Phosphoinositide (PI) 3-kinase contributes to a wide variety of biological actions, including insulin stimulation of glucose transport in adipocytes. Both Akt (protein kinase B), a serine-threonine kinase with a pleckstrin homology domain, and atypical isoforms of protein kinase C (PKC and PKC) have been implicated as downstream effectors of PI 3-kinase. Endogenous or transfected PKC in 3T3-L1 adipocytes or CHO cells has now been shown to be activated by insulin in a manner sensitive to inhibitors of PI 3-kinase (wortmannin and a dominant negative mutant of PI 3-kinase). Overexpression of kinase-deficient mutants of PKC (KD or ⌬NKD), achieved with the use of adenovirus-mediated gene transfer, resulted in inhibition of insulin activation of PKC, indicating that these mutants exert dominant negative effects. Insulin-stimulated glucose uptake and translocation of the glucose transporter GLUT4 to the plasma membrane, but not growth hormone-or hyperosmolarity-induced glucose uptake, were inhibited by KD or ⌬NKD in a dosedependent manner. The maximal inhibition of insulin-induced glucose uptake achieved by the dominant negative mutants of PKC was ϳ50 to 60%. These mutants did not inhibit insulin-induced activation of Akt. A PKC mutant that lacks the pseudosubstrate domain (⌬PD) exhibited markedly increased kinase activity relative to that of the wild-type enzyme, and expression of ⌬PD in quiescent 3T3-L1 adipocytes resulted in the stimulation of glucose uptake and translocation of GLUT4 but not in the activation of Akt. Furthermore, overexpression of an Akt mutant in which the phosphorylation sites targeted by growth factors are replaced by alanine resulted in inhibition of insulin-induced activation of Akt but not of PKC. These results suggest that insulin-elicited signals that pass through PI 3-kinase subsequently diverge into at least two independent pathways, an Akt pathway and a PKC pathway, and that the latter pathway contributes, at least in part, to insulin stimulation of glucose uptake in 3T3-L1 adipocytes.Phosphoinositide (PI) 3-kinase, a lipid kinase composed of an SRC homology 2 (SH2) domain-containing regulatory subunit and a 110-kDa catalytic subunit, catalyzes phosphorylation of the D3 position of PIs (46,48). This enzyme was first identified complexed with SRC kinase and the middle T antigen of polyomavirus and was later found to associate with various tyrosine-phosphorylated proteins in response to stimulation of cells with growth factors or cytokines (46, 48). Activation of PI 3-kinase, either by targeting of the enzyme to the plasma membrane (27) or as a consequence of direct interaction between the SH2 domain of the regulatory subunit and phosphorylated tyrosine residues present within specific motifs (5), results in the triggering of various important biological actions. Thus, with the use of either a dominant negative protein that blocks the interaction between PI 3-kinase and tyrosine-phosphorylated proteins (21,33,41) or pharmacological inhibitors of the enzyme, such as wortmannin or LY294002 (...

Listeria monocytogenes is a bacterial pathogen that invades cultured nonphagocytic cells. Inhibitors and a dominant negative mutation were used to demonstrate that efficient entry requires the phosphoinositide (PI) 3-kinase p85alpha-p110. Infection with L. monocytogenes caused rapid increases in cellular amounts of PI(3, 4)P2 and PI(3,4,5)P3, indicating that invading bacteria stimulated PI 3-kinase activity. This stimulation required the bacterial protein InlB, host cell tyrosine phosphorylation, and association of p85alpha with one or more tyrosine-phosphorylated proteins. This role for PI 3-kinase in bacterial entry may have parallels in some endocytic events.

The transcription factor, signal transducer and activator of transcription-3 (STAT-3) contributes to various physiological processes. Here we show that mice with liver-specific deficiency in STAT-3, achieved using the Cre-loxP system, show insulin resistance associated with increased hepatic expression of gluconeogenic genes. Restoration of hepatic STAT-3 expression in these mice, using adenovirus-mediated gene transfer, corrected the metabolic abnormalities and the alterations in hepatic expression of gluconeogenic genes. Overexpression of STAT-3 in cultured hepatocytes inhibited gluconeogenic gene expression independently of peroxisome proliferator-activated receptor-gamma coactivator-1 alpha (PGC-1 alpha), an upstream regulator of gluconeogenic genes. Liver-specific expression of a constitutively active form of STAT-3, achieved by infection with an adenovirus vector, markedly reduced blood glucose, plasma insulin concentrations and hepatic gluconeogenic gene expression in diabetic mice. Hepatic STAT-3 signaling is thus essential for normal glucose homeostasis and may provide new therapeutic targets for diabetes mellitus.

A wide variety of biological activities including the major metabolic actions of insulin is regulated by phosphatidylinositol (PI) 3-kinase. However, the downstream effectors of the various signaling pathways that emanate from PI 3-kinase remain unclear. Akt (protein kinase B), a serine-threonine kinase with a pleckstrin homology domain, is thought to be one such downstream effector. A mutant Akt (Akt-AA) in which the phosphorylation sites (Thr 308 and Ser 473 ) targeted by growth factors are replaced by alanine has now been shown to lack protein kinase activity and, when overexpressed in CHO cells or 3T3-L1 adipocytes with the use of an adenovirus vector, to inhibit insulin-induced activation of endogenous Akt. Akt-AA thus acts in a dominant negative manner in intact cells. Insulin-stimulated protein synthesis, which is sensitive to wortmannin, a pharmacological inhibitor of PI 3-kinase, was abolished by overexpression of Akt-AA without an effect on amino acid transport into the cells, suggesting that Akt is required for insulin-stimulated protein synthesis. Insulin activation of p70 S6 kinase was inhibited by ϳ75% in CHO cells and ϳ30% in 3T3-L1 adipocytes, whereas insulin-induced activation of endogenous Akt was inhibited by 80 to 95%, by expression of Akt-AA. Thus, Akt activity appears to be required, at least in part, for insulin stimulation of p70 S6 kinase. However, insulin-stimulated glucose uptake in both CHO cells and 3T3-L1 adipocytes was not affected by overexpression of Akt-AA, suggesting that Akt is not required for this effect of insulin. These data indicate that Akt acts as a downstream effector in some, but not all, of the signaling pathways downstream of PI 3-kinase.Akt is a pleckstrin homology (PH) domain-containing protein serine-threonine kinase whose kinase domain shares structural similarity with protein kinase C (PKC) isozymes and cyclic AMP-dependent protein kinase (PKA) (3). Thus, Akt has also been termed RAC-PK (protein kinase related to A and C kinases) (19) and PKB (protein kinase B) (7). Insulin and various other growth factors activate Akt, and this activation is inhibited by pharmacological blockers of phosphatidylinositol (PI) 3-kinase or by a dominant negative mutant of PI 3-kinase (4,14,25). Furthermore, Akt is activated by overexpression of a constitutively active mutant of PI 3-kinase in quiescent cells (11,23). These observations indicate that Akt is a downstream effector of PI 3-kinase.PI 3-kinase, which consists of an 85-kDa regulatory subunit and a 110-kDa catalytic subunit (5), is implicated in various metabolic effects of insulin (18, 59). A dominant negative mutant of PI 3-kinase as well as various pharmacological inhibitors, such as wortmannin and LY294002, have been used to block specific signaling pathways that include this enzyme (6,16,31,39,61). The metabolic actions of insulin that are sensitive to either a dominant negative mutant or pharmacological inhibitors of PI 3-kinase include stimulation of glucose uptake, antilipolysis, activation of fatty acid synthase ...

Cyclic nucleotide phosphodiesterase (PDE) is an important regulator of the cellular concentrations of the second messengers cyclic AMP (cAMP) and cGMP. Insulin activates the 3B isoform of PDE in adipocytes in a phosphoinositide 3-kinase-dependent manner; however, downstream effectors that mediate signaling to PDE3B remain unknown. Insulin-induced phosphorylation and activation of endogenous or recombinant PDE3B in 3T3-L1 adipocytes have now been shown to be inhibited by a dominant-negative mutant of the serine-threonine kinase Akt, suggesting that Akt is necessary for insulin-induced phosphorylation and activation of PDE3B. Serine-273 of mouse PDE3B is located within a motif (RXRXXS) that is preferentially phosphorylated by Akt. A mutant PDE3B in which serine-273 was replaced by alanine was not phosphorylated either in response to insulin in intact cells or by purified Akt in vitro. In contrast, PDE3B mutants in which alanine was substituted for either serine-296 or serine-421, each of which lies within a sequence (RRXS) preferentially phosphorylated by cAMP-dependent protein kinase, were phosphorylated by Akt in vitro or in response to insulin in intact cells. Moreover, the serine-273 mutant of PDE3B was not activated by insulin when expressed in adipocytes. These results suggest that PDE3B is a physiological substrate of Akt and that Akt-mediated phosphorylation of PDE3B on serine-273 is important for insulin-induced activation of PDE3B.Akt is a protein serine-threonine kinase that contains a pleckstrin homology domain and whose kinase domain has structural similarity with those of protein kinase C (PKC) isozymes and cyclic AMP (cAMP)-dependent protein kinase (PKA) (9, 21). Thus, Akt has also been termed protein kinase B. Akt was originally shown to be activated by growth factors such as platelet-derived growth factor and insulin, but later the enzyme was also found to be activated by cytokines and ligands for G protein-coupled receptors (21,33,34). Moreover, expression of polyomavirus middle T antigen as well as cellular stresses such as hyperosmolarity, heat shock, and fluid shear stress also induces activation of Akt (17,27,42). However, the mechanisms by which Akt is activated by these diverse stimuli are not fully understood. The activation of Akt by growth factors or cytokines is blocked by pharmacological or molecular biological inhibitors of phosphoinositide (PI) 3-kinase (7,19,24), indicating that PI 3-kinase is an upstream regulator of Akt, although PI 3-kinase-independent stimuli that induce activation of Akt also appear to exist (27,33,38).Akt is a general mediator of cell survival and protection from apoptosis (9, 21). It has also been suggested to participate in meiosis in oocytes (3), in endocytosis elicited by RAS (5), in differentiation of adipocytes (25), and in various metabolic actions of insulin (23,25,44,45). In spite of the potential importance of Akt in such diverse biological activities, only a few proteins have been identified as physiological substrates of this enzyme. The first iden...