Protein kinase C isoform antagonism controls occludin phosphorylation and tight junction assembly

In the present study, the involvement of the protein kinase C pathway in the regulation of the occludin phosphorylation and tight junction assembly has been investigated. Phorbol 12-myristate 13-acetate and 1,2-dioctanoylglycerol induced the rapid phosphorylation of occludin in MDCK cells cultured in low extracellular calcium medium with a concomitant translocation of occludin to the regions of cell-cell contact. The extent of occludin phosphorylation, as well as the incorporation of occludin and ZO-1 into tight junctions, induced by protein kinase C activators or calcium switch was markedly decreased by the classical and novel protein kinase C inhibitor GF-109203X. Similarly, rottlerin, the inhibitor of novel delta and theta protein kinase C isotypes, blocked both the phosphorylation of occludin and tight junction formation induced by the switch to normal calcium medium. In addition, in vitro experiments showed that recombinant COOH-terminal domain of murine occludin could be directly phosphorylated by purified PKC delta. The application of classical PKC inhibitor Go6976 during calcium switch unexpectedly accelerated the phosphorylation of tight junction protein occludin. The phosphorylation was accompanied by the accelerated incorporation of occludin into newly forming tight junction. Furthermore, Go6976 caused the assembly of tight junctional complex and occludin phosphorylation in cells cultivated in low calcium medium and strongly attenuated the disruption of tight junction complex induced by switch to low calcium medium. A simple procedure for the isolation of highly-phosphorylated occludin forms from rat liver was developed for subsequent identification of phosphorylation sites. Ser371 of rat occludin was identified as an in vivo occludin phosphorylation site by electrospray ionization tandem mass-spectroscopy. Conclusions: TJ assembly is regulated by antagonistic signalling of novel and classical PKC isozymes: the formation of TJ complex is favoured by simultaneous activation of novel PKCs (presumably PKC delta) and inhibition of classical PKC/PKC mu signalling. Occludin was shown to be a target for PKC-mediated signalling: activation of novel PKCs induces the rapid phosphorylation of occludin with a concomitant translocation to the regions of cell-cell contact. Both occludin phosphorylation and incorporation into the newly forming tight junctions was counteracted by the classical PKC signalling. Ser371 was for the first time identified as an in vivo phosphorylation site of Rattus norvegicus liver occludin.

Author dc:creator

• Andreeva, Anna

• Tight junctions
• protein kinase C

Licence dc:rights.uri

• http://www.fu-berlin.de/sites/refubium/rechtliches/Nutzungsbedingungen

Language dc:language

eng

Identifier URI

http://dx.doi.org/10.17169/refubium-17404

OAI record oai:identifier

https://refubium.fu-berlin.de/oai/request?verb=GetRecord&metadataPrefix=dim&identifier=oai%3Arefubium.fu-berlin.de%3Afub188%2F13206