E-cadherin regulates the function of the EphA2 receptor tyrosine kinase.

EphA2 is a member of the Eph family of receptor tyrosine kinases, which are increasingly understood to play critical roles in disease and development. We report here the regulation of EphA2 by E-cadherin. In nonneoplastic epithelia, EphA2 was tyrosine-phosphorylated and localized to sites of cell-cell contact. These properties required the proper expression and functioning of E-cadherin. In breast cancer cells that lack E-cadherin, the phosphotyrosine content of EphA2 was decreased, and EphA2 was redistributed into membrane ruffles. Expression of E-cadherin in metastatic cells restored a more normal pattern of EphA2 phosphorylation and localization. Activation of EphA2, either by E-cadherin expression or antibody-mediated aggregation, decreased cell-extracellular matrix adhesion and cell growth. Altogether, this demonstrates that EphA2 function is dependent on E-cadherin and suggests that loss of E-cadherin function may alter neoplastic cell growth and adhesion via effects on EphA2.

A single amino acid in E-cadherin responsible for host specificity towards the human pathogen Listeria monocytogenes.

Human E-cadherin promotes entry of the bacterial pathogen Listeria monocytogenes into mammalian cells by interacting with internalin (InlA), a bacterial surface protein. Here we show that mouse E-cadherin, although very similar to human E-cadherin (85% identity), is not a receptor for internalin. By a series of domain-swapping and mutagenesis experiments, we identify Pro16 of E-cadherin as a residue critical for specificity: a Pro-->Glu substitution in human E-cadherin totally abrogates interaction, whereas a Glu-->Pro substitution in mouse E-cadherin results in a complete gain of function. A correlation between cell permissivity and the nature of residue 16 in E-cadherins from several species is established. The location of this key specificity residue in a region of E-cadherin not involved in cell-cell adhesion and the stringency of the interaction demonstrated here have important consequences not only for the understanding of internalin function but also for the choice of the animal model to be used to study human listeriosis: mouse, albeit previously widely used, and rat appear as inappropriate animal models to study all aspects of human listeriosis, as opposed to guinea-pig, which now stands as a small animal of choice for future in vivo studies.

E-cadherin mediates adhesion and suppresses cell motility via distinct mechanisms.

Expression of the calcium-dependent adhesion molecule E-cadherin suppresses the invasion of cells in vitro, but the mechanism of this effect is unknown. To investigate this mechanism, we analyzed the effects of expressing E-cadherin in mouse L-cells and rat astrocyte-like WC5 cells. Increased cellular adhesion mediated by E-cadherin reduced invasion in WC5 cells and in some L-cells, but not in others. In all cases, suppression of invasion was correlated with decreased cell movement as assessed in an in vitro wound-filling assay and a transwell motility assay. To define the relationship between adhesion mediated by E-cadherin and suppression of motility, we analyzed the effects of deleting different regions of the E-cadherin cytoplasmic domain. E-cadherin lacking the entire cytoplasmic domain did not mediate calcium-dependent adhesion and did not reduce cell motility when expressed in WC5 cells. E-cadherin lacking a portion of the catenin-binding domain did not associate with the cytoskeleton and did not promote adhesion, yet still suppressed the motility of WC5 cells. In addition, E-cadherin that retains an intact catenin-binding domain, but lacks a juxtamembrane portion of the cytoplasmic domain, mediated effective adhesion, but did not suppress motility. These results indicate E-cadherin mediates adhesion and suppresses cell motility via distinct of E-cadherin plays a key role in suppressing motility.

SRV2, a gene required for RAS activation of adenylate cyclase in yeast.

We have identified a gene, SRV2, mutations of which alleviate stress sensitivity in strains carrying an activated RAS gene. Epistasis analysis suggests that the gene affects accumulation of cAMP in the cell. Direct assays of cAMP accumulation indicate that mutations of the gene diminish the rate of in vivo production of cAMP following stimulation by an activated RAS allele. Null mutations of srv2 result in lethality, which cannot be suppressed by mutational activation of the cAMP-dependent protein kinase. The sequence of the gene indicates that it encodes an adenylate cyclase-associated protein. These results demonstrate that SRV2 protein is required for RAS-activated adenylate cyclase activity, but that it participates in other essential cellular functions as well.

Expression of Rous sarcoma virus transforming protein pp60v-src in Saccharomyces cerevisiae cells.

The Rous sarcoma virus (RSV) pp60v-src protein was expressed in Saccharomyces cerevisiae cells either from a plasmid vector carrying the v-src gene or in yeast cells containing a single-copy v-src gene chromosomally integrated. In both yeast strains, v-src gene transcription is regulated by the galactose-inducible GAL10 promoter. Growth in galactose-containing medium resulted in constitutive expression of pp60v-src in the integrated strain and transient expression of higher levels of pp60v-src in the plasmid-bearing strain. The concentration of pp60v-src in the plasmid-bearing strain at its peak of expression was approximately threefold lower than that found in RSV-transformed mammalian cells. pp60v-src synthesized in yeast cells was phosphorylated in vivo on sites within the amino and carboxyl halves of the molecule. In immune complex kinase assays, the yeast pp60v-src was autophosphorylated on tyrosine and was able to phosphorylate exogenous substrates such as casein and enolase. The specific activity of pp60v-src synthesized in yeast cells was approximately 5- to 10-fold higher than that made in mammalian cells. Induction of pp60v-src caused the death of the plasmid-bearing yeast strain and transient inhibition of growth of the single-copy strain. Concomitantly, this induction resulted in high levels of tyrosine phosphorylation of yeast cell proteins. This indicates that pp60v-src functions as a tyrosine-specific phosphotransferase in yeast cells and suggests that hyperphosphorylation of yeast proteins is inimical to cell growth.