DIFFERENT EFFECTS OF PROTEIN-KINASE INHIBITORS ON THE LOCALIZATION OFJUNCTIONAL PROTEINS AT CELL-CELL CONTACT SITES

The protein kinase inhibitor H-7 prevents the assembly of tight juncti ons in cultured Madin Darby Canine Kidney (MDCK) epithelial cells (Bal da et al. (1991) J. Membr. Biol. 122, 193-202; Nigam et al. (1991) Bio chem. Biophys. Res. Commun. 181, 548-553); however, its mechanism of a ction is unknown. To understand the basis of the activity of H-7 and o ther inhibitors we compared the effect of H-7 on the localization of p roteins belonging to tight junctions and adherens-type junctions (zonu la adhaerens and desmosome), and on the organization of actin microfil aments. Junction assembly was induced in MDCK cells either by the 'Ca2 + switch' procedure or by incubating trypsinized cells at normal extra cellular Ca2+, and the cells were then immunofluorescently labeled wit h antibodies against cingulin, ZO-1, E-cadherin and desmoplakin, and w ith FITC-phalloidin. Here we show by measuring the transepithelial res istance that, in addition to H-7, H-8 and staurosporine can also signi ficantly block the assembly of tight junctions, whereas HA1004 is poor ly active. H-7 inhibited the accumulation of cingulin and ZO-1 in junc tional areas most effectively when added during assembly at normal ext racellular Ca2+. On the other hand, H-7 did not have major effects on the accumulation of E-cadherin and desmoplakin in the regions of cell- cell contact using either assembly protocol. Electron microscopy confi rmed that H-7 does not abolish the formation of adherens-type junction s, suggesting that phosphorylation plays a different role in the assem bly of tight junctions versus adherens-type junctions. Finally, in bot h protocols of junction assembly H-7 caused a major disorganization of actin microfilaments, suggesting that H-7 may prevent TJ assembly thr ough its effect on the cytoskeleton.