Graham Rena

Roles of the forkhead in rhabdomyosarcoma (FKHR) phosphorylation sites in regulating 14-3-3 binding, transactivation and nuclear targetting.

The transcription factor, forkhead in rhabdomyosarcoma (FKHR), is phosphorylated at three amino acid residues (Thr-24, Ser-256 and Ser-319) by protein kinase B (PKB)alpha. In the present study, mutagenesis has been used to study the roles of these phosphorylation events in regulating FKHR function in transfected HEK-293 cells. We find that the overexpression of FKHR[S256A] (where Ser-256-->Ala) blocks PKB activity in cells, preventing phosphorylation of the endogenous substrates FKHRL1 and glycogen synthase kinase-3. Thus some reported effects of overexpression of this and other mutants may be indirect, and result from suppression of the phosphorylation of other sites on FKHR and/or other PKB substrates. For example, we have shown that Thr-24 phosphorylation alone is critical for interaction with 14-3-3 proteins, and that the substitution of Ser-256 with an alanine residue indirectly blocks 14-3-3 protein binding by preventing the phosphorylation of Thr-24. We also found that insulin-like growth factor (IGF)-1 and serum-induced nuclear exclusion of FKHR[S256A] depends on the degree of overexpression of this mutant. Our results indicated that the interaction of FKHR with 14-3-3 proteins was not required for IGF-1-stimulated exclusion of FKHR from the nucleus. We present evidence in support of another mechanism, which depends on the phosphorylation of Ser-256 and may involve the masking of a nuclear localization signal. Finally, we have demonstrated that the failure of IGF-1 to suppress transactivation by FKHR[S256A] is not explained entirely by its failure to bind 14-3-3 proteins or to undergo nuclear exclusion. This result suggests that Ser-256 phosphorylation may also suppress transactivation by FKHR by yet another mechanism, perhaps by disrupting the interaction of FKHR with target DNA binding sites and/or the function of the transactivation domain.

Two novel phosphorylation sites on FKHR that are critical for its nuclear exclusion.

FKHR is phosphorylated by protein kinase B (PKB) at Thr24, Ser256 and Ser319 in response to growth factors, stimulating the nuclear exit and inactivation of this transcription factor. Here we identify two further residues, Ser322 and Ser325, that become phosphorylated in insulin‐like growth factor‐1 (IGF‐1)‐stimulated cells and which are mediated by the phosphatidylinositol 3‐kinase‐dependent PKB‐catalysed phosphorylation of Ser319. Phosphorylation of Ser319 forms a consensus sequence for phosphorylation by CK1, allowing it to phosphorylate Ser322, which in turn primes the CK1‐catalysed phosphorylation of Ser325. IGF‐1 stimulates the phosphorylation of Thr24, Ser256, Ser319, Ser322 and Ser325 in embryonic stem (ES) cells, but not in PDK1−/− ES cells, providing genetic evidence that PDK1 (the upstream activator of PKB) is required for the phosphorylation of FKHR in mammalian cells. In contrast, the phosphorylation of Ser329 is unaffected by IGF‐1 and the phosphorylation of this site is not decreased in PDK1−/− ES cells. The cluster of phosphorylation sites at Ser319, Ser322, Ser325 and Ser329 appears to accelerate nuclear export by controlling the interaction of FKHR with the Ran‐containing protein complex that mediates this process.

D4476, a cell‐permeant inhibitor of CK1, suppresses the site‐specific phosphorylation and nuclear exclusion of FOXO1a

The protein kinase CK1 phosphorylates serine residues that are located close to another phosphoserine in the consensus pSer‐Xaa‐Xaa‐Ser. This specificity generates regions in its target proteins containing two or more neighbouring phosphoserine residues, termed here multisite phosphorylation domains (MPDs). In this paper, we demonstrate that D4476 is a potent and rather selective inhibitor of CK1 in vitro and in cells. In H4IIE hepatoma cells, D4476 specifically inhibits the phosphorylation of endogenous forkhead box transcription factor O1a (FOXO1a) on Ser322 and Ser325 within its MPD, without affecting the phosphorylation of other sites. Our results indicate that these residues are targeted by CK1 in vivo and that the CK1‐mediated phosphorylation of the MPD is required for accelerated nuclear exclusion of FOXO1a in response to IGF‐1 and insulin. D4476 is much more potent and specific than IC261 or CKI‐7, and is therefore the most useful CK1 inhibitor currently available for identifying physiological substrates of CK1.

Anti-Inflammatory Effects of Metformin Irrespective of Diabetes Status

Supplemental Digital Content is available in the text.

Insulin Regulation of Insulin-like Growth Factor-binding Protein-1 Gene Expression Is Dependent on the Mammalian Target of Rapamycin, but Independent of Ribosomal S6 Kinase Activity

Insulin inhibits the expression of the hepatic insulin-like growth factor-binding protein-1 (IGFBP-1) and glucose-6-phosphatase (G6Pase) genes. The signaling pathway that mediates these events requires the activation of phosphatidylinositol 3-kinase, whereas transfection studies have suggested an involvement of Akt (protein kinase B) and FKHR, a transcription factor regulated by Akt. We now demonstrate that insulin repression of endogenous IGFBP-1 gene transcription was blocked by rapamycin or by amino acid starvation. Rapamycin inhibited the mammalian target of rapamycin (mTOR) and the subsequent activation of p70/p85 S6 protein kinase-1 (S6K1) by insulin, whereas amino acid depletion prevented insulin induction of these signaling molecules. Importantly, we demonstrate that insulin regulation of the thymine-rich insulin response element of the IGFBP-1 promoter was also inhibited by rapamycin. However, sustained activation of S6K1 did not repress this promoter. In addition, rapamycin did not affect insulin regulation of G6Pase expression or Akt activation. We propose that these observations indicate that an mTOR-dependent, but S6K-independent mechanism regulates the suppression of IGFBP-1 (but not G6Pase) gene expression by insulin. Therefore, although the insulin-responsive sequence of the G6Pase gene promoter is related to that of the IGFBP-1 promoter, the signaling pathways that mediate suppression of these genes are distinct.

Cellular Responses to the Metal-Binding Properties of Metformin

In recent decades, the antihyperglycemic biguanide metformin has been used extensively in the treatment of type 2 diabetes, despite continuing uncertainty over its direct target. In this article, using two independent approaches, we demonstrate that cellular actions of metformin are disrupted by interference with its metal-binding properties, which have been known for over a century but little studied by biologists. We demonstrate that copper sequestration opposes known actions of metformin not only on AMP-activated protein kinase (AMPK)-dependent signaling, but also on S6 protein phosphorylation. Biguanide/metal interactions are stabilized by extensive π-electron delocalization and by investigating analogs of metformin; we provide evidence that this intrinsic property enables biguanides to regulate AMPK, glucose production, gluconeogenic gene expression, mitochondrial respiration, and mitochondrial copper binding. In contrast, regulation of S6 phosphorylation is prevented only by direct modification of the metal-liganding groups of the biguanide structure, supporting recent data that AMPK and S6 phosphorylation are regulated independently by biguanides. Additional studies with pioglitazone suggest that mitochondrial copper is targeted by both of these clinically important drugs. Together, these results suggest that cellular effects of biguanides depend on their metal-binding properties. This link may illuminate a better understanding of the molecular mechanisms enabling antihyperglycemic drug action.

Black tea polyphenols mimic insulin/insulin-like growth factor-1 signalling to the longevity factor FOXO1a

In vertebrates and invertebrates, relationships between diet and health are controlled by a conserved signalling pathway responsive to insulin‐like ligands. In invertebrate models for example, forkhead transcription factor family O (FOXO) transcription factors in this pathway regulate the rate of aging in response to dietary cues, and in vertebrates, obesity and age‐induced deficits in the same pathway are thought to contribute to dysregulation of hepatic gluconeogenesis through genes such as phosphoenolpyruvate carboxykinase (PEPCK). Recently, we have begun to screen for dietary constituents capable of regulating this pathway in our cell culture model. Here, we identify three black tea theaflavins, theaflavin 3‐O‐gallate, theaflavin 3′‐O‐gallate, theaflavin 3,3′di‐O‐gallate and thearubigins as novel mimics of insulin/IGF‐1 action on mammalian FOXO1a, PEPCK and moreover we provide evidence that the effects on this pathway of the green tea constituent (‐)‐epigallocatechin gallate depend on its ability to be converted into these larger structures. With the exception of water, tea is the most popular drink globally, but despite this, little is known about the biological availability of black tea polyphenols in vivo or the molecular target(s) mediating the effects presented here. Further investigation in these two areas might provide insight into how age‐related metabolic disease may be deferred.

Biomolecular Mode of Action of Metformin in Relation to Its Copper Binding Properties

Metformin (Metf), the most commonly used type 2 diabetes drug, is known to affect the cellular housekeeping of copper. Recently, we discovered that the structurally closely related propanediimidamide (PDI) shows a cellular behavior different from that of Metf. Here we investigate the binding of these compounds to copper, to compare their binding strength. Furthermore, we take a closer look at the electronic properties of these compounds and their copper complexes such as molecular orbital interactions and electrostatic potential surfaces. Our results clearly show that the copper binding energies cannot alone be the cause of the biochemical differentiation between Metf and PDI. We conclude that other factors such as pKa values and hydrophilicity of the compounds play a crucial role in their cellular activity. Metf in contrast to PDI can occur as an anion in aqueous medium at moderate pH, forming much stronger complexes particularly with Cu(II) ions, suggesting that biguanides but not PDI may induce easy oxidation of Cu(I) ions extracted from proteins. The higher hydrophobicity and the lack of planarity of PDI may further differentiate it from biguanides in terms of their molecular recognition characteristics. These different properties could hold the key to metformins mitochondrial activity because they suggest that the drug could act at least in part as a pro-oxidant of accessible protein-bound Cu(I) ions.

Antagonistic effects of phorbol esters on insulin regulation of insulin-like growth factor-binding protein-1 (IGFBP-1) but not glucose-6-phosphatase gene expression.

Glucose-6-phosphatase (G6Pase) and insulin-like growth factor-binding protein-1 (IGFBP-1) genes contain a homologous promoter sequence that is required for gene repression by insulin. Interestingly, this element interacts with members of the forkhead family of transcription factors [e.g. HNF3 (hepatic nuclear factor 3), FKHR (forkhead in rhabdomyosarcoma)] in vitro, while insulin promotes the phosphorylation and inactivation of FKHR in a phosphatidylinositol 3-kinase- and protein kinase B (PKB)-dependent manner. This mechanism has been proposed to underlie insulin action on G6Pase and IGFBP-1 gene transcription. However, we find that treatment of cells with phorbol esters mimics the effect of insulin on G6Pase, but not IGFBP-1, gene expression. Indeed, phorbol ester treatment actually blocks the ability of insulin to repress IGFBP-1 gene expression. In addition, the action of phorbol esters is significantly reduced by inhibition of the p42/p44 mitogen-activated protein (MAP) kinase pathway. However insulin-induced phosphorylation of PKB or FKHR is not affected by the presence of phorbol esters. Therefore we suggest that activation of p42/p44 MAP kinases will reduce the sensitivity of the IGFBP-1 gene promoter, but not the G6Pase gene promoter, to insulin. Importantly, the activation of PKB and phosphorylation of FKHR is not, in itself, sufficient to reduce IGFBP-1 gene expression in the presence of phorbol esters.

The anti-neurodegenerative agent clioquinol regulates the transcription factor FOXO1a

Many diseases of aging including AD (Alzheimers disease) and T2D (Type 2 diabetes) are strongly associated with common risk factors, suggesting that there may be shared aging mechanisms underlying these diseases, with the scope to identify common cellular targets for therapy. In the present study we have examined the insulin-like signalling properties of an experimental AD 8-hydroxyquinoline drug known as CQ (clioquinol). The IIS [insulin/IGF-1 (insulin-like growth factor-1) signalling] kinase Akt/PKB (protein kinase B) inhibits the transcription factor FOXO1a (forkhead box O1a) by phosphorylating it on residues that trigger its exit from the nucleus. In HEK (human embryonic kidney)-293 cells, we found that CQ treatment induces similar responses. A key transcriptional response to IIS is the inhibition of hepatic gluconeogenic gene expression, and, in rat liver cells, CQ represses expression of the key gluconeogenic regulatory enzymes PEPCK (phosphoenolpyruvate carboxykinase) and G6Pase (glucose-6-phosphatase). The effects on FOXO1a and gluconeogenic gene expression require the presence of Zn2+ ions, reminiscent of much earlier studies examining diabetogenic properties of 8-hydroxyquinolines. Comparative investigation of the signalling properties of a panel of these compounds demonstrates that CQ alone exhibits FOXO1a regulation without diabetogenicity. Our results suggest that Zn2+-dependent regulation of FOXOs and gluconeogenesis may contribute to the therapeutic properties of this drug. Further investigation of this signalling response might illuminate novel pharmacological strategies for the treatment of age-related diseases.