Different Modulation of Protein Kinase C Isozymes in Dibutyryl Cyclic AMP‐Differentiated PC12h Cells

Abstract

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 (α, βI, βII, and γ) were analyzed using column chromatography and immunoblotting. Two PKC isozymes, PKC(α) and PKC(βII), were predominantly detected in PC12h cells. When stimulated by dibutyryl cyclic AMP, PKC(α) levels declined in the cytosolic fraction of the cells, whereas PKC(βII) levels increased. Increased PKC(βII) levels were also detected in the particulate fraction, whereas particulate PKC(α) 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(α) activity and a transient increase followed by a decreased level of PKC(βII) activity. This discrepancy between increased PKC(βII) immunoreactivity and reduced PKC(βII) activity suggested the presence of nonactivatable PKC(βII) in cyclic AMP‐treated PC12h extract. These findings indicate that PKC(α) and PKC(βII) 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.

Ca2+‐Dependent and Ca2+‐Independent Protein Kinase C Changes in the Brains of Patients with Alzheimer's Disease

“…Ca 2-dependent PKC assay was determined by using a commercially available PKC enzyme assay system (Amersham, U.K.) as described previously (Cardell et al, 1991;Yamaoka et al, 1993;Uehara-Kunugi et a!., 1994). In brief, the reaction mixtures (75~l) containing 50 mM Tris-HC1 (pH 7.5), 50~zg/ml PS, 150 ng phorbol 12-myristate 13acetate (PMA), 2.5 mM dithiothreitol, 50~M ATP, 0.25 sCi [y-32P]ATP, 0.5 mM calcium acetate, 15 mM magnesium acetate, 75 mM specific substrate peptide, and a brain extract.…”

“…The Ca2-independent PKC assay was performed essentially as described previously (Mizuno et a!., 199! ;Uehara-Kunugi et a!., 1994). In brief, the reaction mixture (75 gil) contained 50 mM Tris-HC1 (pH 7.5), 50~tg/ml CL (or PS, P1), 50 ng/ml PMA, 50~iM ATP, 0.25~iCi [y-32P]ATP, 15 mM magnesium acetate, 50~ig/ml MBP 4_14, 1 mM EGTA, and a brain extract.…”