The invasion suppressor protein, E-cadherin, plays a central role in epithelial cell-cell adhesion. Loss of E-cadherin expression or function in various tumors of epithelial origin is associated with a more invasive phenotype. In this study, by expressing a dominant-negative mutant of E-cadherin (Ec1WVM) in A431 cells, we demonstrated that specific inhibition of E-cadherin-dependent cell-cell adhesion led to the genetic reprogramming of tumor cells. In particular, prolonged inhibition of cell-cell adhesion activated expression of vimentin and repressed cytokeratins, suggesting that the effects of Ec1WVM can be classified as epithelialmesenchymal transition. Both short-term and prolonged expression of Ec1WVM resulted in morphological transformation and increased cell migration though to different extents. Short-term expression of Ec1WVM up-regulated two AP-1 family members, c-jun and fra-1, but was insufficient to induce complete mesenchymal transition. AP-1 activity induced by the short-term expression of Ec1WVM was required for transcriptional up-regulation of AP-1 family members and down-regulation of two other Ec1WVM-responsive genes, S100A4 and igfbp-3. Using a dominant-negative mutant of c-Jun (TAM67) and RNA interference-mediated silencing of c-Jun and Fra-1, we demonstrated that AP-1 was required for cell motility stimulated by the expression of Ec1WVM. In contrast, Ec1WVM-mediated changes in cell morphology were AP-1-independent. Our data suggest that mesenchymal transition induced by prolonged functional inhibition of E-cadherin is a slow and gradual process. At the initial step of this process, Ec1WVM triggers a positive autoregulatory mechanism that increases AP-1 activity. Activated AP-1 in turn contributes to Ec1WVM-mediated effects on gene expression and tumor cell motility. These data provide novel insight into the tumor suppressor function of E-cadherin.

Numerous studies have revealed distinct functions of Fos proteins in different mouse tissues and cell lines. Here, we perform a direct comparison of the features of exogenous c-Fos, Fra-1 and Fra-2 proteins expressed in murine tumor cells of epithelial origin, CSML0. Although transactivation potential of c-Fos is much stronger than that of Fra-1 and Fra-2, all three proteins are capable of modulating transcription of target genes. Moreover, there is a certain degree of specificity in the induction of the transcription of AP-1-responsive genes by different Fos proteins. For instance, c-Fos and Fra-1 but not Fra-2 activated genes of the urokinase system. Additionally, not only a strong transcriptional activator c-Fos, but also Fra-1 induced morphological alterations in CSML0 cells. N-terminal domain of Fra-1 was required for this function. On the other hand, Fra-2 failed to change morphology of CSML0 cells. We therefore conclude that c-Fos, Fra-1 and Fra-2 differently activate transcription of target genes and induce morphological changes in epithelioid carcinoma cells in a manner not directly linked to their transactivation potentials.

Abstract. insulin receptor substrates (irS)-5 and -6 are two recently identified members of the IRS family. We investigated their roles as insulin receptor substrates and compared them with Src-homology-2-containing (Shc) protein, a well-established substrate. Bioluminescence resonance energy transfer (BRET) experiments showed no interaction between the receptor and IRS-5, while interaction with IRS-6 was not enhanced by insulin. By contrast, Shc showed an insulin-induced BRET response, as did a truncated form of IRS-1 (1-262). While Shc was heavily phosphorylated after stimulation of the insulin receptor, IRS-5 and -6 showed very weak phosphorylation levels. These results suggest that, although these two adaptors have previously been proposed as substrates for the insulin receptor, they are poor substrates for the insulin receptor. This calls into question their relevance to insulin signalling. IntroductionInsulin is crucial for the regulation of metabolism, growth and development. Binding of insulin to the insulin receptor (IR) leads to the activation of receptor tyrosine kinase and receptor phosphorylation, which enables the binding of docking proteins such as insulin receptor substrates (IRS)-1, -2, -3 and -4 and Src-homology-2-containing protein (Shc). This in turn leads to their phosphorylation and, thereby, intracellular signalling (1).Until recently, the IRS family included IRS-1, -2, -3 and -4 (2) and three downstream of kinases, Dok-1, -2 and -3. These seven proteins have similar amino-terminal pleckstrin homology (PH) and similar phosphotyrosine binding (PTB) and carboxyl-terminal phosphorylation domains which, when tyrosine-phosphorylated, dock Src-homology-2 (SH2) domain proteins. Despite their similar domain architecture, the Dok proteins can be distinguished from the IRS family based on sequence homology and functional interactions. Recently, two new members of the family were identified: IRS-5 and -6 (also called Dok-4 and -5, respectively). Based on PH and PTB domain sequence identity, IRS-5 and -6 have been shown to be more closely related to each other than to either the IRS or the Dok family (3,4). Dok-6, defined as a novel member of the Dok-4/5 'subclass' of the Dok family (5), and Dok-7 have also recently been identified (6).The physiological processes regulated by the Dok family are poorly understood. IRS family members and other receptor tyrosine kinase-associated adaptor molecules are generally involved in signal amplification. Dok-1, -2 and -3 function primarily as inhibitors of tyrosine kinase signalling (7-9). Dok-4 and -5, however, have been shown to be positively involved in tyrosine kinase signalling (3,10-12), although Dok-4 was suggested to be an inhibitor of tyrosine kinase signalling in epithelial cells (13,14). It has been suggested that Dok-4 and -5 are involved in insulin and related insulin-like growth factor (IGF)-I signalling, and play a role as substrates for the insulin and the IGF-I receptor (3). However, contradictory results have been obtained in other studies (13...

The insulin-receptor substrate family plays important roles in cellular growth, signaling, and survival. We have previously shown the dysregulation of the IGF-axis in clear cell renal cell carcinoma. In this manuscript, we examine the expression of the insulin receptor substrate family in clear cell RCC, and demonstrate that the expression of 2 members of this family are significantly altered in tumors. The most striking finding is that expression of the new IRS family member IRS-5 is significantly upregulated in 90% of examined clear cell RCCs. Studies on this gene has shown that it is regulated through chromatin remodeling in kidney cells. ' 2007 Wiley-Liss, Inc.

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