PKC-DEPENDENT LONG-TERM EFFECT OF PMA ON PROTEIN CELL-SURFACE EXPRESSION IN CACO-2 CELLS

Several recent data indicate that protein traffic is under the control of different phosphorylation pathways. In previous works, we have sho wn that cell surface expression of apical hydrolases and of a basolate ral protein, ''525'' antigen, was impaired in Caco-2 cells treated wit h forskolin, a potent PKA activator (L. Baricault et al., 1995, J. Cel l Sci., 108, 2109-2121). Surprisingly, in these experiments forskolin did not seem to act through PRA activation. These cAMP-independent eff ects of FK may rely on cross-talk between intracellular phosphorylatio n pathways as described recently for PKA and PKC pathways, Therefore, we tested the hypothesis that PRC activation may induce effects compar able to those of FK on three brush border hydrolases as well as on 525 antigen cell surface expression in Caco-2 cells. Using enzymatic acti vity measurements and pulse-chase experiments combined with cell surfa ce biotinylation assays, we show that. long-term treatment with phorbo l la-myristate 13-acetate (PMA) impairs the overall expression of neit her brush border hydrolases nor that of the 525 antigen but decreases total cell surface expression of these proteins. The apical and basola teral delivery pathways are equally affected. Using confocal laser sca nning microscopy we show that the DPP TV and the 525 antigen that were not recovered from the cell surface were sequestrated in Lamp-1-posit ive lysosomal-related vesicles. PMA stimulates PKC translocation even after a 3-week treatment and induces PKC epsilon redistribution to a v esicular- and membrane-associated compartment also labeled with cytoke ratins, These results demonstrate that PMA-dependent PKC activation st rongly impairs protein cell surface targeting. They also suggest that these PKC-dependent effects which are similar to those previously obta ined with FK are relevant to the described cross-talk between PKA- and PKC-dependent phosphorylation pathways. (C) 1997 Academic Press.