Up-regulation of cell surface insulin receptor by protein kinase C-alpha in adrenal chromaffin cells: Involvement of transcriptional and translational events

Our previous study showed that treatment of cultured bovine adrenal chromaf fin cells with phorbol 12,13-dibutyrate (PDBu) or 12-O-tetradecanoylphorbol 13-acetate (TPA) caused a rapid (<15 min) and persistent (>15 h) transloca tion of both conventional (c) protein kinase C-alpha (PKC-alpha) and novel PKC-epsilon (but not atypical PKC-zeta) from cytosol to membranes, whereas thymeleatoxin (TMX) increased the similar but selective membrane associatio n of only cPKC-alpha. In the present study, chronic (greater than or equal to 12 h) treatment of chromaffin cells with PDBu raised cell surface I-125- insulin binding without altering the K-D value it developed in a concentrat ion (EC50 = 1.9 n/M)- and time (t(1/2) = 14.6 h)-dependent manner, reaching its maximum 115% increase at 48 h. Either TPA (30 nM) or TMX (EC50 = 6.4 n M) also increased I-125-insulin binding by 97 or 88%, whereas the biologica lly inactive 4 alpha-TPA had no effect. The increasing effect of PDBu (30 n M for 24 h) on I-125-insulin binding was significantly blocked, even when H 7, an inhibitor of PKC, was added at 8 h after the initiation of PDBu treat ment. Concurrent treatment with brefeldin A, an inhibitor of vesicular tran sport from the trans-Golgi network, cycloheximide, an inhibitor of protein synthesis, or 5,6-dichlorobenzimidazole riboside, an inhibitor of RNA synth esis, abolished the PDBu-induced increment of I-125-insulin binding. Wester n blot analysis, using antibody against the beta-subunit of the insulin rec eptor, showed that treatment with PDBu (30 nM) or TMX (EC50 = 2.3 nM) incre ased levels of insulin receptor precursor (similar to 190 kDa; t(1/2) = 7.1 h) and insulin receptor beta-subunit (t(1/2) = 15.4 h), causing their almo st maximum 52 and 59% rises, respectively, at 24 h. Northern blot analysis revealed that PDBu or TMX increased levels of insulin receptor mRNAs by sim ilar to 35% as soon as 3 h, producing its monophasic peak similar to 76% in creases at 24 h. All of these increasing effects of PDBu and TMX on I-125-i nsulin binding and insulin receptor beta-subunit and insulin receptor mRNA levels were entirely prevented by simultaneous treatment with Go6976, a sel ective inhibitor of cPKC. These results suggest that long-term activation o f cPKC-alpha up-regulates the density of the cell surface insulin receptor via transcriptional/translational events.