HYPERPHOSPHORYLATION OF BETA-CATENIN ON SERINE-THREONINE RESIDUES ANDLOSS OF CELL-CELL CONTACTS INDUCED BY CALYCULIN-A AND OKADAIC ACID INHUMAN EPIDERMAL-CELLS

Phosphorylation and dephosphorylation events may critically control ju nction assembly and stability, as well as regulate the formation of th e cadherin-cytoskeleton complex, thus influencing the adhesive functio n of cells. In the present study, we have used specific activators and inhibitors of protein kinases and phosphatases to analyze the role of protein phosphorylation in the maintenance of epithelial architecture , Okadaic acid and calyculin A cell treatments induced two major effec ts: a dramatic alteration of the keratin network of epidermal cells an d a complete disruption of cell-cell contacts. This loss in cell-cell contacts was not tissue and species restricted and the interactions of keratinocytes with the matrix were not involved, The observed changes were highly specific for these drugs and were obtained in the range o f concentrations corresponding to the inhibition of protein phosphatas e 1 (PP1), They were time- and dose-dependent, and reversible, excludi ng a cytotoxic effect of the drugs. A decrease in electrophoretic mobi lity of beta-catenin, a major protein involved in the regulation of in tercellular adherens junctions, was observed in keratinocytes and fibr oblasts treated with okadaic acid and calyculin A, suggesting a change in the protein phosphorylation level and/or protein conformation. Dat a from beta-catenin immunocomplex autoradiography performed after P-32 in vivo incorporation in untreated and okadaic acid or calyculin A-tr eated Ha-CaT cells, demonstrated a higher level of phosphorylation of beta-catenin in treated cells compared to untreated ones. Analysis of P-32-labeled phosphoaminoacids demonstrated that beta-catenin was excl usively phosphorylated on serine-threonine residues but not on tyrosin e residues. Immunoprecipitations and Western blotting using anti-phosp hoserine and anti-phosphotyrosine antibodies confirmed these data, The change in beta-catenin phosphorylation on serine-threonine residues m ay play a role in the control of the cohesion between epithelial cells and may be involved in the regulation of the transduction signal. (C) 1997 Academic Press.