Brk Expression Research Articles

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.

Correlations between spatiotemporal changes in gene expression and apoptosis underlie wing polyphenism in the ant Pheidole morrisi

Wing polyphenism, which is the ability of a single genome to produce winged and wingless castes in a colony in response to environmental cues, evolved just once and is a universal feature of ants. The gene network underlying wing polyphenism, however, is conserved in the winged castes of different ant species, but is interrupted at different points in the network in the wingless castes of these species. We previously constructed a mathematical model, which predicts that a key gene brinker (brk) mediates the development and evolution of these different "interruption points" in wingless castes of different ant species. According to this model, brk is upregulated throughout the vestigial wing discs of wingless ant castes to reduce growth and induce apoptosis. Here, we tested these predictions by examining the expression of brk, as well as three other genes up- and downstream of brk-decapentaplegic (dpp), spalt (sal), and engrailed (en)-in the winged reproductive and wingless soldier castes in the ant Pheidole morrisi. We show that expression of these genes is conserved in the wing disc of winged castes. Surprisingly, however, we found that brk expression is absent throughout development of the vestigial soldier forewing disc. This absence is correlated with abnormal growth of the soldier forewing disc as revealed by En expression and morphometric analyses. We also discovered that dpp and sal expression change dynamically during the transition from larval-to-prepupal development, and is spatiotemporally correlated with the induction of apoptosis in soldier forewing disc. Our results suggest that, contrary to our predictions, brk may not be a key gene in the network for suppressing wings in soldiers, and its absence may function to disrupt the normal growth of the soldier forewing disc. Furthermore, the dynamic changes in network interruptions we discovered may be important for the induction of apoptosis, and may be a general feature of gene networks that underlie polyphenism.

Breast tumor kinase and extracellular signal-regulated kinase 5 mediate Met receptor signaling to cell migration in breast cancer cells

IntroductionBreast tumor kinase (Brk/protein tyrosine kinase 6 (PTK6)) is a nonreceptor, soluble tyrosine kinase overexpressed in the majority of breast tumors. Previous work has placed Brk downstream of epidermal growth factor receptor (ErbB) activation and upstream of extracellular signal-regulated kinase 5 (ERK5) and p38 mitogen-activated protein (MAP) kinases. Herein we investigate the regulation of Brk kinase activity and cell migration in response to treatment of keratinocytes (HaCaT cells) and breast cancer cell lines (MDA-MB-231 and T47D cells) with hepatocyte growth factor (HGF) and macrophage stimulating protein (MSP), peptide ligands for Met and Ron receptors, respectively.MethodsIn vitro kinase assays were performed to directly measure Brk kinase activity in response to MET and RON ligands. Transfection of Brk-targeted RNAi was used to knock down endogenous Brk or ERK5 in multiple cell lines. Kinase activities (downstream of MET signaling) were assayed by Western blotting using total and phospho-specific antibodies. Boyden chamber assays were used to measure cell migration in response to manipulation of Brk and downstream MET effectors. Rescue experiments were performed by knock down of endogenous Brk using RNAi (targeting the untranslated region (3′-UTR)) and transient transfection (re-expression) of either wild-type or kinase-inactive Brk.ResultsBrk gene silencing revealed that HGF, but not MSP, induced robust Brk-dependent cell migration. Brk and ERK5 copurified in HGF-induced protein complexes, and Brk/ERK5 complexes formed independently of Brk kinase activity. ERK5 was required for breast cancer cell but not keratinocyte cell migration, which became ERK1/2-dependent upon ERK5 knockdown. Notably, rescue experiments indicated that the kinase activity of Brk was not required for HGF-induced cell migration. Further, expression of either wild-type or kinase-inactive Brk in Brk-null MDA-MB-435 cells activated ERK5 and conferred increased HGF-induced cell migration.ConclusionsThese results have identified Brk and ERK5 as important downstream effectors of Met signaling to cell migration. Targeting ERK5 kinase activity or inhibiting the formation of Brk/ERK5 complexes may provide an additional means of blocking cell migration associated with breast cancer progression to metastasis.

Brk/PTK6 & Bcl-x Proteins Contribute to Reduced Sensitivity to Chemotherapy.

Abstract Background: Brk/PTK6 is an intracellular protein tyrosine kinase which is frequently overexpressed in the majority of breast cancers and has been shown to increase the anchorage-independent survival of breast cancer cell lines by altering Bcl-xL:Bcl-xS ratios. A Brk binding partner has also been found to modulate the alternative splicing of the Bcl-x gene, and altered Bcl-xL/xS levels are implicated in cell sensitivity to chemotherapeutic agents. We hypothesised that Brk may decrease cell response to chemotherapy through regulation of Bcl-x.Materials & Methods: To determine the clinical relevance and coexpression of Brk and Bcl-xL/S, quantitative PCR was performed on a clinical breast cancer biopsy material (n= 46). Gene knockdown studies using siRNA for either Brk or Bcl-xL were performed in T47D breast cancer cell line. Cells were subsequently cultured onto a 96 well plate in quadruplicate and subject to dose range of the chemotherapeutic drug, doxorubicin or paclitaxel for 72 hours before being tested for viability using the MTT assay. Protein levels for Brk, Bcl-xL/xS in response to siRNA treatments were determined using western blotting.Results: Quantitative PCR analysis of clinical breast cancer series reveal that Bcl-xL and Bcl-xS expression were associated with high grade tumours (p=0.028 and p=0.009 respectively). Moreover, both Bcl-xL and Bcl-xS were found to be significantly associated with Brk expression within these clinical samples (p<0.0001 and p=0.0026 respectively). T47D breast cancer cells with suppressed Brk levels showed a significant increase in sensitivity to doxorubicin and paclitaxel compared to control cultures (p=0.035 for doxorubicin and p<0.05 for paclitaxel at 0.625μM and 0.25nM respectively). Reduced Brk levels in T47D cells resulted in reduced levels of Bcl-xL protein. T47D cells with suppressed Bcl-xL levels also showed a significant increase in the sensitivity to doxorubicin compared to control cultures (p=0.039 at 0.31μM).Conclusion: These data suggests that Brk expression decreases the sensitivity of currently used chemotherapies through a mechanism that may involve regulation of Bcl-xL/xS protein levels. Thus targeting Brk may represent an important strategy for improving patient responses to chemotherapy. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5068.

Brk Protects Breast Cancer Cells from Autophagic Cell Death Induced by Loss of Anchorage

Brk, a tyrosine kinase expressed in a majority of breast tumors, but not normal mammary tissue, promotes breast carcinoma cell proliferation. Normal epithelial cells are dependent on cell-cell or cell-matrix interactions for survival and undergo apoptosis after disruption of these interactions. Tumor cells are less sensitive to the induction of apoptosis and are predicted to have the potential to disseminate. We investigated whether Brk has further roles in breast tumor progression by relating its expression to tumor grade and demonstrating its role in the regulation of carcinoma cell survival under non-adherent conditions. Brk expression was determined by reverse transcription PCR on RNA extracted from surgical samples of human breast cancers. Breast carcinoma cell survival in suspension culture was examined when Brk protein levels were suppressed by RNA interference. Additionally, the effect of experimentally overexpressing Brk in otherwise Brk-negative breast carcinoma cells was assessed. Brk mRNA expression was notably higher in grade 3 breast tumors, as compared with lower tumor grades. In suspension culture, Brk suppression increased the rate of cell death, as compared with controls, and this cell death program exhibited characteristics of autophagy but not of apoptosis. Conversely, experimental expression of Brk in Brk-negative cells increased cell survival whereas kinase-inactive Brk did not. Therefore, Brk enhances breast carcinoma cell survival in suspension, suggesting a role for Brk in supporting breast cancer cell dissemination.

Brk is coamplified with ErbB2 to promote proliferation in breast cancer

Amplification of the receptor tyrosine kinase ErbB2 is frequently observed in breast cancer. Amplification of erbB2 is also associated with multiple genomic gains and losses; however, the importance of these associated changes is largely unknown. We demonstrate that Brk, a cytoplasmic tyrosine kinase, is coamplified and coexpressed with ErbB2 in human breast cancers. ErbB2 interacts with Brk and increases its intrinsic kinase activity. Expression of Brk enhances the ErbB2-induced activation of Ras/MAPK signaling and cyclin E/cdk2 activity to induce cell proliferation of mammary 3-dimensional acini in culture. In a murine model of breast cancer, expression of Brk was found to shorten the latency of ErbB2-induced tumors by promoting cell proliferation, with no effect on protection from apoptosis. Furthermore, overexpression of Brk conferred resistance to the ability of Lapatinib, an ErbB2 kinase inhibitor, to inhibit ErbB2-induced proliferation. Thus, we identified Brk as a drug target for ErbB2-positive cancers.

Brk expression may affect the differentiation status of breast cancer cells

The breast tumour kinase Brk (PTK6) is found in over two-thirds of breast cancer cell lines and tumours but is not expressed in normal mammary cells. Brk has previously been shown to play a role in regulating proliferation in breast tumour cells [1]. However, in vivo, the site of Brk expression in normal tissues is restricted to nonproliferating cells that are undergoing terminal differentiation such as those in the gut or the skin [2,3]. This led us to hypothesise that Brk expression in breast tumours could be reflective of a differentiation phenotype, especially as a previous study had shown that involucrin, a marker of terminal keratinocyte differentiation, was expressed in a subset of tumours [4]. We therefore examined involucrin expression in breast tumour cells lines and patient biopsy samples. In addition we investigated whether inducers of differentiation in keratinocytes such as prolonged culture in suspension or vitamin D3 treatment could also affect differentiation of breast tumour cells. We found that the expression of Brk in cultured cell lines correlated with involucrin expression. In addition the change in Brk expression, as a result of culture conditions, was accompanied by a change in involucrin levels. Moreover, treatment with vitamin D3 resulted in a decrease in cell numbers in the Brk-positive cell lines relative to the control treatments. The Brk-negative cell line was unaffected by vitamin D3 treatment. These data suggest that Brk and involucrin may be coregulated and that inducers of differentiation such as vitamin D3 could be considered potential therapeutic strategies.

Breast Tumor Kinase (Protein Tyrosine Kinase 6) Regulates Heregulin-Induced Activation of ERK5 and p38 MAP Kinases in Breast Cancer Cells

Total tyrosine kinase activity is often elevated in both cytosolic and membrane fractions of malignant breast tissue and correlates with a decrease in disease-free survival. Breast tumor kinase (Brk; protein tyrosine kinase 6) is a soluble tyrosine kinase that was cloned from a metastatic breast tumor and found to be overexpressed in a majority of breast tumors. Herein, we show that Brk is overexpressed in 86% of invasive ductal breast tumors and coexpressed with ErbB family members in breast cancer cell lines. Additionally, the ErbB ligand, heregulin, activates Brk kinase activity. Knockdown of Brk by stable expression of short hairpin RNA (shRNA) in T47D breast cancer cells decreases proliferation and blocks epidermal growth factor (EGF)- and heregulin-induced activation of Rac GTPase, extracellular signal-regulated kinase (ERK) 5, and p38 mitogen-activated protein kinase (MAPK) but not Akt, ERK1/2, or c-Jun NH(2)-terminal kinase. Furthermore, EGF- and heregulin-induced cyclin D1 expression is dependent on p38 signaling and inhibited by Brk shRNA knockdown. The myocyte enhancer factor 2 transcription factor target of p38 MAPK and ERK5 signaling is also sensitive to altered Brk expression. Finally, heregulin-induced migration of T47D cells requires p38 MAPK activity and is blocked by Brk knockdown. These results place Brk in a novel signaling pathway downstream of ErbB receptors and upstream of Rac, p38 MAPK, and ERK5 and establish the ErbB-Brk-Rac-p38 MAPK pathway as a critical mediator of breast cancer cell migration.

The BRK tyrosine kinase is expressed in high-grade serous carcinoma of the ovary

Background: We identified the BRK tyrosine kinase in a PCR-based screen of tyrosine kinases expressed by ovarian tumors. BRK expression is restricted to normal differentiating epithelial cells and its overexpression may play a role in processes related to tumor development and growth. Its expression in normal ovary and ovarian tumors has not previously been described, and is the focus of this study.Methods: BRK expression levels were determined in 14 normal ovaries and 138 high-grade, late stage serous carcinomas of the ovary by immunohistochemical analysis, and in 19 ovarian cancer cell lines and immortalized ovarian surface epithelium by western blot analysis. Furthermore, BRK/PTK6 gene copy number was determined in 7 primary serous carcinomas by fluorescence in situ hybridization.Results: Immunohistochemical studies indicate that BRK is highly expressed in 97/138 (70%) of high-grade, serous carcinomas of the ovary, but is absent in normal ovarian surface epithelia. BRK is also expressed by 9/19 of ovarian cancer cell lines, but is undetectable in immortalized ovarian surface epithelium. Interestingly, the BRK gene has been mapped to chromosome 20q13.3, a site frequently amplified in ovarian cancers, and associated with poor prognosis. We have determined by fluorescence in situ hybridization (FISH) that BRK is specifically amplified at low levels in 6/7 primary ovarian carcinomas.Conclusions: The amplification of the BRK gene and over-expression of BRK protein in the majority of high-grade serous carcinomas and ovarian cancer cell lines suggest that BRK may play a role in the development and growth of ovarian tumors.

Expression and Oncogenic Role of Brk (PTK6/Sik) Protein Tyrosine Kinase in Lymphocytes

Tyrosine kinases play a fundamental role in cell proliferation, survival, adhesion, and motility and have also been shown to mediate malignant cell transformation. Here we describe constitutive expression of the protein tyrosine kinase Brk in a large proportion of cutaneous T-cell lymphomas and other transformed T- and B-cell populations. The kinase is expressed in the nuclear localization and activated state. Brk expression was also induced in normal T cells on their activation. Introduced expression of the Brk gene resulted in markedly diminished cytokine and growth factor dependence of transfected BaF3 lymphocytes in regard to their in vitro proliferation and survival. Brk also conferred in vivo oncogenicity on the BaF3 cells. siRNA-mediated inhibition of the endogenous Brk in malignant T cells diminished their growth and survival capacity. These findings document inducible expression of Brk in normal T lymphocytes and persistent expression of the activated kinase in malignant T and B cells. Furthermore, our results indicate that Brk may play a key role in lymphomagenesis, hence identifying the kinase as a potential therapeutic target in lymphomas.

The role of brinker in eggshell patterning

Drosophila oogenesis provides a useful system to study signal transduction pathways and their interactions. Through clonal analysis, we found that brinker (brk), a repressor of Dpp signaling, plays an important role in the Drosophila ovary, where its function is essential for dorsal appendage formation. In the absence of brk, operculum fates are specified at the expense of dorsal appendage fates. Brk is expressed by most of the oocyte associated follicle cells, starting from stage 8 of oogenesis. Transforming Growth Factor β (TGFβ) signaling represses brk expression in both the early stage egg chambers and in the anterior follicle cells. In brk mutant follicle cell clones at the dorsal anterior region, Broad Complex (BR-C) expression is down-regulated in a larger domain than in wild type. We show that BR-C is required for dorsal appendage development. In large anterior BR-C mutant clones, dorsal appendages are absent, and instead, the eggshell has an enlarged operculum like region at the anterior. In addition, we show that the Epidermal Growth Factor (EGF) receptor signaling represses the TGFβ signaling in oogenesis by up-regulating brk expression. From our results and previously published data, it appears that anterior follicle cells integrate the levels of EGF receptor activation and TGFβ receptor activation. Operculum fate results when the sum of the level of activation of both pathways reaches a threshold level, and reduction of activity of one pathway can be compensated to some extent by increase in the other pathway.

Identification of STAT3 as a specific substrate of breast tumor kinase

Breast tumor kinase (Brk) is a non-receptor tyrosine kinase distantly related to the Src family kinase. It is expressed in more than 60% of breast tumors, but the biological role of this kinase remains to be determined. Only a limited number of substates have been identified for Brk, and the link of Brk to tumorigenesis remains largely unknown. In this study, we provide evidence that the signal transducer and activator of transcription 3, STAT3, is a physiological target of Brk. Activation of STAT3 previously has been linked to oncogenesis, and results in this study demonstrate that STAT3 is tyrosine phosphorylated and transcriptionally activated in cells expressing endogenous Brk. Signal transducer and activator of transcription 3 is specifically targeted since other STAT members are not responsive to Brk expression. Signal transducer and activator of transcription 3 activation requires the catalytic activity of Brk, and expression of both STAT3 and Brk stimulate cellular proliferation. In addition, we have identified a negative regulator of Brk, the suppressor of cytokine signaling, SOCS3. The SOCS3 protein is known to block signaling mediated by cytokine receptors, and here we find that SOCS3 is able to repress the activity of the Brk non-receptor tyrosine kinase.

Generating and interpreting the Brinker gradient in the Drosophila wing

The transcription factor Brinker (Brk) represses gene expression in the Drosophila wing imaginal disc, where it is expressed in symmetrical lateral-to-medial gradients, a pattern that is established by inverse gradients of the TGF-β, Dpp, which is in turn transduced into graded phosphorylated Mad (pMad, an R-Smad). pMad is part of a complex which directly represses brk. sal and omb are targets of Brk and are, thus, only expressed medially with their domains extending mediolaterally into the region where Brk is graded. omb extends more laterally than sal, indicating that higher levels of Brk are required to repress it. This is supported by our demonstration that higher levels of ectopic Brk are required to completely repress omb than sal. We also show, however, that Mad antagonizes the ability of Brk to repress these genes, indicating that pMad directly activates their expression (as well as repressing brk). Thus, whether a gene is expressed at a particular location may depend not only on how much Brk is present, but also on the level of pMad. We have also investigated the mechanism by which the brk expression gradient is established and show that it is not just a simple readout of the pMad gradient but requires Brk to repress its own expression. In brk mutants, the brk gradient is not established: brk is still off medially and on at high levels laterally, but there is almost no graded expression between these extremes. This Brk negative autoregulation appears to increase the sensitivity of the cells to Dpp/pMad and should also function to stabilize the brk gradient.

The Intracellular Tyrosine Kinase Brk Sensitizes Nontransformed Cells to Inducers of Apoptosis

The intracellular tyrosine kinase Brk is expressed in regenerating epithelial tissues withhighest levels in the gastrointestinal tract and skin. In these tissues, Brk is restricted to epithelialcells that are exiting the cell cycle and undergoing terminal differentiation. While not expressedin mammary gland, Brk expression is often induced in primary breast tumors and breast tumorcell lines. To identify potential oncogenic functions of Brk, we utilized the immortalized Rat1arat fibroblast cell line that is highly sensitive to transformation. We generated Rat1a cell linesthat stably overexpress wild-type and activated Brk, and we analyzed their growth properties andability to undergo apoptosis in response to stress and DNA damage. Overexpression of Brk didnot induce anchorage-independent growth, and no changes in cell morphology or cell cycleprogression were observed. However, when constitutively expressed, Brk sensitized Rat1a cellsto a variety of apoptotic stimuli including serum deprivation and a combination of UV irradiationand serum starvation. These findings indicate that Brk does not promote proliferation ofnontransformed cells, but plays a positive role in the regulation of the apoptotic response toDNA-damage and stress.

Differential expression of the non-receptor tyrosine kinase BRK in oral squamous cell carcinoma and normal oral epithelium

BRK is a non-receptor tyrosine kinase whose functional role is poorly understood. Although it is an epithelial specific kinase, its expression appears to be tissue specific. To date, little is known about BRK expression in human oral epithelium. We investigated expression of BRK in human oral squamous cell carcinomas (OSCC) and normal oral epithelium (NOE) using immunohistochemistry, laser confocal microscopy and Western blotting. The subcellular localization of BRK was identified by confocal microscopy and Western blotting of nuclear and cytoplasmic extracts from these cells. The results indicate that NOE express higher levels of BRK compared with OSCC cells. In NOE and moderately differentiated OSCC cells, BRK was localized in the nucleus and cytoplasm. However, in poorly differentiated OSCC cells, BRK was localized in perinuclear regions. These results suggest that BRK expression differs in normal and OSCC which may reflect a possible functional involvement in OSCC.

Brinker and optomotor-blind act coordinately to initiate development of the L5 wing vein primordium in Drosophila.

The stereotyped pattern of Drosophila wing veins is determined by the action of two morphogens, Hedgehog (Hh) and Decapentaplegic (Dpp), which act sequentially to organize growth and patterning along the anterior-posterior axis of the wing primordium. An important unresolved question is how positional information established by these morphogen gradients is translated into localized development of morphological structures such as wing veins in precise locations. In the current study, we examine the mechanism by which two broadly expressed Dpp signaling target genes, optomotor-blind (omb) and brinker (brk), collaborate to initiate formation of the fifth longitudinal (L5) wing vein. omb is broadly expressed at the center of the wing disc in a pattern complementary to that of brk, which is expressed in the lateral regions of the disc and represses omb expression. We show that a border between omb and brk expression domains is necessary and sufficient for inducing L5 development in the posterior regions. Mosaic analysis indicates that brk-expressing cells produce a short-range signal that can induce vein formation in adjacent omb-expressing cells. This induction of the L5 primordium is mediated by abrupt, which is expressed in a narrow stripe of cells along the brk/omb border and plays a key role in organizing gene expression in the L5 primordium. Similarly, in the anterior region of the wing, brk helps define the position of the L2 vein in combination with another Dpp target gene, spalt. The similar mechanisms responsible for the induction of L5 and L2 development reveal how boundaries set by dosage-sensitive responses to a long-range morphogen specify distinct vein fates at precise locations.

Use of RNA interference to validate Brk as a novel therapeutic target in breast cancer: Brk promotes breast carcinoma cell proliferation.

Brk (PTK6) is a nonreceptor protein tyrosine kinase, which is expressed in over 60% of breast carcinoma tissue samples and breast tumour cell lines, but not normal mammary tissue or benign lesions. Since experimental Brk expression in nontransformed mammary epithelial cells enhances their mitogenic response to epidermal growth factor, it was important to determine the role Brk plays in the proliferation of breast carcinoma cells and validate it as a therapeutic target. We have used RNA interference to efficiently and specifically downregulate Brk protein levels in breast carcinoma cells, and determined that this results in a significant suppression of their proliferation. Additionally, through the expression of a kinase-inactive mutant, we have determined that Brk can mediate promotion of proliferation via a kinase-independent mechanism, potentially functioning as an 'adapter'. These data identify Brk as a novel target for antiproliferative therapy in the majority of breast cancers, and illustrate the power of RNA interference for rapidly validating candidate therapeutic targets.

Conversion of an Extracellular Dpp/BMP Morphogen Gradient into an Inverse Transcriptional Gradient

Morphogen gradients control body pattern by differentially regulating cellular behavior. Here, we analyze the molecular events underlying the primary response to the Dpp/BMP morphogen in Drosophila. Throughout development, Dpp transduction causes the graded transcriptional downregulation of the brinker ( brk) gene. We first provide significance for the brk expression gradient by showing that different Brk levels repress distinct combinations of wing genes expressed at different distances from Dpp-secreting cells. We then dissect the brk regulatory region and identify two separable elements with opposite properties, a constitutive enhancer and a Dpp morphogen-regulated silencer. Furthermore, we present genetic and biochemical evidence that the brk silencer serves as a direct target for a protein complex consisting of the Smad homologs Mad/Medea and the zinc finger protein Schnurri. Together, our results provide the molecular framework for a mechanism by which the extracellular Dpp/BMP morphogen establishes a finely tuned, graded read-out of transcriptional repression.

Cells compete for decapentaplegic survival factor to prevent apoptosis in Drosophila wing development.

During the growth of Drosophila imaginal discs a process called 'cell competition' eliminates slow-proliferating but otherwise viable cells. We report here that cell competition requires the function of the brinker (brk) gene, whose expression is normally repressed by Decapentaplegic (Dpp) signalling but is upregulated in slow-growing Minute/+ cells. Excess brk expression activates the c-Jun amino-terminal kinase pathway, which in turn triggers apoptosis in these cells. We propose that slow-proliferating cells upregulate Brk levels owing to a disadvantage in competing for, or in transducing, the Dpp survival signal. This sequence of events might represent a general mechanism by which weaker cells are eliminated from a growing population, and might serve as a method of controlling cell number and optimizing tissue fitness and hence organ function.

The transcription factor Schnurri plays a dual role in mediating Dpp signaling during embryogenesis.

Decapentaplegic (Dpp), a homolog of vertebrate bone morphogenic protein 2/4, is crucial for embryonic patterning and cell fate specification in Drosophila. Dpp signaling triggers nuclear accumulation of the Smads Mad and Medea, which affect gene expression through two distinct mechanisms: direct activation of target genes and relief of repression by the nuclear protein Brinker (Brk). The zinc-finger transcription factor Schnurri (Shn) has been implicated as a co-factor for Mad, based on its DNA-binding ability and evidence of signaling dependent interactions between the two proteins. A key question is whether Shn contributes to both repression of brk as well as to activation of target genes. We find that during embryogenesis, brk expression is derepressed in shn mutants. However, while Mad is essential for Dpp-mediated repression of brk, the requirement for shn is stage specific. Analysis of brk; shn double mutants reveals that upregulation of brk does not account for all aspects of the shn mutant phenotype. Several Dpp target genes are expressed at intermediate levels in double mutant embryos, demonstrating that shn also provides a brk-independent positive input to gene activation. We find that Shn-mediated relief of brk repression establishes broad domains of gene activation, while the brk-independent input from Shn is crucial for defining the precise limits and levels of Dpp target gene expression in the embryo.