Different effects of protein kinase inhibitors on the localization of junctional proteins at cell-cell contact sites.
The protein kinase inhibitor H-7 prevents the assembly of tight junctions in cultured Madin Darby Canine Kidney (MDCK) epithelial cells (Balda et al. (1991) J. Membr. Biol. 122, 193-202; Nigam et al. (1991) Biochem. Biophys. Res. Commun. 181, 548-553); however, its mechanism of action is unknown. To understand the basis of the activity of H-7 and other inhibitors we compared the effect of H-7 on the localization of proteins belonging to tight junctions and adherens-type junctions (zonula adhaerens and desmosome), and on the organization of actin microfilaments. Junction assembly was induced in MDCK cells either by the 'Ca2+ switch' procedure or by incubating trypsinized cells at normal extracellular Ca2+, and the cells were then immunofluorescently labeled with antibodies against cingulin, ZO-1, E-cadherin and desmoplakin, and with FITC-phalloidin. Here we show by measuring the transepithelial resistance that, in addition to H-7, H-8 and staurosporine can also significantly block the assembly of tight junctions, whereas HA1004 is poorly active. H-7 inhibited the accumulation of cingulin and ZO-1 in junctional areas most effectively when added during assembly at normal extracellular 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 confirmed that H-7 does not abolish the formation of adherens-type junctions, suggesting that phosphorylation plays a different role in the assembly of tight junctions versus adherens-type junctions. Finally, in both protocols of junction assembly H-7 caused a major disorganization of actin microfilaments, suggesting that H-7 may prevent TJ assembly through its effect on the cytoskeleton.
Denisenko
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Dipartimento di Biologia, Universita' di Padova, Italy.
Burighel
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P
Citi
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D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D008841:Actin Cytoskeleton / Q000187:drug effects; D000199:Actins / Q000032:analysis; D000470:Alkaloids / Q000494:pharmacology*; D000818:Animals; D015820:Cadherins / Q000032:analysis; D002118:Calcium / Q000494:pharmacology / Q000502:physiology; D002460:Cell Line; D003598:Cytoskeletal Proteins / Q000032:analysis*; D003599:Cytoskeleton / Q000187:drug effects; D051180:Desmoplakins; D004285:Dogs; D004848:Epithelium; D007365:Intercellular Junctions / Q000737:chemistry / Q000187:drug effects*; D007546:Isoquinolines / Q000494:pharmacology*; D007668:Kidney; D008565:Membrane Proteins / Q000032:analysis; D010750:Phosphoproteins / Q000032:analysis; D010879:Piperazines / Q000494:pharmacology*; D047428:Protein Kinase Inhibitors; D019311:Staurosporine; D013449:Sulfonamides; D062826:Zonula Occludens-1 Protein
D000199:Actins; D000470:Alkaloids; D015820:Cadherins; D003598:Cytoskeletal Proteins; D051180:Desmoplakins; D007546:Isoquinolines; D008565:Membrane Proteins; D010750:Phosphoproteins; D010879:Piperazines; D047428:Protein Kinase Inhibitors; D013449:Sulfonamides; D062826:Zonula Occludens-1 Protein; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; C053177:N-(2-(methylamino)ethyl)-5-isoquinolinesulfonamide; C043204:N-(2-guanidinoethyl)-5-isoquinolinesulfonamide; D019311:Staurosporine; D002118:Calcium