Protein kinase C-alpha regulates insulin action and degradation by interacting with insulin receptor substrate-1 and 14-3-3 epsilon.
Protein kinase C (PKC)-alpha exerts a regulatory function on insulin action. We showed by overlay blot that PKCalpha directly binds a 180-kDa protein, corresponding to IRS-1, and a 30-kDa molecular species, identified as 14-3-3epsilon. In intact NIH-3T3 cells overexpressing insulin receptors (3T3-hIR), insulin selectively increased PKCalpha co-precipitation with IRS-1, but not with IRS-2, and with 14-3-3epsilon, but not with other 14-3-3 isoforms. Overexpression of 14-3-3epsilon in 3T3-hIR cells significantly reduced IRS-1-bound PKCalpha activity, without altering IRS-1/PKCalpha co-precipitation. 14-3-3epsilon 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 14-3-3epsilon by antisense oligonucleotides caused a 3-fold increase of IRS-1-bound PKCalpha activity and a similarly sized reduction of insulin receptor and IRS-1 tyrosine phosphorylation and signaling. In turn, selective inhibition of PKCalpha expression by antisense oligonucleotides reverted the negative effect of 14-3-3epsilon depletion on insulin signaling. Moreover, PKCalpha inhibition was accompanied by a >2-fold decrease of insulin degradation. Similar results were also obtained by overexpressing 14-3-3epsilon. Thus, in NIH-3T3 cells, insulin induces the formation of multimolecular complexes, including IRS-1, PKCalpha, and 14-3-3epsilon. The presence of 14-3-3epsilon in the complex is not necessary for IRS-1/PKCalpha interaction but modulates PKCalpha activity, thereby regulating insulin signaling and degradation.
Oriente
Francesco
F
Dipartimento di Biologia e Patologia Cellulare e Molecolare and Istituto di Endocrinologia ed Oncologia Sperimentale del CNR, Federico II University of Naples, Italy.
Andreozzi
Francesco
F
Romano
Chiara
C
Perruolo
Giuseppe
G
Perfetti
Anna
A
Fiory
Francesca
F
Miele
Claudia
C
Beguinot
Francesco
F
Formisano
Pietro
P
D048948:14-3-3 Proteins / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D015153:Blotting, Western; D015870:Gene Expression; D020868:Gene Silencing; D006006:Glycogen Synthase / Q000378:metabolism; D006801:Humans; D007163:Immunosorbent Techniques; D007328:Insulin / Q000378:metabolism* / Q000494:pharmacology*; D055504:Insulin Receptor Substrate Proteins; D051379:Mice; D041681:NIH 3T3 Cells; D016376:Oligonucleotides, Antisense; D010750:Phosphoproteins / Q000378:metabolism*; D051571:Protein Kinase C-alpha / Q000235:genetics / Q000502:physiology*; D011972:Receptor, Insulin / Q000235:genetics; D011994:Recombinant Proteins; D015398:Signal Transduction; D013936:Thymidine / Q000378:metabolism; D014162:Transfection
D048948:14-3-3 Proteins; C526292:IRS1 protein, human; D007328:Insulin; D055504:Insulin Receptor Substrate Proteins; C526295:Irs1 protein, mouse; D016376:Oligonucleotides, Antisense; D010750:Phosphoproteins; D011994:Recombinant Proteins; D006006:Glycogen Synthase; D011972:Receptor, Insulin; D051571:Protein Kinase C-alpha; D013936:Thymidine