Protein kinase C-zeta and protein kinase B regulate distinct steps of insulin endocytosis and intracellular sorting.
We have investigated the molecular mechanisms regulating insulin internalization and intracellular sorting. Insulin internalization was decreased by 50% upon incubation of the cells with the phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin and LY294002. PI3K inhibition also reduced insulin degradation and intact insulin release by 50 and 75%, respectively. Insulin internalization was reduced by antisense inhibition of protein kinase C-zeta (PKCzeta) expression and by overexpression of a dominant negative PKCzeta mutant (DN-PKCzeta). Conversely, overexpression of PKCzeta increased insulin internalization as a function of the PKCzeta levels achieved in the cells. Expression of wild-type protein kinase B (PKB)-alpha or of a constitutively active form (myr-PKB) did not significantly alter insulin internalization and degradation but produced a 100% increase of intact insulin release. Inhibition of PKB by a dominant negative mutant (DN-PKB) or by the pharmacological inhibitor ML-9 reduced intact insulin release by 75% with no effect on internalization and degradation. In addition, overexpression of Rab5 completely rescued the effect of PKCzeta inhibition on insulin internalization but not that of PKB inhibition on intact insulin recycling. Indeed, PKCzeta bound to and activated Rab5. Thus, PI3K controls different steps within the insulin endocytic itinerary. PKCzeta appears to mediate the PI3K effect on insulin internalization in a Rab5-dependent manner, whereas PKB directs intracellular sorting toward intact insulin release.
Fiory
Francesca
F
Dipartimento di Biologia e Patologia Cellulare e Molecolare L. Califano and Istituto di Endocrinologia ed Oncologia Sperimentale del C.N.R., Università degli Studi di Napoli Federico II, Via S. Pansini, 5, 80131 Naples, Italy.
Oriente
Francesco
F
Miele
Claudia
C
Romano
Chiara
C
Trencia
Alessandra
A
Alberobello
Anna Teresa
AT
Esposito
Iolanda
I
Valentino
Rossella
R
Beguinot
Francesco
F
Formisano
Pietro
P
D016475:3T3 Cells; D000818:Animals; D004705:Endocytosis; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007328:Insulin / Q000378:metabolism*; D008099:Liver / Q000201:enzymology; D051379:Mice; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt; D014162:Transfection; D020696:rab5 GTP-Binding Proteins / Q000502:physiology; D000099221:Protein Kinase C zeta
D004791:Enzyme Inhibitors; D007328:Insulin; D019869:Phosphatidylinositol 3-Kinases; D011493:Protein Kinase C; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D020696:rab5 GTP-Binding Proteins; D000099221:Protein Kinase C zeta