Different modulation of protein kinase C isozymes in dibutyryl cyclic AMP-differentiated PC12h cells

PC12h cells can be differentiated into sympathetic neuron-like cells by various agents, including nerve growth factor, basic fibroblast growth factor, cyclic AMP analogues, and protein kinase C (PKC) activators. To study the involvement of PKC in the process of PC12h cell differentiation by cyclic AMP treatment, PKC isozymes (alpha, beta I, beta II, and gamma) were analyzed using column chromatography and immunoblotting. Two PKC isozymes, PKC(alpha) and PKC(beta II), were predominantly detected in PC12h cells. When stimulated by dibutyryl cyclic AMP, PKC(alpha) levels declined in the cytosolic fraction of the cells, whereas PKC(beta II) levels increased. Increased PKC(beta II) levels were also detected in the particulate fraction, whereas particulate PKC(alpha) levels did not change. The total PKC activity decreased in the cytosolic fraction following cyclic AMP stimulation of PC12h cells, whereas it stayed constant in the particulate fraction. Fractionation on a hydroxyapatite column showed a decreased level of PKC(alpha) activity and a transient increase followed by a decreased level of PKC(beta II) activity. This discrepancy between increased PKC(beta II) immunoreactivity and reduced PKC(beta II) activity suggested the presence of nonactivatable PKC(beta II) in cyclic AMP-treated PC12h extract. These findings indicate that PKC(alpha) and PKC(beta II) are differentially regulated during the differentiation of PC12h cells. In addition, the differentiation of PC12h cells triggered by cyclic AMP seems to involve characteristic alterations of PKC isozymes.