SUMMARY: PMC42-LA cells display an epithelial phenotype: the cells congregate into pavement epithelial sheets in which E-cadherin and ␤-catenin are localized at cell-cell borders. They abundantly express cytokeratins, although 5% to 10% of the cells also express the mesenchymal marker vimentin. Stimulation of PMC42-LA cells with epidermal growth factor (EGF) leads to epithelio-mesenchymal transition-like changes including up-regulation of vimentin and down-regulation of E-cadherin. Vimentin expression is seen in virtually all cells, and this increase is abrogated by treatment of cells with an EGF receptor antagonist. The expression of the mesenchyme-associated extracellular matrix molecules fibronectin and chondroitin sulfate proteoglycan also increase in the presence of EGF. PMC42-LA cells adhere rapidly to collagen I, collagen IV, and laminin-1 substrates and markedly more slowly to fibronectin and vitronectin. EGF increases the speed of cell adhesion to most of these extracellular matrix molecules without altering the order of adhesive preference. EGF also caused a time-dependent increase in the motility of PMC42-LA cells, commensurate with the degree of vimentin staining. The increase in motility was at least partly chemokinetic, because it was evident both with and without chemoattractive stimuli. Although E-cadherin staining at cell-cell junctions disappeared in response to EGF, ␤-catenin persisted at the cell periphery. Further analysis revealed that N-cadherin was present at the cell-cell junctions of untreated cells and that expression was increased after EGF treatment. N-and E-cadherin are not usually coexpressed in human carcinoma cell lines but can be coexpressed in embryonic tissues, and this may signify an epithelial cell population prone to epithelio-mesenchymal-like responses. (Lab Invest 2003, 83:435-448).

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Binding of the protein Raf to the active form of Ras promotes activation of the MAP kinase signaling pathway, triggering cell growth and differentiation. Raf0Arg89 in the center of the binding interface plays an important role determining Ras-Raf binding affinity. We have investigated experimentally and computationally the Raf-R89K mutation, which abolishes signaling in vivo. The binding to @g-35 S#GTP-Ras of a fusion protein between the Raf-binding domain~RBD! of Raf and GST was reduced at least 175-fold by the mutation, corresponding to a standard binding free energy decrease of at least 3.0 kcal0mol. To compute this free energy and obtain insights into the microscopic interactions favoring binding, we performed alchemical simulations of the RBD, both complexed to Ras and free in solution, in which residue 89 is gradually mutated from Arg into Lys. The simulations give a standard binding free energy decrease of 2.9 6 1.9 kcal0mol, in agreement with experiment. The use of numerous runs with three different force fields allows insights into the sources of uncertainty in the free energy and its components. The binding decreases partly because of a 7 kcal0mol higher cost to desolvate Lys upon binding, compared to Arg, due to better solvent interactions with the more concentrated Lys charge in the unbound state. This effect is expected to be general, contributing to the lower propensity of Lys to participate in protein-protein interfaces. Large contributions to the free energy change also arise from electrostatic interactions with groups up to 8 Å away, namely residues 37-41 in the conserved effector domain of Ras including 4 kcal0mol from Ser39 which loses a bifurcated hydrogen bond to Arg89!, the conserved Lys84 and Lys87 of Raf, and 2-3 specific water molecules. This analysis will provide insights into the large experimental database of Ras-Raf mutations.

Mutational analysis of the cRaf-1 Ras binding domain (RBD) identified several point mutants with elevated Ras binding. Detailed examination of the binding kinetics of one mutant (A85K) suggests that it associates with a greater range of isomeric conformers of v-H-Ras than wt-RBD. At limiting v-H-Ras concentrations, saturation binding to A85K-RBD is higher than to wt-RBD. Notably, in assay systems where the RBD concentration is limiting, no difference exists between wt-RBD and A85K-RBD saturation levels in the presence of a sufficiently large molar excess of Ras. The inability of wt-RBD to saturate all bindable Ras/GTP (defined by its binding to A85K-RBD) suggests that Ras/GTP exists as several isoforms and that only a minority of these isoforms are capable of associating with wt-RBD. These findings provide the first experimental evidence in support of functionally distinct Ras/GTP isoforms. We also describe a novel analysis of such isoforms.