Protein kinase C (PKC)-alpha exerts a regulatory function on insulin action. We showed by overlay blot that PKC alpha directly binds a 180-kDa protein, corresponding to IRS-1, and a 30-kDa molecular species, identified as 14-3-3 epsilon. In intact NIH-3T3 cells overexpressing insulin receptors (3T3-hIR), insulin selectively increased PKC alpha coprecipitation with IRS-1, but not with IRS-2, and with 14-3-3 epsilon, but not with other 14-3-3 isoforms. Overexpression of 14-3-3 epsilon in 3T3-hIR cells significantly reduced IRS-1-bound PKC alpha activity, without altering IRS-1/PKC alpha co-precipitation. 14-3-3 epsilon overexpression also increased insulin-stimulated insulin receptor and IRS-1 tyrosine phosphorylation, followed by increased activation of Raf1, ERK1/2, and Akt/protein kinase B. Insulin-induced glycogen synthase activity and thymidine incorporation were also augmented. Consistently, selective depletion of 14-3-3 epsilon by antisense oligonucleotides caused a 3-fold increase of IRS-1-bound PKC alpha activity and a similarly sized reduction of insulin receptor and IRS-1 tyrosine phosphorylation and signaling. In turn, selective inhibition of PKC alpha expression by antisense oligonucleotides reverted the negative effect of 14-3-3 epsilon depletion on insulin signaling. Moreover, PKC alpha inhibition was accompanied by a > 2-fold decrease of insulin degradation. Similar results were also obtained by overexpressing 14-3-3 epsilon. Thus, in NIH-3T3 cells, insulin induces the formation of multimolecular complexes, including IRS-1, PKC alpha, and 14-3-3 epsilon. The presence of 14-3-3 epsilon in the complex is not necessary for IRS-1/PKC alpha interaction but modulates PKC alpha activity, thereby regulating insulin signaling and degradation.

Protein kinase C-alpha regulates insulin action and degradation by interacting with insulin receptor substrate-1 and 14-3-3epsilon.

Miele C;Beguinot F;Formisano P
2005

Abstract

Protein kinase C (PKC)-alpha exerts a regulatory function on insulin action. We showed by overlay blot that PKC alpha directly binds a 180-kDa protein, corresponding to IRS-1, and a 30-kDa molecular species, identified as 14-3-3 epsilon. In intact NIH-3T3 cells overexpressing insulin receptors (3T3-hIR), insulin selectively increased PKC alpha coprecipitation with IRS-1, but not with IRS-2, and with 14-3-3 epsilon, but not with other 14-3-3 isoforms. Overexpression of 14-3-3 epsilon in 3T3-hIR cells significantly reduced IRS-1-bound PKC alpha activity, without altering IRS-1/PKC alpha co-precipitation. 14-3-3 epsilon overexpression also increased insulin-stimulated insulin receptor and IRS-1 tyrosine phosphorylation, followed by increased activation of Raf1, ERK1/2, and Akt/protein kinase B. Insulin-induced glycogen synthase activity and thymidine incorporation were also augmented. Consistently, selective depletion of 14-3-3 epsilon by antisense oligonucleotides caused a 3-fold increase of IRS-1-bound PKC alpha activity and a similarly sized reduction of insulin receptor and IRS-1 tyrosine phosphorylation and signaling. In turn, selective inhibition of PKC alpha expression by antisense oligonucleotides reverted the negative effect of 14-3-3 epsilon depletion on insulin signaling. Moreover, PKC alpha inhibition was accompanied by a > 2-fold decrease of insulin degradation. Similar results were also obtained by overexpressing 14-3-3 epsilon. Thus, in NIH-3T3 cells, insulin induces the formation of multimolecular complexes, including IRS-1, PKC alpha, and 14-3-3 epsilon. The presence of 14-3-3 epsilon in the complex is not necessary for IRS-1/PKC alpha interaction but modulates PKC alpha activity, thereby regulating insulin signaling and degradation.
2005
Istituto di Endocrinologia e Oncologia Sperimentale ''G. Salvatore'' - IEOS
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/20.500.14243/52060
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