PKC-Dependent Long-Term Effect of PMA on Protein Cell Surface Expression in Caco-2 Cells

• Published in: Experimental cell research 1997-03, Vol.231 (2), p.308-318
• Main Authors: Sapin, Catherine, Baricault, Laurent, Trugnan, Germain
• Format: Article
• Language: English
• Subjects: Alkaline Phosphatase - biosynthesis; Alkaline Phosphatase - genetics; Antigens, CD - metabolism; Antigens, Neoplasm - biosynthesis; Antigens, Neoplasm - genetics; Antigens, Surface - biosynthesis; Antigens, Surface - genetics; Caco-2 Cells; CD13 Antigens - biosynthesis; CD13 Antigens - genetics; Cell Polarity; Cyclic AMP-Dependent Protein Kinases - physiology; Dipeptidyl Peptidase 4 - biosynthesis; Dipeptidyl Peptidase 4 - genetics; Endosomes - enzymology; Enzyme Activation - drug effects; Exocytosis - physiology; Gene Expression Regulation, Neoplastic - drug effects; Humans; Lysosomal Membrane Proteins; Membrane Glycoproteins - metabolism; Membrane Proteins - biosynthesis; Membrane Proteins - genetics; Microscopy, Confocal; Microvilli - enzymology; Neoplasm Proteins - biosynthesis; Neoplasm Proteins - genetics; Neoplasm Proteins - physiology; Protein Kinase C - physiology; Tetradecanoylphorbol Acetate - pharmacology
• ISSN: 0014-4827; 1090-2422
• DOI: 10.1006/excr.1997.3488

Several recent data indicate that protein traffic is under the control of different phosphorylation pathways. In previous works, we have shown that cell surface expression of apical hydrolases and of a basolateral protein, “525” antigen, was impaired in Caco-2 cells treated with forskolin, a potent PKA activator (L. Baricaultet al.,1995,J. Cell Sci.,108, 2109–2121). Surprisingly, in these experiments forskolin did not seem to act through PKA activation. These cAMP-independent effects of FK may rely on cross-talk between intracellular phosphorylation pathways as described recently for PKA and PKC pathways. Therefore, we tested the hypothesis that PKC activation may induce effects comparable to those of FK on three brush border hydrolases as well as on 525 antigen cell surface expression in Caco-2 cells. Using enzymatic activity measurements and pulse–chase experiments combined with cell surface biotinylation assays, we show that long-term treatment with phorbol 12-myristate 13-acetate (PMA) impairs the overall expression of neither brush border hydrolases nor that of the 525 antigen but decreases total cell surface expression of these proteins. The apical and basolateral delivery pathways are equally affected. Using confocal laser scanning microscopy we show that the DPP IV and the 525 antigen that were not recovered from the cell surface were sequestrated in Lamp-1-positive lysosomal-related vesicles. PMA stimulates PKC translocation even after a 3-week treatment and induces PKCϵ redistribution to a vesicular- and membrane-associated compartment also labeled with cytokeratins. These results demonstrate that PMA-dependent PKC activation strongly impairs protein cell surface targeting. They also suggest that these PKC-dependent effects which are similar to those previously obtained with FK are relevant to the described cross-talk between PKA- and PKC-dependent phosphorylation pathways.