Search

We have sought to determine the roles of beta-catenin and the Wnt signaling pathway in neurite outgrowth using a model cell system, the Neuro-2a neuroblastoma cell line. Activation of the Wnt signaling pathway disrupts a multiprotein complex that includes beta-catenin, Axin, and glycogen synthase kinase-3 (GSK-3), which would otherwise promote the phosphorylation and degradation of beta-catenin. Stabilized beta-catenin accumulates in the cytosol and in the nucleus; in the nucleus it binds to TCF family transcription factors, forming a bipartite transcriptional activator of Wnt target genes. These events can be mimicked by lithium (Li(+)), which inhibits GSK-3 activity. Both Li(+) and the GSK-3 inhibitor SB415286 induced neurite outgrowth of Neuro-2a cells. Li(+)-induced neurite outgrowth did not require beta-catenin-/TCF-dependent transcription, and increasing levels of beta-catenin either by transfection or using Wnt-3A was not sufficient to induce neurite outgrowth. Interestingly, Axin, which is also a substrate for GSK-3, was destabilized by Li(+) and ectopic expression of Axin inhibited Li(+)-induced neurite outgrowth. Deletion analysis of Axin indicated that this inhibition required the GSK-3 binding site, but not the beta-catenin binding site. Our results suggest that a signaling pathway involving Axin and GSK-3, but not beta-catenin, regulates Li(+)-induced neurite outgrowth in Neuro-2a cells.

alpha-Catenin and beta-catenin link cadherins to the cytoskeleton at adherens junctions. beta-Catenin also associates with members of the T-cell factor (Tcf) family of transcription factors, and mutations in beta-catenin lead to activation of Tcf-dependent transcription and increased cell growth. Although the loss of alpha-catenin expression can also promote cell growth, the role of endogenous alpha-catenin in beta-catenin signaling is unclear. Here we show that loss of alpha-catenin expression in a colon cancer cell line correlates with increased Tcf-dependent transcription. The presence of alpha-catenin in colon cancer cell nuclei suggests that it inhibits transcription directly, and, in agreement with this, ectopic expression of alpha-catenin in the nucleus represses Tcf-dependent transcription. Furthermore, recombinant alpha-catenin disrupts the interaction between the beta-catenin.Tcf complex and DNA. We conclude that alpha-catenin inhibits beta-catenin signaling in the nucleus by interfering with the formation of a beta-catenin. Tcf.DNA complex.

beta-Catenin plays essential roles in cell adhesion, by associating with cadherins, and as a signaling molecule, by interacting with the Tcf/LEF-1 family of transcription factors. In order to study the protein-protein interactions of beta-catenin in living cells, we fused it to green fluorescent protein (GFP). GFP-beta-catenin was incorporated into cell junctions but also accumulated in the nucleus, where it formed rod-like structures. The carboxyl-terminal armadillo repeats of GFP-beta-catenin were sufficient for nuclear localization, but formation of rods required the armadillo repeats and sequences in both the amino- and the carboxyl-terminal domains. Rod formation was prevented by coexpression of N-cadherin, APC, and Tcf-4, which bind to the armadillo repeats of beta-catenin, but not by coexpression of alpha-catenin, although alpha-catenin expression did prevent accumulation of beta-catenin in the nucleus. Interestingly, when alpha-catenin, beta-catenin, and Tcf-4 were coexpressed they colocalized in the nucleus, and this correlated with a decrease in beta-catenin/Tcf-dependent transcriptional activity. These results indicate that binding of beta-catenin to Tcf-4 overrides the function of alpha-catenin to sequester beta-catenin in the cytoplasm and suggest that alpha-catenin can regulate beta-catenin signaling in the nucleus., (Copyright 2000 Academic Press.)

Thrombospondins are a family of extracellular matrix proteins expressed throughout the developing nervous system that promote neurite outgrowth in vitro and help mediate the migration of granule cells across the molecular layer in explants of neonatal cerebellum. The receptors mediating these interactions have not previously been identified. In this study, monoclonal antibodies raised to the integrin alpha 3 beta 1 heterodimer are shown to inhibit neurite outgrowth by rat sympathetic neurons on thrombospondin-1. Alpha 3 beta 1 is found to be expressed on the cell body, neurites, and growth cones of sympathetic neurons in vitro and on sympathetic axons passing through the thrombospondin-rich outer sheath of the superior cervical ganglion in vivo, consistent with its role in mediating axon outgrowth. A receptor-ligand binding assay is used to demonstrate the direct binding of immunopurified alpha 3 beta 1 to thrombospondin-1. These results demonstrate a direct interaction between the integrin alpha 3 beta 1 and thrombospondin-1, which mediates neurite outgrowth in vitro and is likely to mediate the same interactions in vivo.

Three members of the Src family of tyrosine kinases [pp60c-src (Src), p59fyn (Fyn) and pp62c-yes (Yes)] are ubiquitously expressed, and are thus likely to have general roles in growth control. We have previously shown that, after addition of platelet-derived growth factor (PDGF) to quiescent cells, all three kinases become activated and associated with the PDGF receptor. We have now addressed the requirements for this association. First, we have used a baculovirus expression system to show that Fyn associates with the activated PDGF receptor in vitro in the absence of other proteins, demonstrating that the association between the two molecules is direct. Second, by generating cell lines expressing chimeric molecules consisting of Fyn sequences fused to a portion of beta-galactosidase, we found that the SH2 domain of Fyn is necessary for ligand-stimulated association with the PDGF receptor in vivo. Third, those fusion proteins that associated with the PDGF receptor also became phosphorylated in vivo following PDGF treatment, and in in vitro kinase assays, suggesting that the amino-terminal half of Fyn contains the sites of PDGF-stimulated phosphorylation. Partially purified, kinase-negative Fyn also became phosphorylated in the activated PDGF receptor complex in vitro, demonstrating that the PDGF receptor phosphorylates Fyn, rather than the novel phosphorylations occurring by autophosphorylation.

We previously demonstrated that src family tyrosine kinases associate with activated platelet-derived growth factor receptors. We have now investigated the requirement for tyrosine phosphorylation sites in the human platelet-derived growth factor receptor for this binding. Tyrosine 857, but not tyrosine 751, is required for efficient association. Furthermore, even though src family tyrosine kinases associate with receptors lacking tyrosine 751, they are not activated, implying that association does not invariably lead to activation.

We have examined the interaction between the platelet-derived growth factor (PDGF) receptor and three src family tyrosine kinases, pp60c-src, p59fyn, and pp62c-yes. The kinase activities of all three enzymes were elevated after PDGF stimulation of quiescent fibroblasts, coincident with association of the src family kinases with the PDGF receptor and other proteins. The presence of a protein of 81-85 kd in these complexes correlated with the detection of phosphatidylinositol (PI) kinase activity (previously described to associate with both the PDGF receptor and pp60c-src-middle T antigen). These results suggest that the physiological response to PDGF involves interaction of the receptor not only with serine/threonine and lipid kinases and a phospholipase, but also with other tyrosine kinases.