Lm Fletcher

Protein kinase B phosphorylation of PIKfyve regulates the trafficking of GLUT4 vesicles

Insulin-stimulated glucose uptake involves the recruitment of the glucose transporter 4 isoform (GLUT4) from an intracellular location to the plasma membrane of fat and muscle cells. Although the activation of the PI3-kinase/protein kinase B (PKB) pathway is central to this effect of insulin, the key substrates for PKB that are involved require identification. Here we report that serine318 on the FYVE domain-containing PtdIns(3)P 5-kinase (PIKfyve) is a novel substrate for PKB, and show that phosphorylation stimulates the PtdIns(3)P 5-kinase activity of the enzyme. We also demonstrate that PIKfyve is phosphorylated on serine318 in intact cells in response to insulin, in a PI3-kinase-dependent manner, and that PIKfyve colocalises with a highly motile subpopulation of insulin-regulated aminopeptidase (IRAP)/GLUT4 vesicles. Finally, we demonstrate that overexpression of a PIKfyve[S318A] mutant in 3T3-L1 adipocytes enhances insulin-stimulated IRAP/GLUT4 vesicle translocation to the plasma membrane suggesting a role for PKB-dependent phosphorylation of PIKfyve in insulin-regulated IRAP/GLUT4 trafficking. The phosphorylation and activation of PIKfyve by PKB provides a novel signalling paradigm that may link plasma membrane-localised PtdIns(3,4,5)P3 signals via a protein kinase cascade to regulated PtdIns(3,5)P2 production, and thereby to the control of trafficking of other membrane cargos.

Protein kinase B stimulates the translocation of GLUT4 but not GLUT1 or transferrin receptors in 3T3-L1 adipocytes by a pathway involving SNAP-23, synaptobrevin-2, and/or cellubrevin.

An interaction of SNAP-23 and syntaxin 4 on the plasma membrane with vesicle-associated synaptobrevin-2 and/or cellubrevin, known as SNAP (soluble N -ethyl-maleimide-sensitive factorattachment protein) receptors or SNAREs, has been proposed to provide the targeting and/or fusion apparatus for insulin-stimulated translocation of the GLUT4 isoform of glucose transporter to the plasma membrane. By microinjecting 3T3-L1 adipocytes with the Clostridium botulinum toxin B or E, which proteolyzed synaptobrevin-2/cellubrevin and SNAP-23, respectively, we investigated the role of these SNAREs in GLUT4, GLUT1, and transferrin receptor trafficking. As expected, insulin stimulated the translocation of GLUT4, GLUT1, and transferrin receptors to the plasma membrane. By contrast, a constitutively active protein kinase B (PKB-DD) only stimulated a translocation of GLUT4 and not GLUT1 or the transferrin receptor. The GLUT4 response to PKB-DD was abolished by toxins B or E, whereas the insulin-evoked translocation of GLUT4 was inhibited by approximately 65%. These toxins had no significant effect on insulin-stimulated transferrin receptor appearance at the cell surface. Thus, insulin appears to induce GLUT4 translocation via two distinct routes, only one of which involves SNAP-23 and synaptobrevin-2/cellubrevin, and can be mobilized by PKB-DD. The PKB-, SNAP-23-, and synaptobrevin-2/cellubrevin-independent GLUT4 translocation pathway may involve movement through recycling endosomes, together with GLUT1 and transferrin receptors.

Confocal imaging of the subcellular distribution of phosphatidylinositol 3,4,5-trisphosphate in insulin- and PDGF-stimulated 3T3-L1 adipocytes.

The activation of phosphatidylinositol 3-kinase (PI 3-kinase) and production of PtdIns(3,4,5)P(3) is crucial in the actions of numerous extracellular stimuli, including insulin-stimulated glucose uptake. Platelet-derived growth factor (PDGF) also stimulates PI 3-kinase, but only weakly promotes glucose uptake when compared with insulin. Insulin and PDGF have thus been proposed to have differential effects on the subcellular targeting of PI 3-kinase. However, owing to a lack of suitable methodologies, the subcellular localization of the PtdIns(3,4,5)P(3) generated has not been examined. The pleckstrin-homology (PH) domains of the nucleotide exchange factors, ADP-ribosylation factor nucleotide-binding-site opener (ARNO) and general receptor for 3-phosphoinositides (GRP1), which have a high affinity and specificity for PtdIns(3,4,5)P(3), were fused to green fluorescent protein and used to examine the subcellular localization of PtdIns(3,4,5)P(3) generation in living 3T3-L1 adipocytes. PtdIns(3,4,5)P(3) was produced almost exclusively in the plasma membrane in response to both agonists, although the response to insulin was greater in magnitude and occurred in considerably more cells. The results suggest that the greater ability of insulin to stimulate glucose uptake may be the result of its ability to generate significantly more plasma-membrane PtdIns(3, 4,5)P(3) than PDGF. ARNO and GRP1 are nucleotide exchange factors for the small GTP-binding protein ADP-ribosylation factor 6 (ARF6). The inability of a constitutively active GTPase-deficient mutant of ARF6 (ARF6-Q67L; Gln(67)-->Leu) to cause glucose transporter GLUT4 translocation suggests that activation of this pathway is not sufficient to cause GLUT4 translocation.

Nucleo-cytosolic shuttling of FoxO1 directly regulates mouse Ins2 but not Ins1 gene expression in pancreatic beta cells (MIN6).

The Forkhead box transcription factor FoxO1 regulates metabolic gene expression in mammals. FoxO1 activity is tightly controlled by phosphatidylinositol 3-kinase (PI3K) signaling, resulting in its phosphorylation and nuclear exclusion. We sought here to determine the mechanisms involved in glucose and insulin-stimulated nuclear shuttling of FoxO1 in pancreatic β cells and its consequences for preproinsulin (Ins1, Ins2) gene expression. Nuclear-localized endogenous FoxO1 translocated to the cytosol in response to elevated glucose (3 versus 16.7 mm) in human islet β cells. Real-time confocal imaging of nucleo-cytosolic shuttling of a FoxO1-EGFP chimera in primary mouse and clonal MIN6 β cells revealed a time-dependent glucose-responsive nuclear export, also mimicked by exogenous insulin, and blocked by suppressing insulin secretion. Constitutively active PI3K or protein kinase B/Akt exerted similar effects, while inhibitors of PI3K, but not of glycogen synthase kinase-3 or p70 S6 kinase, blocked nuclear export. FoxO1 overexpression reversed the activation by glucose of pancreatic duodenum homeobox-1 (Pdx1) transcription. Silencing of FoxO1 significantly elevated the expression of mouse Ins2, but not Ins1, mRNA at 3 mm glucose. Putative FoxO1 binding sites were identified in the distal promoter of rodent Ins2 genes and direct binding of FoxO1 to the Ins2 promoter was demonstrated by chromatin immunoprecipitation. A 915-bp glucose-responsive Ins2 promoter was inhibited by constitutively active FoxO1, an effect unaltered by simultaneous overexpression of PDX1. We conclude that nuclear import of FoxO1 contributes to the suppression of Pdx1 and Ins2 gene expression at low glucose, the latter via a previously unsuspected and direct physical interaction with the Ins2 promoter.