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PKC-dependent long-term effect of PMA on protein cell surface expression in Caco-2 cells
C Sapin1, L Baricault, G Trugnan
1INSERM, CJF 96-07, Faculté de médecine Saint Antoine, Paris, France.
Abstract:
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. Baricault et 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 PKC epsilon 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.
Insights
Protein kinase C (PKC) activation impairs cell surface targeting of proteins in Caco-2 cells. This effect, similar to forskolin
Area of Science:
- Cell biology
- Molecular biology
- Biochemistry
Background:
- Protein traffic and cell surface expression are regulated by phosphorylation pathways, including protein kinase A (PKA) and protein kinase C (PKC).
- Previous studies showed forskolin (FK), a PKA activator, impaired cell surface expression of apical hydrolases and a basolateral protein in Caco-2 cells, but not via PKA activation.
- Cross-talk between PKA and PKC pathways is known, suggesting FK's cAMP-independent effects might involve PKC.
Purpose of the Study:
- To investigate if PKC activation by phorbol 12-myristate 13-acetate (PMA) affects cell surface expression of brush border hydrolases and the '525' antigen in Caco-2 cells.
- To determine if PKC activation impacts apical and basolateral protein delivery pathways.
- To explore the cellular localization of proteins affected by PKC activation.
Main Methods:
- Enzymatic activity measurements.
- Pulse-chase experiments combined with cell surface biotinylation assays.
- Confocal laser scanning microscopy.
- Analysis of protein kinase C (PKC) translocation and cellular localization.
Main Results:
- Long-term PMA treatment did not alter the overall expression of brush border hydrolases or the '525' antigen.
- PMA treatment significantly decreased the total cell surface expression of these proteins, affecting both apical and basolateral delivery pathways.
- Proteins not recovered at the cell surface were found sequestered in Lamp-1-positive lysosomal-related vesicles.
- PMA induced PKC translocation and redistribution to vesicular and membrane compartments associated with cytokeratins.
Conclusions:
- PMA-dependent PKC activation significantly impairs protein cell surface targeting in Caco-2 cells.
- The observed effects of PKC activation are similar to those previously reported for forskolin, supporting a role for PKA-PKC cross-talk.
- PKC activation leads to sequestration of proteins in lysosomal-related vesicles, disrupting normal protein trafficking.