cell death did not occur through a typical apoptotic mechanism and corresponded with hyperphosphorylation of the Akt and IkappaB proteins. However, inhibition of Akt activation and IkappaB degradation had no effect on Hpr6.6-mediated cell death, suggesting that Hpr6.6 regulates cell death through a novel oxidative damage response pathway. Our work indicates a key regulatory function for Hpr6.6 in epithelial tissues exposed to oxidative damage.     

Interactions of STAP-2 with Brk and STAT3 participate in cell growth of human breast cancer cells

STAP-2 (signal transducing adaptor protein-2) is a recently identified adaptor protein that contains pleckstrin homology (PH) and Src homology 2-like domains, as well as a STAT3-binding motif in its C-terminal region. STAP-2 is also a substrate of breast tumor kinase (Brk). In breast cancers, Brk expression is deregulated and promotes STAT3-dependent cell proliferation. In the present study, manipulated STAP-2 expression demonstrated essential roles of STAP-2 in Brk-mediated STAT3 activation. STAP-2 interacts with both Brk and STAT3. In addition, small interfering RNA-mediated reduction of endogenous STAP-2 expression strongly decreased Brk-mediated STAT3 activation in T47D breast cancer cells. The PH domain of STAP-2 is involved in multiple steps: the binding between Brk and STAP-2, the activation and tyrosine phosphorylation of STAT3, and the activation of Brk. Notably, a STAP-2 PH-Brk fusion protein exhibited robust kinase activity and increased activation and tyrosine phosphorylation of STAT3. Finally, STAP-2 knockdown in T47D cells induced a significant decrease of proliferation, as strong as that of Brk or STAT3 knockdown. Taken together, our findings are likely to inform the development of a novel therapeutic strategy, as well as the determination of novel prognostic values, in breast carcinomas.