Protein kinase C remains functionally active during TPA induced neuronal differentiation of SH‐SY5Y human neuroblastoma cells

Abstract

SH‐SY5Y human neuroblastoma cells can be induced to differentiate into a neuronal phenotype by treatment with 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA). In other cell systems, TPA treatment frequently leads to down‐regulation of protein kinase C (PKC). However, we now report that TPA‐treated and non‐treated SH‐SY5Y cells express PKC‐α, but not PKC‐β and PKC‐γ, mRNA. Furthermore, only a slight down‐regulation of the PKC‐α protein could be seen during prolonged treatment with 16 nM TPA, the concentration giving optimal differentiation. In contrast, a higher concentration of TPA (1.6 μM) results in a poor neuronal differentiation and a complete down‐regulation of PKC‐α. PKC‐α was rapidly translocated to the particulate fraction and remained membrane bound for at least 4 days during treatment with 16 nM TPA. In such cells a sustained increased level of the phosphorylated form of a 80,000 Dalton PKC‐substrate was found. In addition to this sustained augmented phosphorylation, administration of fresh TPA at day 4 caused a small but reproducible further increased level of phosphorylated substrate. When the PKC activity was measured by the histone phosphorylation assay a substantial fraction of the initial enzyme activity could still be detected after 4 days of TPA treatment. Taken together, the data demonstrate that PKC remains functionally active during TPA induced differentiation of SH‐SY5Y cells, which may suggest a continuous role for the enzyme during the differentiation process.