Acylated and unacylated ghrelin promote proliferation and inhibit apoptosis of pancreatic beta-cells and human islets: involvement of 3',5'-cyclic adenosine monophosphate/protein kinase A, extracellular signal-regulated kinase 1/2, and phosphatidyl inositol 3-Kinase/Akt signaling

Among its pleiotropic actions, ghrelin modulates insulin secretion and glucose metabolism. Herein we investigated the role of ghrelin in pancreatic beta-cell proliferation and apoptosis induced by serum starvation or interferon (IFN)-gamma/TNF-alpha, whose synergism is a major cause for beta-cell destruction in type I diabetes. HIT-T15 beta-cells expressed ghrelin but not ghrelin receptor (GRLN-R), which binds acylated ghrelin (AG) only. However, both unacylated ghrelin (UAG) and AG recognized common high-affinity binding sites on these cells. Either AG or UAG stimulated cell proliferation through Galpha(s) protein and prevented serum starvation- and IFN-gamma/TNF-alpha-induced apoptosis. Antighrelin antibody enhanced apoptosis in either the presence or absence of serum but not cytokines. AG and UAG even up-regulated intracellular cAMP. Blockade of adenylyl cyclase/cAMP/protein kinase A signaling prevented the ghrelin cytoprotective effect. AG and UAG also activated phosphatidyl inositol 3-kinase (PI3K)/Akt and ERK1/2, whereas PI3K and MAPK inhibitors counteracted the ghrelin antiapoptotic effect. Furthermore, AG and UAG stimulated insulin secretion from HIT-T15 cells. In INS-1E beta-cells, which express GRLN-R, AG and UAG caused proliferation and protection against apoptosis through identical signaling pathways. Noteworthy, both peptides inhibited cytokine-induced NO increase in either HIT-T15 or INS-1E cells. Finally, they induced cell survival and protection against apoptosis in human islets of Langerhans. These expressed GRLN-R but showed also UAG and AG binding sites. Our data demonstrate that AG and UAG promote survival of both beta-cells and human islets. These effects are independent of GRLN-R, are likely mediated by AG/UAG binding sites, and involve cAMP/PKA, ERK1/2, and PI3K/Akt.     

Mechanisms of glucose-induced expression of pancreatic-derived factor in pancreatic beta-cells

Pancreatic-derived factor (PANDER) is a cytokine-like peptide highly expressed in pancreatic beta-cells. PANDER was reported to promote apoptosis of pancreatic beta-cells and secrete in response to glucose. Here we explored the effects of glucose on PANDER expression, and the underlying mechanisms in murine pancreatic beta-cell line MIN6 and primary islets. Our results showed that glucose up-regulated PANDER mRNA and protein levels in a time- and dose-dependent manner in MIN6 cells and pancreatic islets. In cells expressing cAMP response element-binding protein (CREB) dominant-negative construct, glucose failed to induce PANDER gene expression and promoter activation. Treatment of the cells with calcium chelator [EGTA, 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl)ester (BAPTA/AM)], the voltage-dependent Ca(2+) channel inhibitor (nifedipine), the protein kinase A (PKA) inhibitor (H89), the protein kinase C (PKC) inhibitor (Go6976), or the MAPK kinase 1/2 inhibitor (PD98059), all significantly inhibited glucose-induced PANDER gene expression and promoter activation. Further studies showed that glucose induced CREB phosphorylation through Ca(2+)-PKA-ERK1/2 and Ca(2+)-PKC pathways. Thus, the Ca(2+)-PKA-ERK1/2-CREB and Ca(2+)-PKC-CREB signaling pathways are involved in glucose-induced PANDER gene expression. Wortmannin (phosphatidylinositol 3-kinase inhibitor), ammonium pyrrolidinedithiocarbamate (nuclear factor-kappaB inhibitor and nonspecific antioxidant), and N-acetylcysteine (antioxidant) were also found to inhibit glucose-induced PANDER promoter activation and gene expression. Because there is no nuclear factor-kappaB binding site in the promoter region of PANDER gene, these results suggest that phosphatidylinositol 3-kinase and reactive oxygen species be involved in glucose-induced PANDER gene expression. In conclusion, glucose induces PANDER gene expression in pancreatic beta-cells through multiple signaling pathways. Because PANDER is expressed by pancreatic beta-cells and in response to glucose in a similar way to those of insulin, PANDER may be involved in glucose homeostasis.     

Role of protein kinase C,PI3-kinase and tyrosine kinase in activation of MAP kinase by glucose and agonists of G-protein coupled receptors in INS-1 cells

MAP (mitogen-activated protein) kinase (also called Erk 1/2) plays a crucial role in cell proliferation and differentiation. Its impact on secretory events is less well established. The interplay of protein kinase C (PKC), PI3-kinase and cellular tyrosine kinase with MAP kinase activity using inhibitors and compounds such as glucose, phorbol 12-myristate 13-acetate (PMA) and agonists of G-protein coupled receptors like gastrin releasing peptide (GRP), oxytocin (OT) and glucose-dependent insulinotropic peptide (GIP) was investigated in INS-1 cells, an insulin secreting cell line. MAP kinase activity was determined by using a peptide derived from the EGF receptor as a MAP kinase substrate and [32P]ATP. Glucose as well as GRP, OT and GIP exhibited a time-dependent increase in MAP kinase activity with a maximum at time point 2.5 min. All further experiments were performed using 2.5 min incubations. The flavone PD 098059 is known to bind to the inactive forms of MEK1 (MAPK/ERK-Kinase) thus preventing activation by upstream activators. 20 microM PD 098059 (IC50 = 5 microM) inhibited MAP kinase stimulated by either glucose, GRP, OT, GIP or PMA. Inhibiton ("downregulation") of PKC by a long term (22 h) pretreatment with 1 microM PMA did not influence MAP kinase activity when augmented by either of the above mentioned compound. To investigate whether PI3-kinase and cellular tyrosine kinase are involved in G-protein mediated effects on MAP kinase, inhibitors were used: 100 nM wortmannin (PI3-kinase inhibitor) reduced the effects of GRP, OT and GIP but not that of PMA; 100 microM genistein (tyrosine kinase inhibitor) inhibited the stimulatory effect of either above mentioned compound on MAP kinase activation. Inhibition of MAP kinase by 20 microM PD 098059 did not influence insulin secretion modulated by either compound (glucose, GRP, OT or GIP). [3H]Thymidine incorporation, however, was severely inhibited by PD 098059. Thus MAP kinase is important for INS-1 cell proliferation but not for its insulin secretory response with respect to major initiators and modulators of insulin release. The data indicate that MAP kinase is active and under the control of MAP kinase. PKC is upstream of a genistein-sensitive tyrosine kinase and probably downstream of a PI3-kinase in INS-1 cells.     

Activation of the hexosamine pathway leads to phosphorylation of insulin receptor substrate-1 on Ser307 and Ser612 and impairs the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin insulin biosynthetic pathway in RIN pancreatic beta-cells

Many adverse effects of glucose were attributed to its increased routing through the hexosamine pathway (HBP). There is evidence for an autocrine role of the insulin signaling in beta-cell function. We tested the hypothesis that activation of the HBP induces defects in insulin biosynthesis by affecting the insulin-mediated protein translation signaling. Exposure of human pancreatic islets and RIN beta-cells to glucosamine resulted in reduction in glucose- and insulin-stimulated insulin biosynthesis, which in RIN beta-cells was associated with impairment in insulin-stimulated insulin receptor substrate-1 (IRS-1) phosphorylation at Tyr(608) and Tyr(628), which are essential for engaging phosphatidylinositol 3-kinase (PI 3-kinase). These changes were accompanied by impaired activation of PI 3-kinase, and activation of Akt/mammalian target of rapamycin/phosphorylated heat- and acid-stable protein-1/p70S6 kinase pathway. RIN beta-cells exposed to high glucose exhibited increased c-Jun N-terminal kinase (JNK) and ERK1/2 activity, which was associated with increased IRS-1 phosphorylation at serine (Ser)(307) and Ser(612), respectively, that inhibits coupling of IRS-1 to the insulin receptor and is upstream of the inhibition of IRS-1 tyrosine phosphorylation. Azaserine reverted the stimulatory effects of high glucose on JNK and ERK1/2 activity and IRS-1 phosphorylation at Ser(307) and Ser(612). Glucosamine mimicked the stimulatory effects of high glucose on JNK and ERK1/2 activity and IRS-1 phosphorylation at Ser(307) and Ser(612). Inhibition of JNK and MAPK kinase-1 activity reverted the negative effects of glucosamine on insulin-mediated protein synthesis. These results suggest that activation of the HBP accounts, in part, for glucose-induced phosphorylation at Ser(307) and Ser(612) of IRS-1 mediated by JNK and ERK1/2, respectively. These changes result in impaired coupling of IRS-1 and PI 3-kinase, and activation of the Akt/mammalian target of rapamycin/phosphorylated heat- and acid-stable protein-1/p70S6 kinase pathway.     

Possible role for protein kinase B in the anti-apoptotic effect of prolactin in rat Nb2 lymphoma cells

Prolactin (PRL) is a mitogen for a number of cell types and its action as a survival factor has recently been demonstrated in Nb2 lymphoma cells. However, the intracellular signalling pathways by which PRL promotes the survival of Nb2 cells is unknown. In previous studies, we have shown that PRL caused the activation of phosphatidylinositol 3-kinase (PI3-kinase) and its association with tyrosine phosphorylated fyn. Protein kinase B (PKB), a serine/threonine kinase, is now known to be a downstream component of the PI3-kinase pathway. The aim of the present study was to examine the effect of PRL on the activation of PKB and to find out whether this has any role on the PRL-induced survival of Nb2 cells. Our studies have revealed the phosphorylation and activation of PKB in PRL-stimulated Nb2 cells. We have also observed, using confocal microscopy, translocation of PKB to the membrane of Nb2 cells in response to PRL. These effects were blocked by the PI3-kinase inhibitor, LY294002 (10 microgram/ml). Apoptosis was induced by the general protein kinase inhibitor, staurosporine (STS; 0.1-1 microM), the synthetic glucocorticoid, dexamethasone (Dex; 100 nM) or ionising radiation by exposing Nb2 cells to X-irradiation (IR; 10 Gy). PRL had no effect on STS-induced apoptosis. On the other hand, PRL (100 ng/ml) inhibited apoptosis induced by Dex or IR; this effect of PRL was reversed by the addition of LY294002 (10 microgram/ml). Furthermore, Western blot analysis using phosphospecific PKB antibody on lysates from PRL-treated Nb2 cells showed that phosphorylation of PKB in response to PRL was inhibited by STS (0.5 microM), but not by Dex (100 nM). These results suggest that the PI3-kinase/PKB pathway may mediate the anti-apoptotic effect of PRL in Nb2 cells.     

Macrophage inflammatory protein 1-alpha (MIP-1 alpha ) triggers migration and signaling cascades mediating survival and proliferation in multiple myeloma (MM) cells

Recently, it has been demonstrated that macrophage inflammatory protein 1- alpha (MIP-1 alpha) is crucially involved in the development of osteolytic bone lesions in multiple myeloma (MM). The current study was designed to determine the direct effects of MIP-1 alpha on MM cells. Thus, we were able to demonstrate that MIP-1 alpha acts as a potent growth, survival, and chemotactic factor in MM cells. MIP-1 alpha-induced signaling involved activation of the AKT/protein kinase B (PKB) and the mitogen-activated protein kinase (MAPK) pathway. In addition, inhibition of AKT activation by phosphatidylinositol 3- kinase (PI3-K) inhibitors did not influence MAPK activation, suggesting that there is no cross talk between MIP-1 alpha-dependent activation of the PI3-K/AKT and extracellular-regulated kinase (ERK) pathway. Our data suggest that besides its role in development of osteolytic bone destruction, MIP-1 alpha also directly affects cell signaling pathways mediating growth, survival, and migration in MM cells and provide evidence that MIP-1 alpha might play a pivotal role in the pathogenesis of MM.     

Glucose-dependent insulinotropic polypeptide promotes beta-(INS-1) cell survival via cyclic adenosine monophosphate-mediated caspase-3 inhibition and regulation of p38 mitogen-activated protein kinase

The incretin glucose-dependent insulinotropic polypeptide (GIP) is a major regulator of postprandial insulin secretion in mammals. Recent studies in our laboratory, and others have suggested that GIP is a potent stimulus for protein kinase activation, including the MAPK (ERK1/2) module. Based on these studies, we hypothesized that GIP could regulate cell fate and sought to examine the underlying mechanisms involved in GIP stimulation of cell survival. GIP potentiated glucose-induced beta-(INS-1)-cell growth to levels comparable with GH and GLP-1 while promoting cell survival in the face of serum and glucose-deprivation or treatment with wortmannin or streptozotocin. In the absence of GIP, 50% of cells died after 48 h of serum and glucose withdrawal, whereas 91 +/- 10% of cells remained viable in the presence of GIP [n = 3, P < 0.05; EC50 of 1.24 +/- 0.48 nm GIP (n = 4)]. Effects of GIP on cell survival and inhibition of caspase-3 were mimicked by forskolin, but pharmacological experiments excluded roles for MAPK kinase (Mek)1/2, phosphatidylinositol 3-kinase, protein kinase A, Epac, and Rap 1. Survival effects of GIP were ablated by the inhibitor SB202190, indicating a role for p38 MAPK. Furthermore, caspase-3 activity was also regulated by p38 MAPK, with a lesser role for Mek1/2, based on RNA interference studies. We propose that GIP is able to reverse caspase-3 activation via inhibition of long-term p38 MAPK phosphorylation in response to glucose deprivation (+/-wortmannin). Intriguingly, these findings contrasted with short-term phosphorylation of MKK3/6-->p38 MAPK-->ATF-2 by GIP. Thus, these data suggest that GIP is able to regulate INS-1 cell survival by dynamic control of p38 MAPK phosphorylation via cAMP signaling and lend further support to the notion that GIP regulation of MAPK signaling is critical for its regulation of cell fate.     

Dissociation of cytokine-induced phosphorylation of Bad and activation of PKB/akt: Involvement of MEK upstream of Bad phosphorylation

The phosphatidylinositol 3-kinase (PI3K)-signaling pathway has emerged as an important component of cytokine-mediated survival of hemopoietic cells. Recently, the protein kinase PKB/akt (referred to here as PKB) has been identified as a downstream target of PI3K necessary for survival. PKB has also been implicated in the phosphorylation of Bad, potentially linking the survival effects of cytokines with the Bcl-2 family. We have shown that granulocyte/macrophage colony-stimulating factor (GM-CSF) maintains survival in the absence of PI3K activity, and we now show that when PKB activation is also completely blocked, GM-CSF is still able to stimulate phosphorylation of Bad. Interleukin 3 (IL-3), on the other hand, requires PI3K for survival, and blocking PI3K partially inhibited Bad phosphorylation. IL-4, unique among the cytokines in that it lacks the ability to activate the p21ras–mitogen-activated protein kinase (MAPK) cascade, was found to activate PKB and promote cell survival, but it did not stimulate Bad phosphorylation. Finally, although our data suggest that the MAPK pathway is not required for inhibition of apoptosis, we provide evidence that phosphorylation of Bad may be occurring via a MAPK/ERK kinase (MEK)-dependent pathway. Together, these results demonstrate that although PI3K may contribute to phosphorylation of Bad in some instances, there is at least one other PI3K-independent pathway involved, possibly via activation of MEK. Our data also suggest that although phosphorylation of Bad may be one means by which cytokines can inhibit apoptosis, it may be neither sufficient nor necessary for the survival effect.     

Insulin-like growth factor (IGF) binding protein-3 potentiation of IGF action is mediated through the phosphatidylinositol-3-kinase pathway and is associated with alteration in protein kinase B/AKT sensitivity

Cell-association and processing of insulin-like growth factor binding protein-3 (IGFBP-3) by cultured bovine fibroblasts results in markedly enhanced type I IGF receptor signaling at a step distal to ligand binding. The purpose of the present study was to determine the intracellular mediators of IGFBP-3's potentiating effect. Preincubation of cultured bovine fibroblasts with 50 nM IGFBP-3 had no effect alone, but enhanced by 3- to 4-fold IGF-I-stimulated 3H-aminoisobutryric acid (AIB) uptake. IGFBP-3-induced potentiation was specifically prevented if an inhibitor of phosphatidylinositol 3 (PI3)-kinase activation (LY294002), but not an inhibitor of mitogen-activated protein kinase activation (PD98059), was present during the preincubation period. IGFBP-3 did not directly activate the downstream effector of PI3-kinase, protein kinase B (PKB)/Akt. However, the sensitivity of PKB/Akt to activation by IGF-I was increased by 2- to 4-fold with IGFBP-3 pretreatment. This increased sensitivity was accompanied by altered mobility of PKB/Akt on SDS-polyacrylamide gels, suggestive of a diminished phosphorylation state. Consistent with this, okadaic acid, a potent serine/threonine phosphatase inhibitor, was able to block the potentiation effect of IGFBP-3 and prevent the altered mobility of the PKB/Akt molecule in response to IGFBP-3 treatment. PKB/Akt immunoprecipitated from IGFBP-3-pretreated cells was no longer recognized by an antibody specific for phosphorylated threonine followed by proline. These data indicate that IGFBP-3 modulates type I IGF receptor signaling through an effect on PI-3-kinase pathway substrates and suggest a novel mechanism of dephosphorylation whereby PKB/Akt is transformed into a more sensitive substrate of type I IGF receptor signaling.     

Epidermal growth factor impairs the cytochrome C/caspase-3 apoptotic pathway induced by transforming growth factor beta in rat fetal hepatocytes via a phosphoinositide 3-kinase-dependent pathway

Transforming growth factor beta (TGF-beta)-mediated apoptosis is one of the major death processes in the liver. We have previously shown that epidermal growth factor (EGF) is an important survival signal for TGF-beta-induced apoptosis in fetal hepatocytes (Fabregat et al., FEBS Lett 1996;384:14-18). In this work we have studied the intracellular signaling implicated in the protective effect of EGF. We show here that EGF activates p42 and p44 mitogen-activated protein kinases (MAPK). However, mitogen extracellular kinase (MEK) inhibitors do not block the survival effect of EGF. EGF also activates phosphoinositide 3-kinase (PI 3-kinase) and protein kinase B (PKB/AKT) in these cells. The presence of PI 3-kinase inhibitors blocks the protective effect of EGF on cell viability, DNA fragmentation, and caspase-3 activity. We have found that TGF-beta disrupts the mitochondrial transmembrane potential (DeltaPsi(m))( )and activates the release of cytochrome c, this effect being blocked by EGF, via a PI 3-kinase-dependent pathway. A detailed study on bcl-2 superfamily gene expression shows that TGF-beta produces a decrease in the messenger RNA (mRNA) and protein levels of bcl-x(L), an antiapoptotic member of this family, capable of preventing cytochrome c release. EGF is able to maintain bcl-x(L) levels even in the presence of TGF-beta. PI 3-kinase inhibitors completely block the protective effect of EGF on TGF-beta-induced bcl-x(L )down-regulation. We conclude that PI 3-kinase mediates the survival effect of EGF on TGF-beta-induced death by acting upstream from the mitochondrial changes, i.e., preventing bcl-x(L) down-regulation, cytochrome c release, and activation of caspase-3.     

Involvement of both phosphatidylinositol 3-kinase and p44/p42 mitogen-activated protein kinase pathways in the short-term regulation of pyruvate kinase L by insulin

Pyruvate kinase L (PK-L) is a key regulatory enzyme of the hepatic glycolytic/gluconeogenic pathway that can be dephosphorylated and activated in response to insulin. However, the signaling cascades involved in this insulin effect have not been established. In this work we have investigated the potential involvement of phosphatidylinositol 3-kinase (PI 3-K) and p44/p42 mitogen-activated protein kinase (MAPK) pathways in the short-term modulation of PK-L by insulin in primary cultures of rat hepatocytes. Wortmannin, at a concentration of 100 nM, caused a marked inhibition of the PI 3-K/protein kinase B pathway, which became complete at 500 nM wortmannin. Likewise, wortmannin at 100 and 500 nM, elicited partial and total inhibitions of insulin-mediated activation of PK-L, respectively. However, this PI 3-K inhibitor also reduced insulin-mediated phosphorylation of p44/p42 MAPK in cultured rat hepatocytes, indicating that both the PI 3-K and MAPK pathways could be involved in PK-L activation by insulin. Three facts appear to reinforce this hypothesis: 1) the selective and complete inhibition of the PI 3-K/protein kinase B pathway by LY294002 (50 microM) was accompanied by a partial blockade of insulin-induced PK-L activation; 2) when signaling through the MAPK cascade was selectively suppressed by the presence of PD98059 (50 microM), a 50% reduction of insulin-induced activation of PK-L was observed; and 3) the effect of PD98059 (50 microM) on PK-L activation was reinforced by the additional presence of 100 nM wortmannin. We also observed that the blockade of p70 S6-kinase by rapamycin did not affect the activation of PK-L by insulin. From these findings it can be concluded that both PI 3-K and MAPK pathways, but not p70 S6-kinase, are involved in the short-term activation of PK-L by insulin in rat hepatocytes.     

Regulation of rat Sertoli cell function by FSH: possible role of phosphatidylinositol 3-kinase/protein kinase B pathway

The FSH molecular mechanism of action is best recognized for its stimulation of the adenylyl cyclase/cAMP pathway via activation of a G protein. Recently, links between cAMP, phosphatidylinositol 3-kinase (PI3K) and protein kinase B (PKB) signaling pathways in thyroid and granulosa cells have been observed. The aim of this study was to investigate the possible role of the PI3K/PKB pathway in FSH regulation of Sertoli cell function. Twenty-day-old rat Sertoli cell cultures were used. An increase in phosphorylated PKB (P-PKB) levels in response to FSH and dibutyryl-cAMP was observed. These increments in P-PKB levels were not observed in the presence of two PI3K inhibitors, wortmannin and Ly 294002. Inhibition of protein kinase A (PKA) by H89 did not decrease FSH stimulation of P-PKB levels. Taken together, these results indicate that FSH increases P-PKB levels in a PI3K-dependent and PKA-independent manner in rat Sertoli cells. In addition, wortmannin partially inhibited the ability of FSH to stimulate two well-known parameters of Sertoli cell function - transferrin secretion and lactate production - at doses equal to or lower than 0.1 microM. Related to lactate production, a decrease in FSH stimulation of lactate dehydrogenase activity and of basal and FSH-stimulated glucose uptake was observed in the presence of wortmannin. These metabolic changes were in most cases accompanied by changes in the levels of P-PKB. Altogether, these results suggest a meaningful role of the PI3K/PKB pathway in the mechanism of action of FSH in rat Sertoli cells.     

Inositol polyphosphate multikinase is a physiologic PI3-kinase that activates Akt/PKB

The second messenger phosphatidylinositol (3,4,5)-trisphosphate (PIP(3)), formed by the p110 family of PI3-kinases, promotes cellular growth, proliferation, and survival, in large part by activating the protein kinase Akt/PKB. We show that inositol polyphosphate multikinase (IPMK) physiologically generates PIP(3) as well as water soluble inositol phosphates. IPMK deletion reduces growth factor-elicited Akt signaling and cell proliferation caused uniquely by loss of its PI3-kinase activity. Inhibition of p110 PI3-kinases by wortmannin prevents IPMK phosphorylation and activation. Thus, growth factor stimulation of Akt signaling involves PIP(3) generation through the sequential activations of the p110 PI3-kinases and IPMK. As inositol phosphates inhibit Akt signaling, IPMK appears to act as a molecular switch, inhibiting or stimulating Akt via its inositol phosphate kinase or PI3-kinase activities, respectively. Drugs regulating IPMK may have therapeutic relevance in influencing cell proliferation.     

Phosphatidylinositol 3-kinase functionally compartmentalizes the concurrent G(s) signaling during beta2-adrenergic stimulation

Compartmentation of intracellular signaling pathways serves as an important mechanism conferring the specificity of G protein-coupled receptor (GPCR) signaling. In the heart, stimulation of beta2-adrenoceptor (beta2-AR), a prototypical GPCR, activates a tightly localized protein kinase A (PKA) signaling, which regulates substrates at cell surface membranes, bypassing cytosolic target proteins (eg, phospholamban). Although a concurrent activation of beta2-AR-coupled G(i) proteins has been implicated in the functional compartmentation of PKA signaling, the exact mechanism underlying the restriction of the beta2-AR-PKA pathway remains unclear. In the present study, we demonstrate that phosphatidylinositol 3-kinase (PI3K) plays an essential role in confining the beta2-AR-PKA signaling. Inhibition of PI3K with LY294002 or wortmannin enables beta2-AR-PKA signaling to reach intracellular substrates, as manifested by a robust increase in phosphorylation of phospholamban, and markedly enhances the receptor-mediated positive contractile and relaxant responses in cardiac myocytes. These potentiating effects of PI3K inhibitors are not accompanied by an increase in beta2-AR-induced cAMP formation. Blocking G(i) or Gbetagamma signaling with pertussis toxin or betaARK-ct, a peptide inhibitor of Gbetagamma, completely prevents the potentiating effects induced by PI3K inhibition, indicating that the pathway responsible for the functional compartmentation of beta2-AR-PKA signaling sequentially involves G(i), Gbetagamma, and PI3K. Thus, PI3K constitutes a key downstream event of beta2-AR-G(i) signaling, which confines and negates the concurrent beta2-AR/G(s)-mediated PKA signaling.     

Interleukin 3-dependent survival by the Akt protein kinase

Interleukin 3 (IL-3)-dependent survival of hematopoietic cells is known to rely on the activity of multiple signaling pathways, including a pathway leading to activation of phosphoinositide 3-kinase (PI 3-kinase), and protein kinase Akt is a direct target of PI 3-kinase. We find that Akt kinase activity is rapidly induced by the cytokine IL-3, suggesting a role for Akt in PI 3-kinase-dependent signaling in hematopoetic cells. Dominant-negative mutants of Akt specifically block Akt activation by IL-3 and interfere with IL-3-dependent proliferation. Overexpression of Akt or oncogenic v-akt protects 32D cells from apoptosis induced by IL-3 withdrawal. Apoptosis after IL-3 withdrawal is accelerated by expression of dominant-negative mutants of Akt, indicating that a functional Akt signaling pathway is necessary for cell survival mediated by the cytokine IL-3. Thus Akt appears to be an important mediator of anti-apoptotic signaling in this system.