Engrailed-1 negatively regulates beta-catenin transcriptional activity by destabilizing beta-catenin via a glycogen synthase kinase-3beta-independent pathway.

The Wnt signaling pathway plays a major role in development, and upon deregulation it is implicated in neoplasia. The hallmark of the canonical Wnt signal is the protection of beta-catenin from ubiquitination and proteasomal degradation induced by glycogen synthase kinase (GSK)-3beta inhibition. The stabilized beta-catenin translocates to the nucleus where it binds to T-cell factor/lymphoid enhancer factor (TCF/LEF) transcription factors, activating the expression of Wnt target genes. In the absence of Wnt signal, TCF/LEF bind to Groucho (Gro)/TLE corepressors and repress Wnt target genes. Gro/TLE bind also to Engrailed (En) transcription factors mediating En-repressive activity on En target genes. Here, we present data suggesting that En-1 serves also as a negative regulator of beta-catenin transcriptional activity; however, its repressive effect is independent of Gro/TLE. Our data suggest that En-1 acts by destabilizing beta-catenin via a proteasomal degradation pathway that is GSK-3beta-independent. Moreover, because En-1-mediated beta-catenin degradation is also Siah independent, our data imply that En-1 exerts its repressive effect by a novel mechanism negatively controlling the level of beta-catenin.

The Tat protein of the human immunodeficiency virus type 1 (HIV-1) interacts with the EGF-like repeats of the Notch proteins and the EGF precursor.

Employing the yeast two-hybrid system, the Tat protein of the human immunodeficiency virus (HIV) was shown to interact with a region spanning the EGF-like repeats 1-6 of the mouse Notch1, the human Notch2 and the Drosophila Notch. This observation was confirmed in mammalian cells by demonstrating an interaction between the HIV Tat and the EGF-like repeats 1-6 of the various Notch proteins. The HIV Tat protein interacted also with the full-length mouse Notch1 receptor when co-expressed in mammalian cells. Moreover, the HIV Tat protein interacted also with the EGF-like repeats 1-4-spanning domain of the human EGF precursor. The ability of the HIV Tat protein to interact with the Notch proteins and possibly with other EGF-like repeats-bearing proteins, suggests that such interactions might modulate their physiological functions, thus affecting various AIDS-associated pathologies.

The low density lipoprotein receptor-1, LRP1, interacts with the human frizzled-1 (HFz1) and down-regulates the canonical Wnt signaling pathway.

Members of the low density lipoprotein receptor family (LDLR), LRP5/6, were shown to interact with the Frizzled (Fz) receptors and to function as Wnt coreceptors. Here we show that mLRP4T100, a minireceptor of LRP1, another member of the LDLR family, interacts with the human Fz-1 (HFz1), previously shown to serve as a receptor transmitting the canonical Wnt-3a-induced signaling cascade. However, in contrast to LRP5/6, mLRP4T100, as well as the full-length LRP1, did not cooperate with HFz1 in transmitting the Wnt-3a signaling but rather repressed it. mLRP4T100 inhibitory effect was displayed also by endocytosis-defective mLRP4T100 mutants, suggesting that LRP1 repressive effect is not attributable to LRP1-mediated enhanced HFz1 internalization and subsequent degradation. Enforced expression of mLRP4T100 decreased the capacity of HFz1 cysteine-rich domain (CRD) to interact with LRP6, in contrast to HFz1-CRD/Wnt-3a interaction that was not disrupted by overexpressing mLRP4T100. These data suggest that LRP1, by sequestering HFz1, disrupts the receptor/coreceptor complex formation, leading to the repression of the canonical Wnt signaling. Thus, this study implies that the ability to interact with Fz receptors is shared by several members of the LDLR family. However, whereas some members of the LDLR family, such as LRP5/6, interact with Fz and serve as Wnt coreceptors, others negatively regulate Wnt signaling, presumably by sequestering Fz.

The Tat protein of the caprine arthritis encephalitis virus interacts with the Notch2 EGF-like repeats and the epithelin/granulin precursor.

Using the yeast two-hybrid system, we screened a human placenta cDNA library and identified two proteins that interacted with the Tat protein of the caprine arthritis encephalitis virus (CAEV): the EGF-like repeats 1-6 of the extracellular domain of the human Notch2 receptor and the epithelin/granulin growth factor precursor. This interaction was also confirmed in mammalian cells. Using in vitro mutagenesis assays, we showed that each one of the three cysteine residues located within the cysteine-rich domain of the CAEV Tat protein is essential for the binding of Tat to both the Notch2 and the epithelin/granulin protein. It is thus suggested that the cysteine-rich domain of Tat plays a role in the interaction between the Tat and either Notch2 or the epithelin/granulin domains, both of which exhibit EGF-like-repeat-imposed spatial conformation. It is assumed that such interactions might modulate the physiological functions of Notch2 and epithelin/granulin, thereby affecting various pathologies associated with CAEV.

The human Frizzled 6 (HFz6) acts as a negative regulator of the canonical Wnt. beta-catenin signaling cascade.

Previously we have cloned the human Frizzled 1 (HFz1) and shown that it transmits the Wnt-3a-induced canonical pathway. We also cloned the human Frizzled 6 (HFz6) and show in the present study that, as opposed to HFz1, HFz6 did not activate the canonical Wnt pathway following exposure to various Wnts, whether belonging to the Wnt-1 or to the Wnt-5a group. Moreover we show that HFz6 repressed Wnt-3a-induced canonical signaling when co-expressed with HFz1. HFz6 repressed the canonical Wnt cascade activated also by various Wnt signaling intracellular mediators such as Dishevelled-1, a stabilized beta-catenin(S33Y) mutant, and LiCl-mediated repression of glycogen synthase kinase-3beta activity. Removal of HFz6 N'- or C'-terminal sequences abolished HFz6 repressive activity. As the HFz6 repressive effect was not associated with a decrease in the level of beta-catenin, it is suggested that HFz6 does not affect beta-catenin stabilization, implying that HFz6 transmits a repressive signaling that cross-talks with and inhibits the canonical Wnt pathway downstream of beta-catenin destruction complex. HFz6 did not affect the level of nuclear T-cell factor 4 (TCF4) nor did it affect beta-catenin.TCF4 complex formation. However, electrophoretic mobility shift assays indicated that HFz6 repressed the binding of TCF/lymphoid enhancer factor transcription factors to target DNA. Moreover we present data suggesting that HFz6 activates the transforming growth factor-beta-activated kinase-NEMO-like kinase pathway that blocks TCF/lymphoid enhancer factor binding to target promoters, thereby inhibiting the ability of beta-catenin to activate transcription of Wnt target genes.