Posttranslationale Modifikation und subzelluläre Verteilung von Plakophilin 3 in normalen und Tumorzellen

Cellular adhesion is one hallmark accountable for the evolutionary success of multicellular organisms. Cellular specialization and the development of sophisticated functional units are reflected by the multiple kinds of cells and tissues. Desmosomes and adherens junctions are essential for the assembly and integrity of these tissues and facilitate - inter alia - their functional dynamics. Integral parts of both types of cell junctions are proteins of the armadillo family. Next to cellular adhesion, these proteins implement further cellular functions, which are mostly regulated by specific post-translational modifications. Detailed analyses of some of these proteins describe specific phosphorylation events related to the molecular dynamics. Several studies identified kinases of the Src-family and the epidermal growth factor receptor as responsible enzymes catalyzing reversible phosphorylation of armadillo proteins. In this thesis the desmosomal armadillo protein plakophilin 3 (PKP3) was analyzed for protein modifications occurring in normal and tumor cells. Utilizing two-dimensional gel electrophoresis, it was shown that PKP3 is a target of post-translational modification events, and some of them could be attributed to phosphorylation of tyrosine residues. By screening a phosphotomic database we selected five tyrosine residues and one serine residue that were frequently detected in phosphorylation studies and that may be relevant for functional properties of PKP3. The selected amino acid residues were replaced by mutagenesis experiments and several PKP3 mutants were generated covering single or multiple mutations. Epithelial cells were stably transfected with these PKP3 constructs and analyzed by immunofluorescence staining and differential extraction. So far, the individual modifications studied did not result in a detectable change in PKP3 function or cellular behavior. Furthermore, we generated polyclonal antisera against these phosphorylated PKP3 sequences. Purification of one of these sera revealed that it was suitable for further analyses. Using these antibodies, the phosphorylation of the tyrosine residue Y195 of PKP3 was analyzed by in-vivo and in-vitro experiments. By the application of specific kinase-inhibitors and in-vitro kinase assays we identified PKP3 as a new substrate of c-Src kinase. We found that phosphorylation of this tyrosine residue is tightly regulated and is only detectable after inhibition of cellular tyrosine phosphatases by pervanadate. Incubation of cells with common growth factors like EGF, IGF-1 or HGF surprisingly did not result in a detectable phosphorylation of this specific residue. Interstingly, phosphorylation of Y195 was induced by hydrogen peroxide treatment of cells. This suggests that this modification of PKP3 is at least part of a Src-mediated signalling pathway which is dependent on reactive oxygen species and oxidative stress. Immunofluorescence staining of c-Src over-expressing cells detected Y195-phosphorylated PKP3 at the desmosomal structure. At this particular point further investigations seem to be necessary since tyrosine phosphorylation of PKP3 was associated with altered protein solubility in several experiments, and so far, these findings did not correlate with a specific phosphorylation of PKP3. In conclusion, the present study shows for the first time the existence of a specific tyrosine phosphorylation and an associated signaling pathway for the desmosomal protein PKP3, whose cell biological relevance has to be addressed in future experiments. With regard to malignant tumors, the grade of malignancy of prostate carcinomas was previously correlated with an elevated activity of c-Src kinase and increased PKP3 expression. According to the results obtained in this thesis, adenocarcinomas of prostate were immunohistochemically analyzed for the presence of Y195-phosphorylated PKP3. The specific phosphorylation was found in some poorly differentiated adenocarcinomas at distinct tumour areas along cell borders. Further studies have to elucidate the functional relevance and if the specific PKP3 modification may achieve diagnostic significance.