Jürgen Behrens

Negative Feedback Loop of Wnt Signaling through Upregulation of Conductin/Axin2 in Colorectal and Liver Tumors

ABSTRACT Activation of Wnt signaling through β-catenin/TCF complexes is a key event in the development of various tumors, in particular colorectal and liver tumors. Wnt signaling is controlled by the negative regulator conductin/axin2/axil, which induces degradation of β-catenin by functional interaction with the tumor suppressor APC and the serine/threonine kinase GSK3β. Here we show that conductin is upregulated in human tumors that are induced by β-catenin/Wnt signaling, i.e., high levels of conductin protein and mRNA were found in colorectal and liver tumors but not in the corresponding normal tissues. In various other tumor types, conductin levels did not differ between tumor and normal tissue. Upregulation of conductin was also observed in the APC-deficient intestinal tumors of Min mice. Inhibition of Wnt signaling by a dominant-negative mutant of TCF downregulated conductin but not the related protein, axin, in DLD1 colorectal tumor cells. Conversely, activation of Wnt signaling by Wnt-1 or dishevelled increased conductin levels in MDA MB 231 and Neuro2A cells, respectively. In time course experiments, stabilization of β-catenin preceded the upregulation of conductin by Wnt-1. These results demonstrate that conductin is a target of the Wnt signaling pathway. Upregulation of conductin may constitute a negative feedback loop that controls Wnt signaling activity.

Hakai, a c-Cbl-like protein, ubiquitinates and induces endocytosis of the E-cadherin complex

In epithelial cells, tyrosine kinases induce the tyrosine phosphorylation and ubiquitination of the E-cadherin complex, which induces endocytosis of E-cadherin. With a modified yeast 2-hybrid system, we isolated Hakai, an E-cadherin binding protein, which we have identified as an E3 ubiquitin-ligase. Hakai contains SH2, RING, zinc-finger and proline-rich domains, and interacts with E-cadherin in a tyrosine phosphorylation-dependent manner, inducing ubiquitination of the E-cadherin complex. Expression of Hakai in epithelial cells disrupts cell–cell contacts and enhances endocytosis of E-cadherin and cell motility. Through dynamic recycling of E-cadherin, Hakai can thus modulate cell adhesion, and could participate in the regulation of epithelial–mesenchymal transitions in development or metastasis.

Plexin-B1 Directly Interacts with PDZ-RhoGEF/LARG to Regulate RhoA and Growth Cone Morphology

Plexins are widely expressed transmembrane proteins that, in the nervous system, mediate repulsive signals of semaphorins. However, the molecular nature of plexin-mediated signal transduction remains poorly understood. Here, we demonstrate that plexin-B family members associate through their C termini with the Rho guanine nucleotide exchange factors PDZ-RhoGEF and LARG. Activation of plexin-B1 by semaphorin 4D regulates PDZ-RhoGEF/LARG activity leading to RhoA activation. In addition, a dominant-negative form of PDZ-RhoGEF blocks semaphorin 4D-induced growth cone collapse in primary hippocampal neurons. Our study indicates that the interaction of mammalian plexin-B family members with the multidomain proteins PDZ-RhoGEF and LARG represents an essential molecular link between plexin-B and localized, Rho-mediated downstream signaling events which underly various plexin-mediated cellular phenomena including axonal growth cone collapse.

Biochemical interactions in the wnt pathway

The wnt signal transduction pathway is involved in many differentiation events during embryonic development and can lead to tumor formation after aberrant activation of its components. The cytoplasmic component beta-catenin is central to the transmission of wnt signals to the nucleus: in the absence of wnts beta-catenin is constitutively degraded in proteasomes, whereas in the presence of wnts beta-catenin is stabilized and associates with HMG box transcription factors of the LEF/TCF family. In tumors, beta-catenin degradation is blocked by mutations of the tumor suppressor gene APC (adenomatous polyposis coli), or of beta-catenin itself. As a consequence, constitutive TCF/beta-catenin complexes are formed and activate oncogenic target genes. This review discusses the mechanisms that silence the pathway in cells that do not receive a wnt signal and goes on to describe the regulatory steps involved in the activation of the pathway.

Control of β-Catenin Signaling in Tumor Development

Abstract: The wnt signal transduction pathway is involved in various differentiation events during embryonic development and leads to tumor formation when aberrantly activated. The wnt signal is transmitted to the nucleus by the cytoplasmic component β‐catenin: in the absence of wnts, β‐catenin is constitutively degraded in proteasomes, whereas in the presence of wnts β‐catenin is stabilized and can associate with HMG box transcription factors of the LEF/TCF family. The LEF/TCF/β‐catenin complexes activate specific wnt target genes. In tumors, β‐catenin degradation is blocked by mutations of β‐catenin or of the tumor suppressor gene product APC. As a consequence, β‐catenin is stabilized, constitutive complexes with LEF/TCF factors are formed, and oncogenic target genes, such as c‐myc, cyclin D1, and c‐jun, are activated. Thus, control of β‐catenin is a major regulatory event in normal wnt signaling and during tumor formation. It has been found that a multiprotein complex assembled by the cytoplasmic component conductin induces degradation of cytoplasmic β‐catenin. The complex includes APC, the serine/threonine kinase GSK3beta;, and β‐catenin, which bind to conductin at distinct domains. In colon carcinoma cells, forced expression of conductin downregulates β‐catenin, whereas in normal cells mutants of conductin that are deficient in complex formation stabilize β‐catenin. Fragments of APC that contain a conductin‐binding domain also block β‐catenin degradation. In Xenopus embryos, conductin inhibits the wnt pathway. In situ hybridization analysis shows that conductin is expressed in various embryonal tissues known to be regulated by wnts, such as the developing brain, mesenchyme below the epidermis, lung mesenchyme, and kidney. It is suggested that conductin controls wnt signaling by assembling the essential components of the β‐catenin degradation pathway. Alterations of conductin function may lead to tumor formation.

The canonical Wnt signalling pathway and its APC partner in colon cancer development.

The flat surface of the colon is covered by an epithelium composed of four differentiated cell types (enterocytes, enteroendocrine, goblet and Paneth cells) that invaginates at regular intervals to form crypts (fig 1). The bottom of the crypts is occupied by a few stem cells that give rise to actively dividing precursor cells that populate the bottom two-thirds of the crypts.1 Proliferation occurs under the influence of growth factors from the Wnt family that may be produced by the underlying stromal cells underneath the stem cell compartment or by the epithelial cells themselves. The precursors migrate upward in an ordered fashion, which is also controlled by Wnt factors,2 and they stop proliferation when they reach the top third of the crypt, probably because they are too far from the Wnt source. Meanwhile, they continue their migration movement and colonise the surface of the colon. After about a week, epithelial cells undergo apoptosis and are shed in the lumen of the gut. The Paneth cells constitute an exception, as they migrate downward and occupy the very bottom of the crypt. Thus, the epithelium of the colon is under perpetual renewal. Wnt growth factors activate a cascade of intracellular events, which is known as the canonical Wnt pathway that ultimately leads to the expression of a genetic program controlling the co-ordinated expansion, fate and sorting of the epithelial cell population. In colorectal cancer, epithelial cells initially proliferate inappropriately because they acquired mutations in components of the pathway, thereby mimicking the effect of a permanent Wnt stimulation. Thus, the mutated cell recapitulates a progenitor-like phenotype, independent of its position in the epithelium.3 Canonical Wnt signalling has received considerable attention from cancer researchers over the years, because of its essential role in the homeostasis of the colonic epithelium and its deregulation …

Nucleo-cytoplasmic distribution of β-catenin is regulated by retention

β-catenin is the central signalling molecule of the canonical Wnt pathway, where it activates target genes in a complex with LEF/TCF transcription factors in the nucleus. The regulation of β-catenin activity is thought to occur mainly on the level of protein degradation, but it has been suggested that β-catenin nuclear localization and hence its transcriptional activity may additionally be regulated via nuclear import by TCF4 and BCL9 and via nuclear export by APC and axin. Using live-cell microscopy and fluorescence recovery after photobleaching (FRAP), we have directly analysed the impact of these factors on the subcellular localization of β-catenin, its nucleo-cytoplasmic shuttling and its mobility within the nucleus and the cytoplasm. We show that TCF4 and BCL9/Pygopus recruit β-catenin to the nucleus, and APC, axin and axin2 enrich β-catenin in the cytoplasm. Importantly, however, none of these factors accelerates the nucleo-cytoplasmic shuttling of β-catenin, i.e. increases the rate of β-catenin nuclear import or export. Moreover, the cytoplasmic enrichment of β-catenin by APC and axin is not abolished by inhibition of CRM-1-dependent nuclear export. TCF4, APC, axin and axin2 move more slowly than β-catenin in their respective compartment, and concomitantly decrease β-catenin mobility. Together, these data indicate that β-catenin interaction partners mainly regulate β-catenin subcellular localization by retaining it in the compartment in which they are localized, rather than by active transport into or out of the nucleus.

Hot spots in beta-catenin for interactions with LEF-1, conductin and APC.

Interactions between β-catenin and LEF-1/TCF, APC and conductin/axin are essential for wnt-controlled stabilization of β-catenin and transcriptional activation. The wnt signal transduction pathway is important in both embryonic development and tumor progression. We identify here amino acid residues in β-catenin that distinctly affect its binding to LEF-1/TCF, APC and conductin. These residues form separate surface clusters, termed hot spots, along the armadillo superhelix of β-catenin. We also show that complementary charged and hydrophobic amino acids are required for formation of the bipartite β-catenin–LEF-1 transcription factor. Moreover, we demonstrate that conductin/axin binding to β-catenin is essential for β-catenin degradation, and that APC acts as a cofactor of conductin/axin in this process. Binding of APC to conductin/axin activates the latter and occurs between their SAMP and RGS domains, respectively.

RB and c-Myc Activate Expression of the E-Cadherin Gene in Epithelial Cells through Interaction with Transcription Factor AP-2

ABSTRACT E-cadherin plays a pivotal role in the biogenesis of the first epithelium during development, and its down-regulation is associated with metastasis of carcinomas. We recently reported that inactivation of RB family proteins by simian virus 40 large T antigen (LT) in MDCK epithelial cells results in a mesenchymal conversion associated with invasiveness and a down-regulation of c-Myc. Reexpression of RB or c-Myc in such cells allows the reexpression of epithelial markers including E-cadherin. Here we show that both RB and c-Myc specifically activate transcription of the E-cadherin promoter in epithelial cells but not in NIH 3T3 mesenchymal cells. This transcriptional activity is mediated in both cases by the transcription factor AP-2. In vitro AP-2 and RB interaction involves the N-terminal domain of AP-2 and the oncoprotein binding domain and C-terminal domain of RB. In vivo physical interaction between RB and AP-2 was demonstrated in MDCK and HaCat cells. In LT-transformed MDCK cells, LT, RB, and AP-2 were all coimmunoprecipitated by each of the corresponding antibodies, and a mutation of the RB binding domain of the oncoprotein inhibited its binding to both RB and AP-2. Taken together, our results suggest that there is a tripartite complex between LT, RB, and AP-2 and that the physical and functional interactions between LT and AP-2 are mediated by RB. Moreover, they define RB and c-Myc as coactivators of AP-2 in epithelial cells and shed new light on the significance of the LT-RB complex, linking it to the dedifferentiation processes occurring during tumor progression. These data confirm the important role for RB and c-Myc in the maintenance of the epithelial phenotype and reveal a novel mechanism of gene activation by c-Myc.

Apoptosis-induced cleavage of beta-catenin by caspase-3 results in proteolytic fragments with reduced transactivation potential.

β-Catenin is a member of the Armadillo repeat protein family with a dual cellular function as a component of both the adherens junction complex and the Wnt/wingless signaling pathway. Here we show that β-catenin is proteolytically cleaved during anoikis and staurosporine-induced apoptosis. Cleavage of β-catenin was found to be caspase-dependent. Five cleavage products of β-catenin were identified in vivo and after in vitrocleavage by caspase-3. Amino acid sequencing and mass spectrometry analysis indicated two caspase-3 cleavage sites at the C terminus and three further sites at the N terminus, whereas the central Armadillo repeat region remained unaffected. All β-catenin cleavage products were still able to associate with E-cadherin and α-catenin and were found to be enriched in the cytoplasm. Functional analysis revealed that β-catenin deletion constructs resembling the observed proteolytic fragments show a strongly reduced transcription activation potential when analyzed in gene reporter assays. We therefore conclude that an important role of the β-catenin cleavage during apoptosis is the removal of its transcription activation domains to prevent its transcription activation potential.