Abstract. Calcium-dependent cell-cell adhesion is mediated by the cadherin family of cell adhesion proteins. Transduction of cadherin adhesion into cellular reorganization is regulated by cytosolic proteins, termed ct-, ~-, and qc-catenin (plakoglobin), that bind to the cytoplasmic domain of cadherins and link them to the cytoskeleton. Previous studies of cadherin/catenin complex assembly and organization relied on the coimmunoprecipitation of the complex with cadherin antibodies, and were limited to the analysis of the Triton X-100 (TX-100)-soluble fraction of these proteins. These studies concluded that only one complex exists which contains cadherin and all of the catenins. We raised antibodies specific for each catenin to analyze each protein independent of its association with E-cadherin. Extracts of Madin-Darby canine kidney epithelial cells were sequentially immunoprecipitated and immunoblotted with each antibody, and the results showed that there were complexes of E-cadherin/ot-catenin, and either/3-catenin or plakoglobin in the TX-100-soluble fraction. We analyzed the assembly of cadherin/catenin complexes in the TX-100-soluble fraction by [35S]methionine pulse-chase labeling, followed by sucrose density gradient fractionation of proteins. Immediately after synthesis, E-cadherin, H-catenin, and plakoglobin cosedimented as complexes. t~-Catenin was not associated with these complexes after synthesis, but a subpopulation of a-catenin joined the complex at a time coincident with the arrival of E-cadherin at the plasma membrane. The arrival of E-cadherin at the plasma membrane coincided with an increase in its insolubility in TX-100, but extraction of this insoluble pool with 1% SDS disrupted the cadherin/catenin complex. Therefore, to examine protein complex assembly in both the TX-100-soluble and -insoluble fractions, we used [35S]methionine labeling followed by chemical cross-linking before cell extraction. Analysis of cross-linked complexes from cells labeled to steady state indicates that, in addition to cadherin/catenin complexes, there were cadherin-independent pools of catenins present in both the TX-100-soluble and -insoluble fractions. Metabolic labeling followed by chase showed that immediately after synthesis, cadherin//3-catenin, and cadherin/ plakoglobin complexes were present in the TX-100-soluble fraction. Approximately 50% of complexes were titrated into the TX-100-insoluble fraction coincident with the arrival of the complexes at the plasma membrane and the assembly of t~-catenin. Subsequently, >90% of labeled cadherin, but no additional labeled catenin complexes, entered the TX-100-insoluble fraction. These results indicate that catenins either interact with cadherin complexes synthesized at different times, or there was exchange of catenins between labeled and unlabeled cadherin complexes. In addition, cross-linking revealed additional proteins associated with catenin complexes. We propose a model in which the assembly of cadherin/catenin complexes is regulated by the exchange of catenin...

The integrin-linked kinase (ILK) is an ankyrin repeat containing serine-threonine protein kinase that can interact directly with the cytoplasmic domains of the ␤1 and ␤3 integrin subunits and whose kinase activity is modulated by cell-extracellular matrix interactions. Overexpression of constitutively active ILK results in loss of cell-cell adhesion, anchorage-independent growth, and tumorigenicity in nude mice. We now show that modest overexpression of ILK in intestinal epithelial cells as well as in mammary epithelial cells results in an invasive phenotype concomitant with a down-regulation of Ecadherin expression, translocation of ␤-catenin to the nucleus, formation of a complex between ␤-catenin and the high mobility group transcription factor, LEF-1, and transcriptional activation by this LEF-1͞␤-catenin complex. We also find that LEF-1 protein expression is rapidly modulated by cell detachment from the extracellular matrix, and that LEF-1 protein levels are constitutively up-regulated at ILK overexpression. These effects are specific for ILK, because transformation by activated H-ras or v-src oncogenes do not result in the activation of LEF-1͞␤-catenin. The results demonstrate that the oncogenic properties of ILK involve activation of the LEF-1͞␤-catenin signaling pathway, and also suggest ILK-mediated cross-talk between cellmatrix interactions and cell-cell adhesion as well as components of the Wnt signaling pathway.The integrin-linked kinase (ILK) was identified from a yeast two-hybrid genetic screen by using as bait the cytoplasmic domain of the ␤ 1 integrin subunit (1). ILK can interact with ␤ 1 and ␤ 3 integrins (1). ILK is a novel ankyrin-repeat containing serinethreonine kinase (1), which also contains sequence motifs found in pleckstrin homology domains capable of interacting with phosphoinositide lipids. The kinase activity of ILK can be modulated by interaction of cells with components of the extracellular matrix (1) or by integrin clustering. The activation or inhibition of ILK activity is cell-type dependent and can be modified by growth factors (M. Delcommenne and S. D., unpublished results). Overexpression of ILK in epithelial cells results in the stimulation of anchorage-independent cell growth (1) and cell cycle progression (2). The latter is caused by the constitutive up-regulation of expression of cyclin D 1 and cyclin A, resulting in the hyperphosphorylation of the retinoblastoma protein (2). Overexpression of ILK in epithelial cells also results in the induction of tumorigenicity in nude mice (3), indicating that ILK is a protooncogene.

The Wnt-1 proto-oncogene induces the accumulation of ␤-catenin and plakoglobin, two related proteins that associate with and functionally modulate the cadherin cell adhesion proteins. Here we have investigated the effects of Wnt-1 expression on the tumor suppressor protein APC, which also associates with catenins. Expression of Wnt-1 in two different cell lines greatly increased the stability of APC-catenin complexes. The steady-state levels of both catenins and APC were elevated by Wnt-1, and the half-lives of both ␤-catenin and plakoglobin associated with APC were also markedly increased. The stabilization of catenins by Wnt-1 was primarily the result of a selective increase in the amount of uncomplexed, monomeric ␤-catenin and plakoglobin, detected both by affinity precipitation and size-exclusion chromatography of cell extracts. Exogenous expression of ␤-catenin was possible in cells already responding to Wnt-1 but not in the parental cells, suggesting that Wnt-1 inhibits an essential regulatory mechanism for ␤-catenin turnover. APC has the capacity to oppose this Wnt-1 effect in experiments in which overexpression of the central region of APC significantly reduced the size of the monomeric pool of ␤-catenin induced by Wnt-1. Thus, the Wnt-1 signal transduction pathway leads to the accumulation of monomeric catenins and stabilization of catenin complex formation with both APC and cadherins.