Protein Kinase C-ζ and Protein Kinase B Regulate Distinct Steps of Insulin Endocytosis and Intracellular Sorting

Abstract

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-(PKC ) expression and by overexpression of a dominant negative PKC mutant (DN-PKC ). Conversely, overexpression of PKC increased insulin internalization as a function of the PKC levels achieved in the cells. Expression of wild-type protein kinase B (PKB)-␣ 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 PKC inhibition on insulin internalization but not that of PKB inhibition on intact insulin recycling. Indeed, PKC bound to and activated Rab5. Thus, PI3K controls different steps within the insulin endocytic itinerary. PKC appears to mediate the PI3K effect on insulin internalization in a Rab5-dependent manner, whereas PKB directs intracellular sorting toward intact insulin release.Insulin binding is followed by tyrosine phosphorylation of insulin receptor (IR) 1 and of its intracellular substrates (1). Thereafter, insulin signaling impinges on at least three major enzymatic systems, the Ras/extracellular signal-regulated kinases, the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB), and the protein kinase C (PKC) signal transduction pathways. These systems contribute to insulin regulation of cell metabolism, differentiation, and growth (2).In addition, hormone-bound IR is rapidly removed from the cell surface and internalized within clathrin-coated vesicles (3). Internalized insulin is then either degraded or released intact in the extracellular medium (4, 5). IR endocytosis is necessary for insulin clearance from the circulation (6, 7), down-regulation of surface binding sites (8), and transduction of specific biological effects (9 -11). Defective insulin internalization may also cause insulin resistance in animal models (7). It is now clear that endocytosis of insulin-IR complexes is triggered by receptor tyrosine kinase activation (12, 13). Receptor autophosphorylation on tyrosine residues allows the migration of IR toward coated pits (14), but it may not be sufficient for inducing internalization (15,16). Nevertheless, the molecular mechanisms, downstream receptor kinase activation, which control the internalization and the subsequent intracellular itinerary, have not been elucidated.PI3K is necessary for the internalization of several growth factor receptors (17-19). PI3K and its downst...

Type-3 metabotropic glutamate receptors regulate chemoresistance in glioma stem cells, and their levels are inversely related to survival in patients with malignant gliomas

“…The actions of LY294002 and LY341495 were less than additive (Figure 3a), suggesting that mGlu3 receptor blockade facilitates cytotoxicity by limiting the activation of the PtdIns-3-K pathway. This hypothesis was supported by the use of GSCs expressing a constitutively active form of the PtdIns-3-K substrate, Akt 15 (see Supplementary Figure 3). In these cells, in which the PtdIns-3-K pathway was active in spite of mGlu3 receptor blockade, the synergism between LY341495 and temozolomide was largely attenuated (Figure 3b).…”

“…This suggests that, at least under our conditions, other endogenously active receptors coupled to PtdIns-3-K could not rescue temozolomide-induced MGMT expression when mGlu3 receptors were blocked. NF-kB, which is activated by the PtdIns-3-K/Akt pathway, 15 is one of the strongest inducers of MGMT expression. In recombinant cells, NF-kB/p65 homodimers enhance MGMT expression sevenfold more than AP1/ c-jun, and, remarkably, induction of MGMT by NF-kB is unaffected by the methylation state of the MGMT gene promoter.…”

Insulin-stimulated Interaction between Insulin Receptor Substrate 1 and p85α and Activation of Protein Kinase B/Akt Require Rab5

“…Insulin receptors were found to be internalized upon insulin binding, and the rate of internalization was decreased by overexpression of the dominant negative form of Rab5a (14). Therefore, internalized IR was analyzed as described under "Experimental Procedures."…”

“…However, down-regulation of all three Rab5 isoforms significantly decreased the internalization of both EGF and Tfn (32). Experiments utilizing dominant negative Rab5A suggest a requirement for Rab5 activity in both IR and EGF internalization (14,33).…”

“…After binding to insulin, the activated receptor complex is internalized into endosomes from which receptors are either recycled back to the PM or transported to lysosomes for degradation (14,15). Thus, receptor internalization represents a mechanism for insulin degradation and receptor deactivation.…”