Although Src expression and activity are often elevated in colon cancer, the precise consequences of overexpression of the non-catalytic Src homology (SH) domains, or enhanced catalytic activity, are unknown. We show that, in KM12C colon cancer cells, elevated Src activity causes the components of adherens junctions, including vinculin, to be redistributed to Src-induced integrin adhesion complexes. Specifically, elevated Src activity blocks proper assembly of cell cell contacts after cells are switched from media containing a low level of calcium to media containing a high level of calcium, and E-cadherin remains internalized. In contrast, although elevated expression of the non-catalytic domains of Src is sufficient to induce assembly of integrin adhesion complexes, it does not induce disorganization of E-cadherin-associated intercellular contacts. Surprisingly, Src-induced disruption of E-cadherin localization requires specific integrin signalling, because E-cadherin redistribution is blocked by loss of cell-matrix interaction, or by inhibitory antibodies to alpha(v) or beta(1) integrin subunits. Furthermore, phosphorylation of the integrin-regulated focal adhesion kinase (FAK) on Src-specific sites is required for Src-induced de-regulation of E-cadherin, demonstrating interdependence between integrin-induced signals and cadherin-associated adhesion changes induced by Src.

Nalidixic acid, enoxacin, and other antibacterial 4-quinolones inhibit DNA gyrase activity by interrupting DNA breakage and reunion by A subunits of the A2B2 gyrase complex. Despite their clinical importance, the mode of quinolone action and mechanisms of resistance are poorly understood at the molecular level. Using a DNA fragment enrichment procedure, we isolated the gyrA gene from a uropathogenic Escherichia coli strain that encodes a gyrase A protein cross-resistant to a variety of quinolones. When complemented with gyrase B subunit, the purified A protein reconstituted DNA supercoiling activity 100-fold more resistant to inhibition by enoxacin than the susceptible enzyme and failed to mediate quinolone-dependent DNA cleavage. Nucleotide sequence analysis revealed that the gene differed at 58 nucleotide positions compared with the K-12 gyrA sequence. The 875-amino-acid residue-resistant gyrase A protein differed at three positions from its wild-type E. coli K-12 counterpart: tryptophan, glutamate, and serine replaced serine, aspartate, and alanine residues at positions 83, 678, and 828, respectively. By genetic analysis of chimeric gyrA genes in a gyrA(Ts) background, we showed that the Ser-83-*Trp mutation in the gyrase A protein was solely responsible for high-level bacterial resistance to nalidixic acid and fluoroquinolones.Bacterial DNA is maintained in a negatively supercoiled state by DNA gyrase, an ATP-dependent type II DNA topoisomerase (10). Gyrase is essential for cell viability, being implicated in a range of DNA transactions, including DNA replication and recombination, and in the control of gene expression (8,35). Gyrase catalyzes DNA supercoiling by an interesting and unusual mechanism. It passes a duplex DNA segment through a transient double-stranded DNA break made within a 120-to 150-base-pair (bp) loop of DNA wrapped on the surface of the tetrameric A2B2 gyrase complex. DNA strand passage is the salient mechanistic feature that allows DNA supercoiling and ATP-independent DNA relaxation by gyrase and also accounts for its catenation or decatenation and DNA unknotting activities (3,16,18).Antibacterial quinolones such as oxolinic acid inhibit DNA supercoiling by gyrase in vitro and rapidly arrest DNA replication in vivo (2, 9, 12, 28). Addition of detergent to gyrase-DNA complexes formed in the presence of oxolinic acid results in site-specific double-stranded DNA breakage and covalent attachment of the gyrase A subunits to each 5'-phosphate end via tyrosine 7,9,15,22,31). Thus, the A subunits appear to promote DNA breakage and reunion during catalysis, a process interrupted by quinolone inhibitors. In contrast, the B subunits bind ATP and are the locus of action of coumarin antibiotics such as novobiocin (11,20,30). The A and B subunits can be individually purified but must be combined to generate the topoisomerase activities of gyrase (14, 19

Despite the importance of epithelial cell contacts in determining cell behavior, we still lack a detailed understanding of the assembly and disassembly of intercellular contacts. Here we examined the role of the catalytic activity of the Src family kinases at epithelial cell contacts in vitro. Like E- and P-cadherin, Ca(2+) treatment of normal and tumor-derived human keratinocytes resulted in c-Yes (and c-Src and Fyn), as well as their putative substrate p120(CTN), being recruited to cell-cell contacts. A tyrosine kinase inhibitor with selectivity against the Src family kinases, PD162531, and a dominant-inhibitory c-Src protein that interferes with the catalytic function of the endogenous Src kinases induced cell-cell contact and E-cadherin redistribution, even in low Ca(2+), which does not normally support stable cell-cell adhesion. Time-lapse microscopy demonstrated that Src kinase inhibition induced stabilization of transiently formed intercellular contacts in low Ca(2+). Furthermore, a combination of E- and P-cadherin-specific antibodies suppressed cell-cell contact, indicating cadherin involvement. As a consequence of contact stabilization, normal cells were unable to dissociate from an epithelial sheet formed at high density and repair a wound in vitro, although individual cells were still motile. Thus, cadherin-dependent contacts can be stabilized both by high Ca(2+) and by inhibiting Src activity in low (0.03 mM) Ca(2+) in vitro.