Vinculin but not alpha-actinin is a target of PKC phosphorylation during junctional assembly induced by calcium

The establishment of the junctional complex in epithelial cells requires th e presence of extracellular calcium, and is controlled by a network of reac tions involving C-proteins, phospholipase C and protein kinase C, Since pot ential candidates for phosphorylation are the tight junction associated pro teins ZO1, ZO2 and ZO3, in a previous work we specifically explored these m olecules but found no alteration in their phosphorylation pattern. To conti nue the search for the target of protein kinase C, in the present work we h ave studied the subcellular distribution and phosphorylation of vinculin an d alpha-actinin, two actin binding proteins of the adherent junctions. We f ound that during the junctional sealing induced by Ca2+, both proteins move towards the cell periphery and, while there is a significant increase in t he phosphorylation of vinculin, alpha-actinin remains unchanged. The increa sed phosphorylation of vinculin is due to changes in phosphoserine and phos phothreonine content and seems to be regulated by protein kinase C, since: (1) DiC8 (a kinase C stimulator) added to monolayers cultured without calci um significantly increases the vinculin phosphorylation level; (2) H7 and c alphostin C (both protein kinase C inhibitors) completely abolish this Incr ease during a calcium switch; (3) inhibition of phosphorylation during a ca lcium switch blocks the subcellular redistribution of vinculin and a-actini n, These results therefore suggest that vinculin phosphorylation by protein kinase C is a crucial step in the correct assembly of the epithelial junct ional complex.