Involvement of desmoplakin phosphorylation in the regulation of desmosomesby 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, Immunofluoresc ence and electron microscopy showed that PKC activation upon short exposure to 12-O-tetradecanoylphorbol 13-acetate (TPA) resulted in a reduction of i ntercellular contacts, splitting of desmosomes and dislocation of desmosoma l components from the cell periphery towards the cytoplasm, As determined b y immunoblot analysis of Triton X-100-soluble and -insoluble pools of prote ins, 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, Immu noprecipitation experiments and immunoblotting with anti-phosphoserine, ant i-phosphothreonine and anti-phosphotyrosine antibodies revealed that desmop lakin was mainly phosphorylated on serine and tyrosine residues in both tre ated 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 m ay be a mechanism by which PKC mediates desmosome disassembly, Consistent w ith the loss of PKC activity, we also showed that down-modulation of the ki nase (in response to prolonged TPA treatment) or its specific inhibition (b y GF109203X) had opposite effects and increased desmosome formation, Taken together, these results clearly demonstrate an important role for PKC in th e regulation of desmosomal junctions in HeLa cells, and identify serine pho sphorylation of desmoplakin as a crucial event in this pathway.