TY - JOUR

T1 - Vinculin but not alpha-actinin is a target of PKC phosphorylation during junctional assembly induced by calcium

AU - Perez-Moreno, M.

AU - Avila, A.

AU - Islas, S.

AU - Sanchez, S.

AU - González-Mariscal, L

PY - 1998

Y1 - 1998

N2 - The establishment of the junctional complex in epithelial cells requires the presence of extracellular calcium, and is controlled by a network of reactions involving G-proteins, phospholipase C and protein kinase C. Since potential candidates for phosphorylation are the tight junction associated proteins ZO1, ZO2 and ZO3, in a previous work we specifically explored these molecules but found no alteration in their phosphorylation pattern. To continue the search for the target of protein kinase C, in the present work we have studied the subcellular distribution and phosphorylation of vinculin and alpha-actinin, two actin binding proteins of the adherent junctions. We found that during the junctional sealing induced by Ca2+, both proteins move towards the cell periphery and, while there is a significant increase in the phosphorylation of vinculin, alpha-actinin remains unchanged. The increased phosphorylation of vinculin is due to changes in phosphoserine and phosphothreonine content and seems to be regulated by protein kinase C, since: (1) DiC8 (a kinase C stimulator) added to monolayers cultured without calcium significantly increases the vinculin phosphorylation level; (2) H7 and calphostin C (both protein kinase C inhibitors) completely abolish this increase during a calcium switch; (3) inhibition of phosphorylation during a calcium switch blocks the subcellular redistribution of vinculin and alpha-actinin. These results therefore suggest that vinculin phosphorylation by protein kinase C is a crucial step in the correct assembly of the epithelial junctional complex.

AB - The establishment of the junctional complex in epithelial cells requires the presence of extracellular calcium, and is controlled by a network of reactions involving G-proteins, phospholipase C and protein kinase C. Since potential candidates for phosphorylation are the tight junction associated proteins ZO1, ZO2 and ZO3, in a previous work we specifically explored these molecules but found no alteration in their phosphorylation pattern. To continue the search for the target of protein kinase C, in the present work we have studied the subcellular distribution and phosphorylation of vinculin and alpha-actinin, two actin binding proteins of the adherent junctions. We found that during the junctional sealing induced by Ca2+, both proteins move towards the cell periphery and, while there is a significant increase in the phosphorylation of vinculin, alpha-actinin remains unchanged. The increased phosphorylation of vinculin is due to changes in phosphoserine and phosphothreonine content and seems to be regulated by protein kinase C, since: (1) DiC8 (a kinase C stimulator) added to monolayers cultured without calcium significantly increases the vinculin phosphorylation level; (2) H7 and calphostin C (both protein kinase C inhibitors) completely abolish this increase during a calcium switch; (3) inhibition of phosphorylation during a calcium switch blocks the subcellular redistribution of vinculin and alpha-actinin. These results therefore suggest that vinculin phosphorylation by protein kinase C is a crucial step in the correct assembly of the epithelial junctional complex.

KW - Actinin

KW - Animals

KW - Binding Sites

KW - Calcium

KW - Cell Line

KW - Dogs

KW - Microscopy, Fluorescence

KW - Phosphorylation

KW - Protein Kinase C

KW - Serine

KW - Subcellular Fractions

KW - Threonine

KW - Tight Junctions

KW - Vinculin

KW - Journal Article

KW - Research Support, Non-U.S. Gov't

U2 - 10.1242/jcs.111.23.3563

DO - 10.1242/jcs.111.23.3563

M3 - Journal article

C2 - 9811570

VL - 111

SP - 3563

EP - 3571

JO - Journal of Cell Science

JF - Journal of Cell Science

SN - 0021-9533

IS - 23

ER -