Involvement of desmoplakin phosphorylation in the regulation of desmosomes by protein kinase C, in HeLa cells.
In the present study, we have examined how modulation of protein kinase C (PKC) activity affected desmosome organization in HeLa cells. Immunofluorescence and electron microscopy showed that PKC activation upon short exposure to 12-O-tetradecanoylphorbol 13-acetate (TPA) resulted in a reduction of intercellular contacts, splitting of desmosomes and dislocation of desmosomal components from the cell periphery towards the cytoplasm. As determined by immunoblot analysis of Triton X-100-soluble and -insoluble pools of proteins, these morphological changes were not correlated with modifications in the extractability of both desmoglein and plakoglobin, but involved almost complete solubilization of the desmosomal plaque protein, desmoplakin. Immunoprecipitation experiments and immunoblotting with anti-phosphoserine, antiphosphothreonine and anti-phosphotyrosine antibodies revealed that desmoplakin was mainly phosphorylated on serine and tyrosine residues in both treated and untreated cells. While phosphotyrosine content was not affected by PKC activation, phosphorylation on serine residues was increased by about two-fold. This enhanced serine phosphorylation coincided with the increase in the protein solubility, suggesting that phosphorylation of desmoplakin may be a mechanism by which PKC mediates desmosome disassembly. Consistent with the loss of PKC activity, we also showed that down-modulation of the kinase (in response to prolonged TPA treatment) or its specific inhibition (by GF 109203X) had opposite effects and increased desmosome formation. Taken together, these results clearly demonstrate an important role for PKC in the regulation ofdesmosomal junctions in HeLa cells, and identify serine phosphorylation of desmoplakin as a crucial event in this pathway.
Amar
L S
LS
Laboratoire de Biologie-Odontologie, Université Paris VII, Institut Biomédical des Cordeliers, France.
Shabana
A H
AH
Oboeuf
M
M
Martin
N
N
Forest
N
N
D000818:Animals; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D051182:Desmogleins; D051180:Desmoplakins; D003896:Desmosomes / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D008301:Maleimides / Q000494:pharmacology; D051379:Mice; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D012995:Solubility; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D051185:gamma Catenin
D003598:Cytoskeletal Proteins; C495294:DSP protein, human; D051182:Desmogleins; D051180:Desmoplakins; C495295:Dsp protein, mouse; D004791:Enzyme Inhibitors; D007211:Indoles; D008301:Maleimides; D051185:gamma Catenin; D011493:Protein Kinase C; C070515:bisindolylmaleimide I; D013755:Tetradecanoylphorbol Acetate