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-α, -δ, and -ζ 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-α, -δ, and -ζ 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-α and -δ, 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-ζ. In lysates of NIH-3T3 cells expressing the R1152Q/K1153A IRs (3T3Mut), insulin-induced coprecipitation of PKC-α, -δ, and -ζ 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-α and/or -δ with the IR appears to control its intracellular routing. The turn on of the insulin signaling mechanism by the insulin receptor (IR)1 involves a complex network of protein-protein interactions (1). Insulin-bound receptors phosphorylate a variety of docking proteins which include the IRS and Shc systems. Once phosphorylated, the docking proteins recruit and activate multiple insulin effectors (1). By employing the IRSs to engage Src homology 2 domain proteins, the IR avoids the stoichiometric constraints encountered by receptors which directly recruit these signaling molecules to their autophosphorylation sites (1, 2). The IRSs widen the connection and the tuning opportunities of the insulin signaling (3). There is evidence, however, that certain insulin bioeffects also follow the direct interaction of the IR with major insulin effectors. These effectors include phosphatidylinositol 3-kinase (4, 5) and, possibly, protein kinase C (PKC) (6, 7). PKCs represent a family of structurally and functionally related serine/threonine kinases derived from multiple genes as well as from alternative splicing of single mRNA transcripts (8, 9). The individual isoforms differ in their regulatory domains and in their dependence on Ca2+, as well as in their tissue distribution and intracellular localization (10, 11). PKCs appear to play a dual role in the insulin signaling network. First, PKCs control insulin-dependent receptor kinase activation (12-15) and may regulate IRS-1 signaling as well (12, 13). Second, at least in certain cells and tissues, insulin activation of PKCs is required to evoke insulin effects on glucose transport and its intracellular metabolism (16, 17). Current evidence (6) indicates that chimeric receptors consisting of the EGF receptor extracellular domain fused to the cytoplasmic domain of the IR form stable complexes with PKC-α following EGF binding. It has also been reported that insulin increases PKC activity in Tyr(P) Ab precipitates from KB cells (22). However, the molecular mechanisms of PKC activation in response to insulin as well as the role of each individual PKC isoform in insulin signal transduction is still unsettled. In previous reports (23) we demonstrated that Arg1152 and Lys1153 in the regulatory domain of the IR kinase are crucial for enabling IR phosphorylation by PKC. A peptide encoding the receptor sequence surrounding these residues inhibited phosphorylation of IR by PKC. In contrast, a mutant peptide in which the Arg and Lys were substituted by neutral amino acids exhibited no inhibitory effects, suggesting that IR phosphorylation by PKC follows direct IR-PKC interaction (23). In the present work we have shown that insulin controls IR association with PKC-α, -δ, and -ζ. In turn, in the NIH-3T3 cells, PKC-α and/or -δ association with IR appears crucial for enabling proper intracellular sorting of the receptor to the insulin degradative route.

In NIH-3T3 fibroblasts, insulin receptor interaction with specific protein kinase C isoforms controls receptor intracellular routing / Formisano, Pietro; Oriente, Francesco; Miele, C; Caruso, M; Auricchio, Renata; Vigliotta, G; Condorelli, Gerolama; Beguinot, Francesco. - In: THE JOURNAL OF BIOLOGICAL CHEMISTRY. - ISSN 0021-9258. - 273:21(1998), pp. 13197-13202. [10.1074/jbc.273.21.13197]

In NIH-3T3 fibroblasts, insulin receptor interaction with specific protein kinase C isoforms controls receptor intracellular routing

FORMISANO, PIETRO;ORIENTE, FRANCESCO;AURICCHIO, RENATA;CONDORELLI, GEROLAMA;BEGUINOT, FRANCESCO
1998

Abstract

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-α, -δ, and -ζ 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-α, -δ, and -ζ 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-α and -δ, 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-ζ. In lysates of NIH-3T3 cells expressing the R1152Q/K1153A IRs (3T3Mut), insulin-induced coprecipitation of PKC-α, -δ, and -ζ 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-α and/or -δ with the IR appears to control its intracellular routing. The turn on of the insulin signaling mechanism by the insulin receptor (IR)1 involves a complex network of protein-protein interactions (1). Insulin-bound receptors phosphorylate a variety of docking proteins which include the IRS and Shc systems. Once phosphorylated, the docking proteins recruit and activate multiple insulin effectors (1). By employing the IRSs to engage Src homology 2 domain proteins, the IR avoids the stoichiometric constraints encountered by receptors which directly recruit these signaling molecules to their autophosphorylation sites (1, 2). The IRSs widen the connection and the tuning opportunities of the insulin signaling (3). There is evidence, however, that certain insulin bioeffects also follow the direct interaction of the IR with major insulin effectors. These effectors include phosphatidylinositol 3-kinase (4, 5) and, possibly, protein kinase C (PKC) (6, 7). PKCs represent a family of structurally and functionally related serine/threonine kinases derived from multiple genes as well as from alternative splicing of single mRNA transcripts (8, 9). The individual isoforms differ in their regulatory domains and in their dependence on Ca2+, as well as in their tissue distribution and intracellular localization (10, 11). PKCs appear to play a dual role in the insulin signaling network. First, PKCs control insulin-dependent receptor kinase activation (12-15) and may regulate IRS-1 signaling as well (12, 13). Second, at least in certain cells and tissues, insulin activation of PKCs is required to evoke insulin effects on glucose transport and its intracellular metabolism (16, 17). Current evidence (6) indicates that chimeric receptors consisting of the EGF receptor extracellular domain fused to the cytoplasmic domain of the IR form stable complexes with PKC-α following EGF binding. It has also been reported that insulin increases PKC activity in Tyr(P) Ab precipitates from KB cells (22). However, the molecular mechanisms of PKC activation in response to insulin as well as the role of each individual PKC isoform in insulin signal transduction is still unsettled. In previous reports (23) we demonstrated that Arg1152 and Lys1153 in the regulatory domain of the IR kinase are crucial for enabling IR phosphorylation by PKC. A peptide encoding the receptor sequence surrounding these residues inhibited phosphorylation of IR by PKC. In contrast, a mutant peptide in which the Arg and Lys were substituted by neutral amino acids exhibited no inhibitory effects, suggesting that IR phosphorylation by PKC follows direct IR-PKC interaction (23). In the present work we have shown that insulin controls IR association with PKC-α, -δ, and -ζ. In turn, in the NIH-3T3 cells, PKC-α and/or -δ association with IR appears crucial for enabling proper intracellular sorting of the receptor to the insulin degradative route.
1998
In NIH-3T3 fibroblasts, insulin receptor interaction with specific protein kinase C isoforms controls receptor intracellular routing / Formisano, Pietro; Oriente, Francesco; Miele, C; Caruso, M; Auricchio, Renata; Vigliotta, G; Condorelli, Gerolama; Beguinot, Francesco. - In: THE JOURNAL OF BIOLOGICAL CHEMISTRY. - ISSN 0021-9258. - 273:21(1998), pp. 13197-13202. [10.1074/jbc.273.21.13197]
File in questo prodotto:
Non ci sono file associati a questo prodotto.

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11588/346448
Citazioni
  • ???jsp.display-item.citation.pmc??? ND
  • Scopus 41
  • ???jsp.display-item.citation.isi??? 41
social impact