3-Phosphoinositide-dependent protein kinase-1 (PDK1) interacts stereoselectively with the d-enantiomer of PtdIns(3,4,5)P3 (KD 1.6 nM) and PtdIns(3,4)P2 (KD 5.2 nM), but binds with lower affinity to PtdIns3P or PtdIns(4,5)P2. The binding of PtdIns(3,4,5)P3 to PDK1 was greatly decreased by making specific mutations in the pleckstrin homology (PH) domain of PDK1 or by deleting it. The same mutations also greatly decreased the rate at which PDK1 activated protein kinase Balpha (PKBalpha) in vitro in the presence of lipid vesicles containing PtdIns(3,4,5)P3, but did not affect the rate at which PDK1 activated a PKBalpha mutant lacking the PH domain in the absence of PtdIns(3,4,5)P3. When overexpressed in 293 or PAE cells, PDK1 was located at the plasma membrane and in the cytosol, but was excluded from the nucleus. Mutations that disrupted the interaction of PtdIns(3,4,5)P3 or PtdIns(4,5)P2 with PDK1 abolished the association of PDK1 with the plasma membrane. Growth-factor stimulation promoted the translocation of transfected PKBalpha to the plasma membrane, but had no effect on the subcellular distribution of PDK1 as judged by immunoelectron microscopy of fixed cells. This conclusion was also supported by confocal microscopy of green fluorescent protein-PDK1 in live cells. These results, together with previous observations, indicate that PtdIns(3,4,5)P3 plays several roles in the PDK1-induced activation of PKBalpha. First, it binds to the PH domain of PKB, altering its conformation so that it can be activated by PDK1. Secondly, interaction with PtdIns(3,4,5)P3 recruits PKB to the plasma membrane with which PDK1 is localized constitutively by virtue of its much stronger interaction with PtdIns(3,4,5)P3 or PtdIns(4,5)P2. Thirdly, the interaction of PDK1 with PtdIns(3,4,5)P3 facilitates the rate at which it can activate PKB.

Insulin regulates the rate of expression of many hepatic genes, including PEPCK, glucose-6-phosphatase (G6Pase), and glucose-6-phosphate dehydrogenase (G6PDHase). The expression of these genes is also abnormally regulated in type 2 diabetes. We demonstrate here that treatment of hepatoma cells with 5-aminoimidazole-4-carboxamide riboside (AICAR), an agent that activates AMP-activated protein kinase (AMPK), mimics the ability of insulin to repress PEPCK gene transcription. It also partially represses G6Pase gene transcription and yet has no effect on the expression of G6PDHase or the constitutively expressed genes cyclophilin or -actin. Several lines of evidence suggest that the insulin-mimetic effects of AICAR are mediated by activation of AMPK. Also, insulin does not activate AMPK in H4IIE cells, suggesting that this protein kinase does not link the insulin receptor to the PEPCK and G6Pase gene promoters. Instead, AMPK and insulin may lie on distinct pathways that converge at a point upstream of these 2 gene promoters. Investigation of the pathway by which AMPK acts may therefore give insight into the mechanism of action of insulin. Our results also suggest that activation of AMPK would inhibit hepatic gluconeogenesis in an insulin-independent manner and thus help to reverse the hyperglycemia associated with type 2 diabetes. Diabetes 4 9 :8 9 6-903, 2000

The regulatory and catalytic properties of the three mammalian isoforms of protein kinase B (PKB) have been compared. All three isoforms (PKBalpha, PKBbeta and PKBgamma) were phosphorylated at similar rates and activated to similar extents by 3-phosphoinositide-dependent protein kinase-1 (PDK1). Phosphorylation and activation of each enzyme required the presence of PtdIns(3,4,5)P3 or PtdIns(3,4)P2, as well as PDK1. The activation of PKBbeta and PKBgamma by PDK1 was accompanied by the phosphorylation of the residues equivalent to Thr308 in PKBalpha, namely Thr309 (PKBbeta) and Thr305 (PKBgamma). PKBgamma which had been activated by PDK1 possessed a substrate specificity identical with that of PKBalpha and PKBbeta towards a range of peptides. The activation of PKBgamma and its phosphorylation at Thr305 was triggered by insulin-like growth factor-1 in 293 cells. Stimulation of rat adipocytes or rat hepatocytes with insulin induced the activation of PKBalpha and PKBbeta with similar kinetics. After stimulation of adipocytes, the activity of PKBbeta was twice that of PKBalpha, but in hepatocytes PKBalpha activity was four-fold higher than PKBbeta. Insulin induced the activation of PKBalpha in rat skeletal muscle in vivo, with little activation of PKBbeta. Insulin did not induce PKBgamma activity in adipocytes, hepatocytes or skeletal muscle, but PKBgamma was the major isoform activated by insulin in rat L6 myotubes (a skeletal-muscle cell line).

The mechanism by which leptin increases ATP-sensitive K ؉ (K ATP ) channel activity was investigated using the insulin-secreting cell line, CRI-G1. Wortmannin and LY 294002, inhibitors of phosphoinositide 3-kinase (PI3-kinase), prevented activation of K ATP channels by leptin. The inositol phospholipids phosphatidylinositol bisphosphate and phosphatidylinositol trisphosphate (PtdIns(3,4,5)P 3 ) mimicked the effect of leptin by increasing K ATP channel activity in whole-cell and insideout current recordings. LY 294002 prevented phosphatidylinositol bisphosphate, but not PtdIns(3,4,5)P 3 , from increasing K ATP channel activity, consistent with the latter lipid acting as a membrane-associated messenger linking leptin receptor activation and K ATP channels. Signaling cascades, activated downstream from PI 3-kinase, utilizing PtdIns(3,4,5)P 3 as a second messenger and commonly associated with insulin and cytokine action (MAPK, p70 ribosomal protein-S6 kinase, stress-activated protein kinase 2, p38 MAPK, and protein kinase B), do not appear to be involved in leptin-mediated activation of K ATP channels in this cell line. Although PtdIns(3,4,5)P 3 appears a plausible and attractive candidate for the messenger that couples K ATP channels to leptin receptor activation, direct measurement of PtdIns(3,4,5)P 3 demonstrated that insulin, but not leptin, increased global cellular levels of PtdIns(3,4,5)P 3 . Possible mechanisms to explain the involvement of PI 3-kinases in K ATP channel regulation are discussed.The hormone leptin, secreted by adipocytes, has a major influence on body weight homeostasis (1, 2). Although the hypothalamus is considered the main target for leptin, particularly with respect to body weight regulation, it is clear that this hormone has distinct actions on other peripheral, target organs. There have been several reports that leptin reduces insulin secretion from pancreatic beta cells (3-6), although this view is not shared by all investigators (7). One mechanism proposed to explain the leptin-induced reduction in insulin secretion is via activation of ATP-sensitive K ϩ (K ATP ) channels (8, 9). This increase in potassium current results in beta cell hyperpolarization, reduced calcium entry, and hence decreased insulin secretion. In addition, there are features common to both insulin-secreting cells and leptin-sensitive hypothalamic neurones (10, 11), most notably glucose responsiveness and the presence of K ATP channels, which are activated by exposure of the cells to leptin. The apparent involvement of both leptin receptors and K ATP channel activation in key systems involved in metabolic homeostasis has led us to examine the likely signal transduction pathways underlying this effect.The leptin receptor belongs to the class I cytokine receptor superfamily (1, 2), members of which are thought to signal via janus-tyrosine kinases. Activated janus-tyrosine kinases can mediate signaling via insulin receptor substrate proteins (12-14), which following tyrosine phosphorylation become docking sites for Sr...

Glucose-6-phosphatase plays an important role in the regulation of hepatic glucose production, and insulin suppresses glucose-6-phosphatase gene expression. Recent studies indicate that protein kinase B and Forkhead proteins contribute to insulin-regulated gene expression in the liver. Here, we examined the role of protein kinase B and Forkhead proteins in mediating effects of insulin on glucose-6-phosphatase promoter activity. Transient transfection studies with reporter gene constructs demonstrate that insulin suppresses both basal and dexamethasone/cAMP-induced activity of the glucose-6-phosphatase promoter in H4IIE hepatoma cells. Both effects are partially mimicked by coexpression of protein kinase B␣. Coexpression of the Forkhead transcription factor FKHR stimulates the glucose-6-phosphatase promoter activity via interaction with an insulin response unit (IRU), and this activation is suppressed by protein kinase B. Coexpression of a mutated form of FKHR that cannot be phosphorylated by protein kinase B abolishes the regulation of the glucose-6-phosphatase promoter by protein kinase B and disrupts the ability of insulin to regulate the glucose-6-phosphatase promoter via the IRU. Mutation of the insulin response unit of the glucose-6-phosphatase promoter also prevents the regulation of promoter activity by FKHR and protein kinase B but only partially impairs the ability of insulin to suppress both basal and dexamethasone/ cAMP-stimulated promoter function. Taken together, these results indicate that signaling by protein kinase B to Forkhead proteins can account for the ability of insulin to regulate glucose-6-phosphatase promoter activity via the IRU and that other mechanisms that are independent of the IRU, protein kinase B, and Forkhead proteins also are important in mediating effects of in insulin on glucose-6-phosphatase gene expression.Glucose-6-phosphatase (Glc-6-Pase) 1 catalyzes the hydrolysis of glucose 6-phosphate to glucose, which is the terminal step of both hepatic gluconeogenesis and glycogen breakdown. Glc-6-Pase is induced in starved and diabetic animals (1, 2). In vitro models have shown that glucocorticoids and cAMP induce Glc-6-Pase gene expression. This effect is opposed by insulin, which also is able to reduce basal expression of the Glc-6-Pase gene (3-6). Identification of the signaling events that connect the insulin receptor to the Glc-6-Pase promoter, leading to the subsequent repression of gene transcription, is of particular interest because Glc-6-Pase plays a key role in the regulation of hepatic glucose production and blood glucose homeostasis.In H4IIE hepatoma cells, activation of class 1a phosphoinositide 3-kinase (PI 3-kinase), but not of the Ras/Raf/MAP kinase pathway, is necessary for the suppression of Glc-6-Pase promoter activity by insulin (6). The formation of PtdIns(3,4,5)P 3 catalyzed by PI 3-kinase has been shown to increase the activity of 3-phosphoinositide-dependent protein kinase-1 (PDK1) and to result in a conformational change in PKB which renders it susceptible...

The protein G M , which targets protein phosphatase 1 (PP1) to the glycogen particles and sarcoplasmic reticulum (SR) of striated muscles, is known to be phosphorylated at Ser48 and Ser67 in vitro by adenosine 3P P,5P P cyclic monophosphatedependent protein kinase (PKA) and at Ser48 by MAP kinaseactivated protein kinase-1 (MAPKAP-K1, also called p90 RSK). The phosphorylation of Ser48 increases the rate at which the glycogen-associated PP1.G M complex dephosphorylates (activates) glycogen synthase, but the phosphorylation of Ser67 has the opposite effect, suppressing the activity of PP1 toward glycogen-bound substrates. The phosphorylation of Ser67 overrides the activating effect of Ser48 phosphorylation because it dissociates PP1 from G M . Here, we use two phospho-specific antibodies to demonstrate that the SR-associated form of G M , as well as the glycogen-associated form of G M , becomes phosphorylated at Ser48 and Ser67 in response to adrenaline, supporting the view that the PKA-mediated regulation of the PP1.G M complex plays a role in the adrenergic control of glycogen metabolism and SR function. In contrast, Ser48 is not phosphorylated significantly in response to insulin, and neither is Ser67. Thus the phosphorylation of G M at Ser48 by MAPKAP-K1 or other insulin-stimulated protein kinases is not involved in the activation of glycogen synthase by insulin.z 2000 Federation of European Biochemical Societies.

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