beta-catenin is a target for the ubiquitin-proteasome pathway.

beta-catenin is a central component of the cadherin cell adhesion complex and plays an essential role in the Wingless/Wnt signaling pathway. In the current model of this pathway, the amount of beta-catenin (or its invertebrate homolog Armadillo) is tightly regulated and its steady-state level outside the cadherin-catenin complex is low in the absence of Wingless/Wnt signal. Here we show that the ubiquitin-dependent proteolysis system is involved in the regulation of beta-catenin turnover. beta-catenin, but not E-cadherin, p120(cas) or alpha-catenin, becomes stabilized when proteasome-mediated proteolysis is inhibited and this leads to the accumulation of multi-ubiquitinated forms of beta-catenin. Mutagenesis experiments demonstrate that substitution of the serine residues in the glycogen synthase kinase 3beta (GSK3beta) phosphorylation consensus motif of beta-catenin inhibits ubiquitination and results in stabilization of the protein. This motif in beta-catenin resembles a motif in IkappaB (inhibitor of NFkappaB) which is required for the phosphorylation-dependent degradation of IkappaB via the ubiquitin-proteasome pathway. We show that ubiquitination of beta-catenin is greatly reduced in Wnt-expressing cells, providing the first evidence that the ubiquitin-proteasome degradation pathway may act downstream of GSK3beta in the regulation of beta-catenin.     

Dissecting the pathways that destabilize mutant p53: The proteasome or autophagy?

One fundamental feature of mutant forms of p53 consists in their accumulation at high levels in tumors. At least in the case of neomorphic p53 mutations, which acquire oncogenic activity, stabilization is a driving force for tumor progression. It is well documented that p53 mutants are resistant to proteasome-dependent degradation compared with wild-type p53, but the exact identity of the pathways that affect mutant p53 stability is still debated. We have recently shown that macroautophagy (autophagy) provides a route for p53 mutant degradation during restriction of glucose. Here we further show that in basal conditions of growth, inhibition of autophagy with chemical inhibitors or by downregulation of the essential autophagic genes ATG1/Ulk1, Beclin-1 or ATG5, results in p53 mutant stabilization. Conversely, overexpression of Beclin-1 or ATG1/Ulk1 leads to p53 mutant depletion. Furthermore, we found that in many cell lines, prolonged inhibition of the proteasome does not stabilize mutant p53 but leads to its autophagic-mediated degradation. Therefore, we conclude that autophagy is a key mechanism for regulating the stability of several p53 mutants. We discuss plausible mechanisms involved in this newly identified degradation pathway as well as the possible role played by autophagy during tumor evolution driven by mutant p53.     

p73beta, a variant of p73, enhances Wnt/beta-catenin signaling in Saos-2 cells

The Wnt/beta-catenin pathway and p53 are very common targets for genetic alterations in colorectal cancer, and relationships between them have been reported. Here, we describe the relation between Wnt/beta-catenin signaling and the p53-related gene p73. p73, but not p53, activated a promoter containing the Tcf-binding sequence in Saos-2 cells, and the degree of activation was positively correlated with that on a p53-responsive promoter. Moreover, p73beta enhanced Wnt/beta-catenin signaling synergistically with Wnt-3a or exogenously expressed beta-catenin, unlike p53, and the enhancement was not caused by the accumulation of beta-catenin. These results show that the effects of p73 on Wnt/beta-catenin signaling differ from those of p53.     

Siah-1 mediates a novel beta-catenin degradation pathway linking p53 to the adenomatous polyposis coli protein

The adenomatous polyposis coli (APC) tumor-suppressor protein, together with Axin and GSK3beta, forms a Wnt-regulated signaling complex that mediates phosphorylation-dependent degradation of beta-catenin by the proteasome. Siah-1, the human homolog of Drosophila seven in absentia, is a p53-inducible mediator of cell cycle arrest, tumor suppression, and apoptosis. We have now found that Siah-1 interacts with the carboxyl terminus of APC and promotes degradation of beta-catenin in mammalian cells. The ability of Siah-1 to downregulate beta-catenin signaling was also demonstrated by hypodorsalization of Xenopus embryos. Unexpectedly, degradation of beta-catenin by Siah-1 was independent of GSK3beta-mediated phosphorylation and did not require the F box protein beta-TrCP. These results indicate that APC and Siah-1 mediate a novel beta-catenin degradation pathway linking p53 activation to cell cycle control.     

Ectopic Wnt signal determines the eyeless phenotype of zebrafish masterblind mutant

masterblind (mbl) is a zebrafish mutation characterised by the absence or reduction in size of the telencephalon, optic vesicles and olfactory placodes. We show that inhibition of Gsk3beta in zebrafish embryos either by overexpression of dominant negative dn gsk3beta mRNA or by lithium treatment after the midblastula transition phenocopies mbl. The loss of anterior neural tissue in mbl and lithium-treated embryos is preceded by posteriorization of presumptive anterior neuroectoderm during gastrulation, which is evident from the anterior shift of marker genes Otx2 and Wnt1. Heterozygous mbl embryos showed increased sensitivity to inhibition of GSK3beta by lithium or dn Xgsk3beta that led to the loss of eyes. Overexpression of gsk3beta mRNA rescued eyes and the wild-type fgf8 expression of homozygous mbl embryos. emx1 that delineates the telencephalon is expanded and shifted ventroanteriorly in mbl embryos. In contrast to fgf8, the emx1 expression domain was not restored upon overexpression of gsk3beta mRNA. These experiments place mbl as an antagonist of the Wnt pathway in parallel or upstream of the complex consisting of Axin, APC and Gsk3beta that binds and phosphorylates beta-catenin, thereby destabilising it. mbl maps on LG 3 close to a candidate gene axin1. In mbl we detected a point mutation in the conserved minimal Gsk3beta-binding domain of axin1 leading to a leucine to glutamine substitution at position 399. Overexpression of wild-type axin1 mRNA rescued mbl completely, demonstrating that mutant axin1 is responsible for the mutant phenotype. Overexpression of mutant L399Q axin1 in wild-type embryos resulted in a dose-dependent dominant negative activity as demonstrated by the loss of telencephalon and eyes. We suggest that the function of Axin1/Mbl protein is to antagonise the Wnt signal and in doing so to establish and maintain the most anterior CNS. Our findings provide new insights into the mechanisms by which the Wnt pathway generates anteroposterior polarity of the neural plate.     

Hypertonic stress activates glycogen synthase kinase 3beta-mediated apoptosis of renal medullary interstitial cells, suppressing an NFkappaB-driven cyclooxygenase-2-dependent survival pathway

The survival of renal medullary interstitial cells (RMICs) requires their adaptation to rapid shifts in ambient tonicity normally occurring in the renal medulla. Previous studies determined that cyclooxygenase-2 (COX 2) activation is critical for this adaptation. The present studies find that these adaptive mechanisms are dampened by the simultaneous activation of an apoptotic pathway linked to a glycogen synthase kinase 3beta (GSK 3beta). Inhibition of GSK 3 by LiCl or specific small molecule GSK inhibitors increased RMIC survival following hypertonic stress, and transduction of RMICs with a constitutively active GSK 3beta (AdGSK 3betaA9) significantly increased apoptosis, consistent with a proapoptotic role of GSK 3beta. Following GSK 3beta inhibition, increased survival was accompanied by increased COX 2 expression and COX 2 reporter activity. In contrast, GSK 3beta overexpression reduced COX 2 reporter activity. Importantly, enhanced RMIC survival produced by GSK 3beta inhibition was completely dependent on COX 2 because it was abolished by a COX 2-specific inhibitor, SC58236. The signaling pathway by which GSK 3beta suppresses COX 2 expression was then explored. GSK 3beta inhibition increased both NFkappaB and beta-catenin activity associated with decreased IkappaB and increased beta-catenin levels. The increase in COX 2 following GSK 3beta inhibition was entirely blocked by NFkappaB inhibition using mutant IkappaB adenovirus. However, adenoviral overexpression of beta-catenin did not increase COX 2 levels. These findings suggest that GSK 3beta negatively regulates COX 2 expression and that GSK 3beta inhibitors protect RMICs from hypertonic stress via induction of NFkappaB-COX 2-dependent pathway.     

Downregulation of wild-type p53 protein by HER-2／neu mediated PI3K pathway activation in human breast cancer cells： its effect on cell proliferation and implication for therapy

Overexpression and activation of HER-2/neu (also known as c-erbB-2), a proto-oncogene, was found in about 30% of human breast cancers, promoting cancer growth and making cancer cells resistant to chemo- and radio-therapy.Wild-type p53 is crucial in regulating cell growth and apoptosis and is found to be mutated or deleted in 60-70% of human cancers. And some cancers with a wild-type p53 do not have normal p53 function, suggesting that it is implicated in a complex process regulated by many factors. In the present study, we showed that the overexpression of HER-2/neu could decrease the amount of wild-type p53 protein via activating PI3K pathway, as well as inducing MDM2 nuclear translocation in MCF7 human breast cancer cells. Blockage of PI3K pathway with its specific inhibitor LY294002 caused G1-S phase arrest, decreased cell growth rate and increased chemo- and radio-therapeutic sensitivity in MCF7 cells expressing wild-type p53. However, it did not increase the sensitivity to adriamycin in MDA-MB-453 breast cancer cells containing mutant p53. Our study indicates that blocking PI3K pathway activation mediated by HER-2/neu overexpression may be useful in the treatment of breast tumors with HER-2/neu overexpression and wild-type p53.     

A mode of regulation of beta-catenin signaling activity in Xenopus embryos independent of its levels

The signaling activity of beta-catenin is thought to be regulated by phosphorylation of a cluster of N-terminal serines, putative sites for GSK3beta. In the prevailing model in the literature, GSK3beta-dependent phosphorylation of these sites targets beta-catenin for ubiquitin-mediated degradation. Wnt signaling inhibits GSK3beta activity and this blocks degradation, allowing beta-catenin to accumulate and signal. We show here that beta-catenin activity is not regulated solely by protein stability. Mutations in the putative GSK3beta phosphorylation sites of beta-catenin enhance its signaling activity, but this cannot be accounted for by accumulation of either total or cadherin-free protein. Instead, the mutant protein has a threefold higher specific activity than the wild type both in vivo and in an in vitro signaling assay. We conclude that the N-terminal serines convey a layer of regulation upon beta-catenin signaling in addition to the effects these sites exert upon protein stability.