Differential Expression and Subcellular Localization of Protein Kinase C α, β, γ, δ, and ɛ Isoforms in SH‐SY5Y Neuroblastoma Cells: Modifications During Differentiation

Abstract: A decrease in protein kinase C activity caused either by treatment with inhibitors, such as staurosporine or H‐7, or by prolonged exposure to phorbol diesters has been proposed to be involved in the early events of SH‐SY5Y neuroblastoma cell differentiation. Because eight distinct isoforms of protein kinase C with discrete subcellular and tissue distributions have been described, we determined which isoforms are present in SH‐SY5Y cells and studied their modifications during differentiation. The α, β, δ, and ɛ isoforms were present in SH‐SY5Y cells, as well as in rat brain. Protein kinase C‐α and ‐β1 were the most abundant isoforms in SH‐SY5Y cells, and immunoreactive protein kinase C‐δ and ‐ɛ were present in much smaller amounts than in rat brain. Subcellular fractionation and immunocytochemistry demonstrated that all four isoforms are distributed bimodally in the cytoplasm and the membranes. Immunocytochemical analysis showed that the α isoform is associated predominantly with the plasma membrane and the processes extended during treatment with 12‐tetradecanoyl‐13‐acetyl‐β‐phorbol or staurosporine, and that protein kinase C‐ɛ is predominantly membrane‐bound. Its localization did not change during differentiation. Western blots of total SH‐SY5Y cell extracts and of subcellular fractions probed with isoform‐specific polyclonal antibodies showed that when SH‐SY5Y cells acquired a morphologically differentiated phenotype, protein kinase C‐α and ‐ɛ decreased, and protein kinase C‐β1, did not change. These data suggest distinct roles for the different protein kinase C isoforms during neuronal differentiation, as well as possible involvement of protein kinase α and ɛ in neuritogenesis.