Localisation of Protein Kinase C in Apoptosis and Neurite Outgrowth

Sammanfattning: Protein kinase C (PKC) is a family of serine/threonine kinases, which are subgrouped into classical (a, bI, bII, g), novel (d, e, h, q) and atypical (z, i/l) isoforms. One major aim of this thesis work was to investigate if altered levels of PKC isoforms influence the apoptotic responses of malignant cell-lines. We show that overexpression of PKCd or PKCq renders SK-N-BE(2) neuroblastoma cells sensitive to apoptosis induced by phorbol esters or C2-ceramide. Moreover, overexpression of PKCa, PKCd or PKCe sensitises both androgen-dependent and androgen-independent prostate cancer cells to phorbol ester-induced cell-death. The apoptotic effects of PKCd and PKCq in neuroblastoma cells are independent on the catalytic activity of the enzymes and the isolated regulatory domain (RD) of PKCq induces apoptosis in neuroblastoma cells. Induction of apoptosis depends on the localisation of PKCqRD to the Golgi complex, which is mediated by the C1b domain of the protein. Mutation of a single amino acid residue, Met-267 in PKCqC1b, blocks both the Golgi localisation and the apoptotic effect of the PKCqRD. Previous studies have shown that PKCe induces neurites in neuroblastoma cells. Here we report that treatment with cell-permeable C2-ceramide inhibits PKCe-induced neurite formation, conceivably by relocating the protein from the cytosol to the perinuclear region. Mutation of Asp-257 and Met-278 (the latter corresponding to Met-267 in PKCq) in PKCe blocks the C2-ceramide induced translocation of PKCe. Furthermore, the mutated variant of PKCe still induces neurites after C2-ceramide treatment. Thus, the specific subcellular localisation of PKCq and PKCe are important for their biological activities.