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Up‐Regulation of Cell Surface Insulin Receptor by Protein Kinase C‐α in Adrenal Chromaffin Cells
Author(s) -
Yamamoto Ryuichi,
Kobayashi Hideyuki,
Yanagita Toshihiko,
Yokoo Hiroki,
Kurose Takeshi,
Shiraishi Seiji,
Minami Shinichi,
Matsukura Shigeru,
Wada Akihiko
Publication year - 2000
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.2000.750672.x
Subject(s) - protein kinase c , medicine , cycloheximide , endocrinology , insulin , chromaffin cell , insulin receptor , receptor , cytosol , biology , brefeldin a , phorbol , chemistry , kinase , microbiology and biotechnology , golgi apparatus , cell , adrenal medulla , biochemistry , protein biosynthesis , catecholamine , enzyme , insulin resistance
Our previous study showed that treatment of cultured bovine adrenal chromaffin cells with phorbol 12, 13‐dibutyrate (PDBu) or 12‐ O ‐tetradecanoylphorbol 13‐acetate (TPA) caused a rapid (<15 min) and persistent (>15 h) translocation of both conventional (c) protein kinase C‐α (PKC‐α) and novel PKC‐ε (but not atypical PKC‐ζ) from cytosol to membranes, whereas thymeleatoxin (TMX) increased the similar but selective membrane association of only cPKC‐α. In the present study, chronic (≥12 h) treatment of chromaffin cells with PDBu raised cell surface 125 I‐insulin binding without altering the K D value ; it developed in a concentration (EC 50 = 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 n M ) or TMX (EC 50 = 6.4 n M ) also increased 125 I‐insulin binding by 97 or 88%, whereas the biologically inactive 4α‐TPA had no effect. The increasing effect of PDBu (30 n M for 24 h) on 125 I‐insulin binding was significantly blocked, even when H7, an inhibitor of PKC, was added at 8 h after the initiation of PDBu treatment. Concurrent treatment with brefeldin A, an inhibitor of vesicular transport from the trans ‐Golgi network, cycloheximide, an inhibitor of protein synthesis, or 5,6‐dichlorobenzimidazole riboside, an inhibitor of RNA synthesis, abolished the PDBu‐induced increment of 125 I‐insulin binding. Western blot analysis, using antibody against the β‐subunit of the insulin receptor, showed that treatment with PDBu (30 n M ) or TMX (EC 50 = 2.3 n M ) increased levels of insulin receptor precursor (~190 kDa ; t 1/2 = 7.1 h) and insulin receptor β‐subunit ( t 1/2 = 15.4 h), causing their almost maximum 52 and 59% rises, respectively, at 24 h. Northern blot analysis revealed that PDBu or TMX increased levels of insulin receptor mRNAs by ~35% as soon as 3 h, producing its monophasic peak ~76% increases at 24 h. All of these increasing effects of PDBu and TMX on 125 I‐insulin binding and insulin receptor β‐subunit and insulin receptor mRNA levels were entirely prevented by simultaneous treatment with Gö6976, a selective inhibitor of cPKC. These results suggest that long‐term activation of cPKC‐α up‐regulates the density of the cell surface insulin receptor via transcriptional/translational events.