No abstract

Cadherin-mediated cell-cell adhesion is dynamically modulated during epithelial-mesenchymal transition triggered by activation of receptor tyrosine kinases (RTK) in epithelial cells. Several cadherin-binding proteins have been identified that control cell-cell adhesion. However, the mechanisms by which intercellular adhesion and cell motility are coregulated are still unknown. Here, we delineate a hitherto uncharted cooperation between RTKs, RhoA GTPase, and p120 catenin in instructing a motile behavior to epithelial cells. We found that expression of an N-terminus-deleted p120 catenin in a variety of epithelial cell types, including primary keratinocytes, effectively competes for endogenous p120 at cadherin binding sites and abrogates EGFstimulated cell motility as well as HGF-induced cell scattering. The deleted mutant also inhibits the PI3K-dependent RhoA activation ensuing receptor activation. Conversely, we also show that the ectopic expression of full-length p120 in epithelial cells promotes cytoskeletal changes, stimulates cell motility, and activates RhoA. Both motogenic response to p120 and RhoA activation require coactivation of signaling downstream of RTKs as they are suppressed by ablation of the Ras/PI3K pathway. These studies demonstrate that p120 catenin is a necessary target of RTKs in regulating cell motility and help define a novel pathway leading to RhoA activation, which may contribute to the early steps of metastatic invasion.

No abstract

We report the application of a strategy to inactivate cellular proteins in vertebrate cells based on the intracellular expression of immunoglobulin genes. We have selected, in this instance, the p21 protein, encoded by the ras proto-oncogene, as a target protein. The variable regions of the neutralizing anti-p21ras monoclonal antibody Y13-259 were cloned in vectors for the expression of either the whole antibody molecule or its single-chain Fv fragment (ScFv) derivative. In order to target the recombinant antibodies to the cytosol, their hydrophobic leader sequence for secretion was mutated or deleted. When these proteins are expressed in the cytosol of Xenopus laevis oocytes they colocalize with the endogenous p21ras protein in the cytoplasmic face of the oocyte plasma membrane, and they markedly inhibit the H1 kinase activity induced by insulin. Moreover, cytosolic anti-p21ras ScFv fragments block the ensuing meiotic maturation. Thus the intracellular expression of both whole antibodies and antibody domains can be used to block a biological function.

Poly-A+ mRNA from Xenopus laevis oocytes, partially enriched for r-protein coding capacity has been used as starting material for preparing a cDNA bank in plasmid pBR322. The clones containing sequences specific for r-proteins have been selected by translation of the complementary mRNAs. Clones for six different r-proteins have been identified and utilized as probes for studying their genomic organization. Two gene copies per haploid genome were found for r-proteins L1, L14, S19, and four-five for protein S1, S8 and L32. Moreover a population polymorphism has been observed for the genomic regions containing sequences for r-protein S1, S8 and L14.