In NIH-3T3 fibroblasts, insulin receptor interaction with specific protein kinase C isoforms controls receptor intracellular routing.
Insulin increased protein kinase C (PKC) activity by 2-fold in both membrane preparations and insulin receptor (IR) antibody precipitates from NIH-3T3 cells expressing human IRs (3T3hIR). PKC-alpha, -delta, and -zeta were barely detectable in IR antibody precipitates of unstimulated cells, while increasing by 7-, 3.5-, and 3-fold, respectively, after insulin addition. Preexposure of 3T3hIR cells to staurosporine reduced insulin-induced receptor coprecipitation with PKC-alpha, -delta, and -zeta by 3-, 4-, and 10-fold, respectively, accompanied by a 1.5-fold decrease in insulin degradation and a similar increase in insulin retroendocytosis. Selective depletion of cellular PKC-alpha and -delta, by 24 h of 12-O-tetradecanoylphorbol-13-acetate (TPA) exposure, reduced insulin degradation by 3-fold and similarly increased insulin retroendocytosis, with no change in PKC-zeta. In lysates of NIH-3T3 cells expressing the R1152Q/K1153A IRs (3T3Mut), insulin-induced coprecipitation of PKC-alpha, -delta, and -zeta with the IR was reduced by 10-, 7-, and 3-fold, respectively. Similar to the 3T3hIR cells chronically exposed to TPA, untreated 3T3Mut featured a 3-fold decrease in insulin degradation, with a 3-fold increase in intact insulin retroendocytosis. Thus, in NIH-3T3 cells, insulin elicits receptor interaction with multiple PKC isoforms. Interaction of PKC-alpha and/or -delta with the IR appears to control its intracellular routing.
Formisano
P
P
Dipartimento di Biologia e Patologia Cellulare e Molecolare "L. Califano" and Centro di Endocrinologia ed Oncolgia Sperimentale del Consiglio Nazionale delle Ricerche (CNR), "Federico II" University of Naples Medical School, Naples, Italy.
Oriente
F
F
Miele
C
C
Caruso
M
M
Auricchio
R
R
Vigliotta
G
G
Condorelli
G
G
Beguinot
F
F
D016475:3T3 Cells; D000818:Animals; D001692:Biological Transport; D015536:Down-Regulation; D005347:Fibroblasts / Q000201:enzymology / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism*; D051379:Mice; D011233:Precipitin Tests; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011972:Receptor, Insulin / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism
D007527:Isoenzymes; D011994:Recombinant Proteins; D011972:Receptor, Insulin; D011493:Protein Kinase C