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Snail1 transcriptional factor plays a key role in the control of epithelial to mesenchymal transition and fibroblast activation. As a consequence, Snail1 expression and function is regulated at multiple levels from gene transcription to protein modifications, affecting its interaction with specific cofactors. In this review, we describe the different elements that control Snail1 expression and its activity both as transcriptional repressor or activator. Ministerio de Economía, Industria y Competitividad, Gobierno de España: SAF2016-76461-R.

The transcription factor Snail1 induces epithelial-to-mesenchymal transition (EMT) in tumor epithelial cells, a process associated with the emergence of stemness, invasion and cancer malignancy. Here, we review recent reports indicating that Snail1 also regulates mesenchymal plasticity and paracrine signaling and propose that Snail1 orchestrates the generation of cancer stem cells (CSCs) and cancer-associated fibroblasts (CAFs). Our view supports the current models for tumorigenesis that consider stemness and tumor microenvironment as retroactive actors for metastasis formation, revealing Snail1 as a regulator of these metastatic forces. This view offers new perspectives for understanding and targeting metastasis.

Crosstalk between tumor and stromal cells in the tumor microenvironment alter its properties in ways that facilitate the invasive behavior of tumor cells. Here, we demonstrate that cancer-associated fibroblasts (CAF) increase the stiffness of the extracellular matrix (ECM) and promote anisotropic fiber orientation, two mechanical signals generated through a Snail1/RhoA/αSMA–dependent mechanism that sustains oriented tumor cell migration and invasiveness. Snail1-depleted CAF failed to acquire myofibroblastic traits in response to TGFβ, including RhoA activation, αSMA-positive stress fibers, increased fibronectin fibrillogenesis, and production of a stiff ECM with oriented fibers. Snail1 expression in human tumor–derived CAF was associated with an ability to organize the ECM. In coculture, a relatively smaller number of Snail1-expressing CAF were capable of imposing an anisotropic ECM architecture, compared with nonactivated fibroblasts. Pathologically, human breast cancers with Snail1+ CAF tended to exhibit desmoplastic areas with anisotropic fibers, lymph node involvement, and poorer outcomes. Snail1 involvement in driving an ordered ECM was further confirmed in wound-healing experiments in mice, with Snail1 depletion preventing the anisotropic organization of granulation tissue and delaying wound healing. Overall, our results showed that inhibiting Snail1 function in CAF could prevent tumor-driven ECM reorganization and cancer invasion. Cancer Res; 75(2); 284–95. ©2014 AACR.

Beyond inducing epithelial-to-mesenchymal transcription (EMT), transcriptional factors of the Snail, ZEB and Twist families (EMT-TFs) control global plasticity programmes affecting cell stemness and fate. Literature addressing the reactivation of these factors in adult tumour cells is very extensive, as they enable cancer cell plasticity and fuel both tumour initiation and metastatic spread. Incipient data reveal that EMT-TFs are also expressed in fibroblasts, providing these with additional properties. Here, I will review recent reports on the expression of EMT-TFs in cancer-associated fibroblasts (CAFs). The new model suggests that EMT-TFs can be envisioned as essential metastasis and chemoresistance-promoting molecules, thereby enabling coordinated plasticity programmes in parenchyma and stroma-tumour compartments.

40 p.-6 fig., Snail1 transcriptional factor is essential for triggering epithelial-to-mesenchymal transition (EMT) and inducing tumor cell invasion. We report here an additional, EMTindependent action of Snail1 on tumor invasion: its expression in cancer-associated fibroblasts is necessary for enhancement by these cells on epithelial cells tumor invasion. Snail1 expression in fibroblast requires signals derived from tumor cells such as TGF-; reciprocally, in fibroblasts Snail1 organizes a complex program that favors collective invasion of epithelial cells at least in part by the secretion of diffusible signaling molecules, such as prostaglandin E2. The capability of human or murine tumor-derived cancer associated fibroblasts to promote tumor invasion is associated to Snail1 expression and obliterated by Snail1 depletion. In vivo experiments show that Snail1 depletion in mice prevents the invasion of breast tumors and epithelial tumor cells co-xenografted with Snail1-depleted fibroblasts originate tumors with lower invasion than those transplanted with control fibroblasts. Therefore, these results demonstrate that the role of Snail1 in tumor invasion is not limited to EMT but dependent on its expression in stromal fibroblasts where it orchestrates its activation and the crosstalk with epithelial tumor cells. Moreover, they point to the interference of Snail expression as a promising target for preventing the action of stromal fibroblasts on tumor progression., This work was funded by grants from Fundación Científica de la Asociación Española contra el Cáncer to AGH, JIC and FB, and Ministerio de Economía(SAF2013-48849-C2-1-R) and Fundació La Marató de TV3 (120130) to AGH. We also thank the support from Instituto Carlos III (RD12/0036/005 and RD12/0036/004,1 part of the Plan Nacional I+D+I and cofounded by the ISCIII-Subdirección General de Evaluación and Fondo Europeo de Desarrollo Regional-FEDER) and Generalitat de Catalunya (2014 SGR 32).

Mortality in cancer is strongly associated with the capacity of tumor cells to spread and critically affect other tissues and organs. Genetic mutations accumulated by tumor cells and cross-signaling between tumor and host cells underlie the formation of metastasis. Cancer-activated fibroblasts (CAFs), which are host fibroblasts activated by tumor signaling, can alter tumor cell behavior by both paracrine signaling (secreting diffusible molecules) and mechanical signaling (modifying the composition and organization of the stroma). These fibroblasts resemble myofibroblasts (MFs) of the granulation tissue generated during wound healing, which produce a rigid desmoplastic stroma rich in signaling molecules and cross-linked extracellular fibers. Desmoplasia favors malignant tumor cell properties such as mobility, stemness, and even resistance to pharmacological insults [1].

Expression of Snail transcriptional factor is a determinant in the acquisition of a mesenchymal phenotype by epithelial tumor cells. However, the regulation of the transcription of this gene is still unknown. We describe here the characterization of a human SNAIL promoter that contains the initiation of transcription and regulates the expression of this gene in tumor cells. This promoter was activated in cell lines in response to agents that induce Snail transcription and the mesenchymal phenotype, as addition of the phorbol ester PMA or overexpression of integrin-linked kinase (ILK) or oncogenes such as Ha-ras or v-Akt. Although other regions of the promoter were required for a complete stimulation by Akt or ILK, a minimal fragment (-78/+59) was sufficient to maintain the mesenchymal specificity. Activity of this minimal promoter and SNAIL RNA levels were dependent on ERK signaling pathway. NFkappaB/p65 also stimulated SNAIL transcription through a region located immediately upstream the minimal promoter, between -194 and -78. These results indicate that Snail transcription is driven by signaling pathways known to induce epithelial to mesenchymal transition, reinforcing the role of Snail in this process.

E-cadherin protein plays a key role in the establishment and maintenance of adherent junctions. Recent evidence implicates the transcription factor Snail in the blockage of E-cadherin expression in fibroblasts and some epithelial tumor cells through direct binding to three E-boxes in the E-cadherin promoter. Transfection of Snail into epithelial cells leads to a more fibroblastic phenotype. Cells expressing Snail presented a scattered flattened phenotype with low intercellular contacts. Other epithelial markers like Cytokeratin 18 or MUC1 were also repressed. The effects of Snail on MUC1 transcription were mediated by two E-boxes present in the proximal promoter. Snail also induced expression of the mesenchymal markers fibronectin and LEF1 and the transcription repressor ZEB1. ZEB1 and Snail had a similar pattern of expression in epithelial cell lines, and both were induced by overexpression of ILK1, a kinase that causes the loss of E-cadherin and the acquisition of a fibroblastic phenotype. Snail overexpression in several cell lines raised ZEB1 RNA levels and increased the activity of ZEB1 promoter. ZEB1 could also repress E-cadherin and MUC1 promoters but less strongly than Snail. However, since ZEB1 expression persisted after Snail was down-regulated, ZEB1 may regulate epithelial genes in several tumor cell lines.

Loss of functional adenomatous polyposis coli (APC) protein results in the stabilization of cytosolic beta-catenin and activation of genes that are responsive to Lef/Tcf family transcription factors. We have recently shown that an independent cell adhesion and integrin linked kinase (ILK)-dependent pathway can also activate beta-catenin/LEF mediated gene transcription and downregulate E-cadherin expression. In addition, ILK activity and expression are elevated in adenomatous polyposis and colon carcinomas. To examine the role of this pathway in the background of APC mutations we inhibited ILK activity in APC-/- human colon carcinoma cell lines. In all cases, inhibition of ILK resulted in substantial inhibition of TCF mediated gene transcription and inhibition of transcription and expression of the TCF regulated gene, cyclin D1. Inhibition of ILK resulted in decreased nuclear beta-catenin expression, and in the inhibition of phosphorylation of GSK-3 and stimulation of its activity, leading to accelerated degradation of beta-catenin. In addition, inhibition of ILK suppressed cell growth in culture as well as growth of human colon carcinoma cells in SCID mice. Strikingly, inhibition of ILK also resulted in the transcriptional stimulation of E-cadherin expression and correlated with the inhibition of gene transcription of snail, a repressor of E-cadherin gene expression. Overexpression of ILK caused a stimulation of expression of snail, but snail expression was found not to be regulated by beta-catenin/Tcf. These data demonstrate that ILK can regulate beta-catenin/TCF and snail transcription factors by distinct pathways. We propose that inhibition of ILK may be a useful strategy in the control of progression of colon as well as other carcinomas. Oncogene (2001) 20, 133 - 140.

The adhesion protein E-cadherin plays a central part in the process of epithelial morphogenesis. Expression of this protein is downregulated during the acquisition of metastatic potential at late stages of epithelial tumour progression. There is evidence for a transcriptional blockage of E-cadherin gene expression in this process. Here we show that the transcription factor Snail, which is expressed by fibroblasts and some E-cadherin-negative epithelial tumour cell lines, binds to three E-boxes present in the human E-cadherin promoter and represses transcription of E-cadherin. Inhibition of Snail function in epithelial cancer cell lines lacking E-cadherin protein restores the expression of the E-cadherin gene.

Alterations in the transcriptional activity of the beta-catenin-Tcf complex have been associated with the earlier stages of colonic transformation. We show here that the activation of protein kinase C by the phorbol ester PMA in several intestinal cell lines increases the levels of beta-catenin detected in the nucleus and augments the transcriptional activity mediated by beta-catenin. The response to PMA was not related to modifications in the cytosolic levels of beta-catenin and was observed not only in cells with wild-type adenomatous polyposis coli protein (APC) but also in APC-deficient cells. Binding assays in vitro revealed that PMA facilitates the interaction of the beta-catenin with the nuclear structure. Our results therefore show that beta-catenin-mediated transcription can be regulated independently of the presence of APC.

2-Deoxyglucose uptake was enhanced in ts371 KiMuSV-NRK cells when growing at the permissive temperature to allow the expression of a transforming p21 ras protein. This change is due to a decrease in the Km by approximately 2.5-fold without affecting the Vmax of the transporter. The amount of the GLUT1 glucose transporter dit not increase as deduced from immunoblot experiments on total membranes. Nevertheless, ras-transformed GLUT1 displays a higher molecular mass due to an increased N-glycosylation of the protein. Experiments made in tunicamycin-treated cells indicates that a higher glycosylation is responsible for the increase in 2-deoxyglucose uptake in ras-transformed cells.

Heregulin receptors are unable to mediate the rapid internalization of bound ligand as demonstrated in cells transfected with chimeric or wild-type ErbB-2, -3, or -4 receptors (Baulida et al., 1996, J. Biol. Chem. 271, 5251-5257; Pinkas-Kramanski et al., 1996, EMBO J. 15, 2452-2467). This observation is now extended to include mammary carcinoma cell lines (SK-BR-3 and MDA-543) which express endogenous ErbB-2 and ErbB-3 receptors. Also, the fate of receptor-bound heregulin is examined. While receptor-bound heregulin is not rapidly internalized, the ligand is subject to a slow process of inactivation and degradation, which requires heregulin incubation at 37 degrees C with cells that express heregulin receptors. The degradation of heregulin is blocked to a significant extent by chloroquine, an inhibitor of endosome fusion with lysosomes, indicating that heregulin is slowly internalized and degraded. However, this process is not sufficiently rapid to produce ligand-dependent down-regulation of heregulin receptors.

Snail1 is a transcriptional repressor of E-cadherin that triggers epithelial–mesenchymal transition (EMT). Here, we report assisted Snail1 interaction with the promoter of a typical mesenchymal gene, fibronectin (FN1), both in epithelial cells undergoing EMT and in fibroblasts. Together with Snail1, the p65 subunit of NF-κB and PARP1 bound to the FN1 promoter. We detected nuclear interaction of these proteins and demonstrated the requirement of all three for FN1 transcription. Moreover, other genes involved in cell movement mimic FN1 expression induced by Snail1 or TGF-β1 treatment and recruit p65NF-κB and Snail1 to their promoters. The molecular cooperation between Snail1 and NF-κB in transcription activation provides a new insight into how Snail1 can modulate a variety of cell programs.

The epithelial to mesenchymal transition (EMT) consists of a rapid change of cell phenotype, characterized by the loss of epithelial traits and the acquisition of a more motile phenotype reminiscent of a fibroblast. The study of this process has received considerable attention because of its potential role in the acquisition of several cancer traits, particularly in cell invasion. In this article we describe the current knowledge of the molecular mechanisms governing this transition. In particular we discuss how initiation of EMT is dependent on the mutually exclusive levels of the transmembrane cell to cell adhesion molecule E-cadherin and its transcriptional repressor Snail1 and how Snail1 and other E-cadherin transcriptional repressors drive the EMT process. We focus on several new aspects of Snail1 regulation and propose a model for understanding the initiation and progression of this transition, based on the existence of feed-back mechanisms that limit or amplify the response to extracellular cues.

Fibronectins are cell-secreted glycoproteins that modulate cell attachment, spreading, migration, morphology, differentiation and oncogenic transformation. Fibronectin expression is activated during EMT (epithelial–mesenchymal transition) and is a hallmark of mesenchymal cells. It is shown in the present study that a transcription factor previously unrelated with EMT, TFCP2c/LSF/LBP-1c, was translocated to the nucleus and bound to the fibronectin promoter upon EMT induction by Snail1. Consequently, the interference of TFCP2c/LSF/LBP-1c's activity prevented fibronectin expression. Moreover, TFCP2c/LSF/LBP-1c was detected in nuclei of embryonic dermal mesenchymal cells adjacent to the hair bud, a cell population that expresses endogenous nuclear Snail1 and fibronectin. Therefore we indicate a new molecular role for TFCP2c/LSF/LBP-1c in fibronectin expression.

The transcription factor SNAIL1 is a master regulator of epithelial to mesenchymal transition. SNAIL1 is a very unstable protein, and its levels are regulated by the E3 ubiquitin ligase beta-TrCP1 that interacts with SNAIL1 upon its phosphorylation by GSK-3beta. Here we show that SNAIL1 polyubiquitylation and degradation may occur in conditions precluding SNAIL1 phosphorylation by GSK-3beta, suggesting that additional E3 ligases participate in the control of SNAIL1 protein stability. In particular, we demonstrate that the F-box E3 ubiquitin ligase FBXl14 interacts with SNAIL1 and promotes its ubiquitylation and proteasome degradation independently of phosphorylation by GSK-3beta. In vivo, inhibition of FBXl14 using short hairpin RNA stabilizes both ectopically expressed and endogenous SNAIL1. Moreover, the expression of FBXl14 is potently down-regulated during hypoxia, a condition that increases the levels of SNAIL1 protein but not SNAIL1 mRNA. FBXL14 mRNA is decreased in tumors with a high expression of two proteins up-regulated in hypoxia, carbonic anhydrase 9 and TWIST1. In addition, Twist1 small interfering RNA prevents hypoxia-induced Fbxl14 down-regulation and SNAIL1 stabilization in NMuMG cells. Altogether, these results demonstrate the existence of an alternative mechanism controlling SNAIL1 protein levels relevant for the induction of SNAIL1 during hypoxia.

11 pages, 7 figures.-- et al., E-cadherin and its transcriptional repressor Snail1 (Snai1) are two factors that control epithelial phenotype. Expression of Snail1 promotes the conversion of epithelial cells to mesenchymal cells, and occurs concomitantly with the downregulation of E-cadherin and the upregulation of expression of mesenchymal genes such as those encoding fibronectin and LEF1. We studied the molecular mechanism controlling the expression of these genes in mesenchymal cells. Forced expression of E-cadherin strongly downregulated fibronectin and LEF1 RNA levels, indicating that E-cadherin-sensitive factors are involved in the transcription of these genes. E-cadherin overexpression decreased the transcriptional activity of the fibronectin promoter and reduced the interaction of beta-catenin and NF-kappaB with this promoter. Similar to beta-catenin, NF-kappaB was found, by co-immunoprecipitation and pull-down assays, to be associated with E-cadherin and other cell-adhesion components. Interaction of the NF-kappaB p65 subunit with E-cadherin or beta-catenin was reduced when adherens junctions were disrupted by K-ras overexpression or by E-cadherin depletion using siRNA. These conditions did not affect the association of p65 with the NF-kappaB inhibitor IkappaBalpha. The functional significance of these results was stressed by the stimulation of NF-kappaB transcriptional activity, both basal and TNF-alpha-stimulated, induced by an E-cadherin siRNA. Therefore, these results demonstrate that E-cadherin not only controls the transcriptional activity of beta-catenin but also that of NF-kappaB. They indicate too that binding of this latter factor to the adherens junctional complex prevents the transcription of mesenchymal genes., G. S. was supported by a fellowship from the UAB; M.E. and D.C. from the Ministerio de Educación; C.A., from the FIS (Fondo de Investigaciones Sanitarias), and M.P. from the AGAUR (Agència de Gestió d’Ajuts Universitaris i de Recerca). S.P. was supported by a La Cierva contract. This research was funded by FIS grants 01/3060 and 03/0925 to J.B., SAF2006-00339 to A.G.H., BFU2006-03203 to M.D., and SAF2004-01015 to A.M. Partial support through grants from the Instituto Carlos III (RTICCC, C03710), Comunidad de Madrid (S-GEN-0266-2006) and the Generalitat de Catalunya (2005SGR00970) is also appreciated.

Presenilin1 (PS1) is a component of the gamma-secretase complex mutated in cases of Familial Alzheimer's disease (FAD). PS1 is synthesized as a 50 kDa peptide subsequently processed to two 29 and 20 kDa subunits that remain associated. Processing of PS1 is inhibited by several mutations detected in FAD patients. PS1 acts as negative modulator of beta-catenin.Tcf-4 transcriptional activity. In this article we show that in murine embryonic fibroblasts (MEFs) the mechanisms of action of the processed and non-processed forms of PS1 on beta-catenin.Tcf-4 transcription are different. Whereas non-processed PS1 inhibits beta-catenin.Tcf-4 activity through a mechanism independent of gamma-secretase and associated with the interaction of this protein with plakoglobin and Tcf-4, the effect of processed PS1 is prevented by gamma-secretase inhibitors, and requires its interaction with E- or N-cadherin and the generation of cytosolic terminal fragments of these two cadherins, which in turn destabilize the beta-catenin transcriptional cofactor CBP. Accordingly, the two forms of PS1 interact differently with E-cadherin or beta-catenin and plakoglobin: whereas processed PS1 binds E-cadherin with high affinity and beta-catenin or plakoglobin weakly, the non-processed form behaves inversely. Moreover, contrarily to processed PS1, that decreases the levels of c-fos RNA, non-processed PS1 inhibits the expression c-myc, a known target of beta-catenin.Tcf-4, and does not block the activity of other transcriptional factors requiring CBP. These results indicate that prevention of PS1 processing in FAD affects the mechanism of repression of the transcriptional activity dependent on beta-catenin.

The Snail gene product is a transcriptional repressor of E-cadherin expression and an inducer of the epithelial-to-mesenchymal transition in several epithelial tumor cell lines. This report presents data indicating that Snail function is controlled by its intracellular location. The cytosolic distribution of Snail depended on export from the nucleus by a CRM1-dependent mechanism, and a nuclear export sequence (NES) was located in the regulatory domain of this protein. Export of Snail was controlled by phosphorylation of a Ser-rich sequence adjacent to this NES. Modification of this sequence released the restriction created by the zinc finger domain and allowed nuclear export of the protein. The phosphorylation and subcellular distribution of Snail are controlled by cell attachment to the extracellular matrix. Suspended cells presented higher levels of phosphorylated Snail and an augmented extranuclear localization with respect to cells attached to the plate. These findings show the existence in tumor cells of an effective and fine-tuning nontranscriptional mechanism of regulation of Snail activity dependent on the extracellular environment.

et al., The β-catenin signaling pathway is deregulated in nearly all colon cancers. Nonhypercalcemic vitamin D3 (1α,25-dehydroxyvitamin D3) analogues are candidate drugs to treat this neoplasia. We show that these compounds promote the differentiation of human colon carcinoma SW480 cells expressing vitamin D receptors (VDRs) (SW480-ADH) but not that of a malignant subline (SW480-R) or metastasic derivative (SW620) cells lacking VDR. 1α,25(OH)2D3 induced the expression of E-cadherin and other adhesion proteins (occludin, Zonula occludens [ZO]-1, ZO-2, vinculin) and promoted the translocation of β-catenin, plakoglobin, and ZO-1 from the nucleus to the plasma membrane. Ligand-activated VDR competed with T cell transcription factor (TCF)-4 for β-catenin binding. Accordingly, 1α,25(OH)2D3 repressed β-catenin–TCF-4 transcriptional activity. Moreover, VDR activity was enhanced by ectopic β-catenin and reduced by TCF-4. Also, 1α,25(OH)2D3 inhibited expression of β-catenin–TCF-4-responsive genes, c-myc, peroxisome proliferator-activated receptor δ, Tcf-1, and CD44, whereas it induced expression of ZO-1. Our results show that 1α,25(OH)2D3 induces E-cadherin and modulates β-catenin–TCF-4 target genes in a manner opposite to that of β-catenin, promoting the differentiation of colon carcinoma cells., H.G. Pálmer and J.M. González-Sancho were recipients of fellowships from the Comunidad Autónoma de Madrid. This work was supported by a grant from the Plan Nacional de Investigación y Desarrollo (SAF98-0060), Ministerio de Ciencia y Tecnología of Spain.

A431 cells were treated with epidermal growth factor (EGF) to study the mechanism by which this factor accelerates the glycolytic flux. After EGF treatment, fructose-2,6-bisphosphate (Fru-2,6-P2) levels rose up to 2-fold. This change correlated with an increase in phosphofructokinase-2 activity, which was not due to a change in the transcription or translation of the enzyme, neither in the amount of enzyme. PK-C does not appear to be involved in the signalling mechanism since EGF was equally potent in PK-C depleted cells than in control cells. The increase in Fru-2,6-P2 levels was lower and more transient in cells treated with EGF in a calcium-free medium than in the presence of the cation, and it was restored by the addition of calcium to the medium. These results suggest a possible role for calcium-mediated pathways in the control of Fru-2,6-P2 levels in A431 cells.

The use of NIH3T3 fibroblasts overexpressing different mutations of the EGF receptor shows that regulation of fructose-2,6-bisphosphate (Fru-2,6-P2) metabolism by EGF is mediated by the kinase activity of the EGF receptor and suggests a PLCγ1-mediated mechanism. The effect of several extracellular matrix components on glucose metabolism was assessed by incubating A431 cells and NIH3T3 fibroblasts with heparin, laminin, fibronectin, collagen and PG-I and PG-II proteoglycans and measuring the levels of Fru-2,6-P2. Laminin increased the levels of Fru-2,6-P2 and heparin decreased the levels of the metabolite, whereas the other molecules did not have any effect. No effect of laminin or heparin in glucose uptake by the cell was observed. Laminin was able to modulate the effects of EGF on Fru-2,6-P2 concentration, suggesting cross-talk between these agents.

The 180-kDa transmembrane tyrosine kinase ErbB-4 is a receptor for the growth factor heregulin. 125I-Heregulin binding to NIH 3T3 cells overexpressing the ErbB-4 receptor is rapidly decreased by 12-O-tetradecanoylphorbol-13-acetate (TPA) pretreatment. Immunologic analysis demonstrates that TPA treatment of cells induces the proteolytic cleavage of ErbB-4, producing an 80-kDa cytoplasmic domain fragment, which contains a low level of phosphotyrosine, and a 120-kDa ectodomain fragment, which is released into the extracellular medium. Cleavage of ErbB-4 was also enhanced by other protein kinase C activators, i.e. platelet-derived growth factor, ionomycin, and synthetic diacylglycerol, while protein kinase C inhibition or down-regulation suppressed the TPA stimulation of ErbB-4 degradation. TPA did not induce the degradation of related receptors (ErbB-1, ErbB-2, and ErbB-3) in the EGF receptor family. The phorbol ester-induced cleavage of ErbB-4 occurs within or close to the ectodomain, as the 80-kDa cytoplasmic domain fragment is recognized by antibody to the ErbB-4 carboxyl terminus and is membrane-associated. Coprecipitation experiments show that, while the 80-kDa ErbB-4 fragment is associated with the SH2-containing molecules PLC-gamma1 and Shc, TPA did not induce the phosphorylation of these substrates in intact cells. In addition, kinase assays in vitro indicate that the 80-kDa fragment is not an active tyrosine kinase. These results show that protein kinase C negatively regulates heregulin signaling through the ErbB-4 receptor by the activation of a selective proteolytic mechanism.