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Phosphoinositide 3-kinase-dependent Ras activation by tauroursodesoxycholate in rat liver.

ABSTRACT: Ursodesoxycholic acid, widely used for the treatment of cholestatic liver disease, causes choleretic, anti-apoptotic and immunomodulatory effects. Here the effects on choleresis of its taurine conjugate tauroursodesoxycholate (TUDC), which is present in the enterohepatic circulation, were correlated with the activation of important elements of intracellular signal transduction in cultured rat hepatocytes and perfused rat liver. TUDC induced a time- and concentration-dependent activation of the small GTP-binding protein Ras and of phosphoinositide 3-kinase (PI 3-kinase) in cultured hepatocytes. Ras activation was dependent on PI 3-kinase activity, without the involvement of protein kinase C- and genistein-sensitive tyrosine kinases. Ras activation by TUDC was followed by an activation of the mitogen-activated protein kinases extracellular-signal-regulated kinase-1 (Erk-1) and Erk-2. In perfused rat liver, PI 3-kinase inhibitors largely abolished the stimulatory effect of TUDC on taurocholate excretion, suggesting an important role for a PI 3-kinase/Ras/Erk pathway in the choleretic effect of TUDC.

SUBMITTER: Kurz AK

PROVIDER: S-EPMC1221243 | biostudies-other | 2000 Aug

REPOSITORIES: biostudies-other

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Phosphoinositide-dependent protein kinase (PDK) activity regulates phosphatidylinositol 3,4,5-trisphosphate-dependent and -independent protein kinase B activation and chemotaxis.

Project description:Chemotactic cells must sense shallow extracellular gradients and produce localized intracellular responses. We previously showed that the temporal and spatial activation of two protein kinase B (PKB) homologues, PkbA and PkbR1, in Dictyostelium discoideum by phosphorylation of activation loops (ALs) and hydrophobic motifs had important roles in chemotaxis. We found that hydrophobic motif phosphorylation depended on regulation of TorC2 (target of rapamycin complex 2); however, the regulation of AL phosphorylation remains to be determined at a molecular level. Here, we show that two PDK (phosphoinositide-dependent protein kinase) homologues, PdkA and PdkB, function as the key AL kinases. Cells lacking both PdkA and PdkB are defective in PKB activation, chemotaxis, and fruiting body formation upon nutrient deprivation. The pleckstrin homology domain of PdkA is sufficient to localize it to the membrane, but transient activation of PdkA is independent of PIP(3) as well as TorC2 and dispensable for full function. These results confirm the importance of the TorC2-PDK-PKB pathway in chemotaxis and point to a novel mechanism of regulation of PDKs by chemoattractant.

| S-EPMC2832944 | biostudies-literature

Drosophila phosphoinositide-dependent kinase-1 regulates apoptosis and growth via the phosphoinositide 3-kinase-dependent signaling pathway.

Project description:Phosphoinositide-dependent kinase-1 (PDK-1) is a central mediator of the cell signaling between phosphoinositide 3-kinase (PI3K) and various intracellular serine/threonine kinases including Akt/protein kinase B (PKB), p70 S6 kinases, and protein kinase C. Recent studies with cell transfection experiments have implied that PDK-1 may be involved in various cell functions including cell growth and apoptosis. However, despite its pivotal role in cellular signalings, the in vivo functions of PDK-1 in a multicellular system have rarely been investigated. Here, we have isolated Drosophila PDK-1 (dPDK-1) mutants and characterized the in vivo roles of the kinase. Drosophila deficient in the dPDK-1 gene exhibited lethality and an apoptotic phenotype in the embryonic stage. Conversely, overexpression of dPDK-1 increased cell and organ size in a Drosophila PI3K-dependent manner. dPDK-1 not only could activate Drosophila Akt/PKB (Dakt1), but also substitute the in vivo functions of its mammalian ortholog to activate Akt/PKB. This functional interaction between dPDK-1 and Dakt1 was further confirmed through genetic analyses in Drosophila. On the other hand, cAMP-dependent protein kinase, which has been proposed as a possible target of dPDK-1, did not interact with dPDK-1. In conclusion, our findings provide direct evidence that dPDK-1 regulates cell growth and apoptosis during Drosophila development via the PI3K-dependent signaling pathway and demonstrate our Drosophila system to be a powerful tool for elucidating the in vivo functions and targets of PDK-1.

| S-EPMC33436 | biostudies-literature

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Interaction of Ras with phosphoinositide 3-kinase gamma.

Project description:Phosphoinositide 3-kinase gamma (PI3Kgamma) can be activated in vitro by both alpha and betagamma subunits of heterotrimeric G-proteins and does not interact with p85, the regulatory subunit of PI3Kalpha. Here we demonstrate the binding of Ras to PI3Kgamma in vitro. An N-terminal region of PI3Kgamma was identified as a binding site for Ras. After co-expression with PI3Kgamma in COS-7 cells, Ras induced only a modest increase in PI3K activity compared with the stimulation of PI3Kalpha by Ras in the same cells.

| S-EPMC1218747 | biostudies-other

A Complex Interplay of Anionic Phospholipid Binding Regulates 3'-Phosphoinositide-Dependent-Kinase-1 Homodimer Activation.

Project description:3'-Phosphoinositide-dependent-Kinase-1 (PDK1) is a master regulator whereby its PI3-kinase-dependent dysregulation in human pathologies is well documented. Understanding the direct role for PtdIns(3,4,5)P3 and other anionic phospholipids in the regulation of PDK1 conformational dynamics and its downstream activation remains incomplete. Using advanced quantitative-time-resolved imaging (Fluorescence Lifetime Imaging and Fluorescence Correlation Spectroscopy) and molecular modelling, we show an interplay of antagonistic binding effects of PtdIns(3,4,5)P3 and other anionic phospholipids, regulating activated PDK1 homodimers. We demonstrate that phosphatidylserine maintains PDK1 in an inactive conformation. The dysregulation of the PI3K pathway affects the spatio-temporal and conformational dynamics of PDK1 and the activation of its downstream substrates. We have established a new anionic-phospholipid-dependent model for PDK1 regulation, depicting the conformational dynamics of multiple homodimer states. We show that the dysregulation of the PI3K pathway perturbs equilibrium between the PDK1 homodimer conformations. Our findings provide a role for the PtdSer binding site and its previously unrewarding role in PDK1 downregulation, suggesting a possible therapeutic strategy where the constitutively active dimer conformer of PDK1 may be rendered inactive by small molecules that drive it to its PtdSer-bound conformer.

| S-EPMC6787260 | biostudies-literature

PPAR? activation acts cooperatively with 3-phosphoinositide-dependent protein kinase-1 to enhance mammary tumorigenesis.

Project description:Peroxisome proliferator-activated receptor? (PPAR?) is a transcription factor that is associated with metabolic gene regulation and inflammation. It has been implicated in tumor promotion and in the regulation of 3-phosphoinositide-dependent kinase-1 (PDK1). PDK1 is a key regulator of the AGC protein kinase family, which includes the proto-oncogene AKT/PKB implicated in several malignancies, including breast cancer. To assess the role of PDK1 in mammary tumorigenesis and its interaction with PPAR?, transgenic mice were generated in which PDK1 was expressed in mammary epithelium under the control of the MMTV enhancer/promoter region. Transgene expression increased pT308AKT and pS9GSK3?, but did not alter phosphorylation of mTOR, 4EBP1, ribosomal protein S6 and PKC?. The transgenic mammary gland also expressed higher levels of PPAR? and a gene expression profile resembling wild-type mice maintained on a diet containing the PPAR? agonist, GW501516. Both wild-type and transgenic mice treated with GW501516 exhibited accelerated rates of tumor formation that were more pronounced in transgenic animals. GW501516 treatment was accompanied by a distinct metabolic gene expression and metabolomic signature that was not present in untreated animals. GW501516-treated transgenic mice expressed higher levels of fatty acid and phospholipid metabolites than treated wild-type mice, suggesting the involvement of PDK1 in enhancing PPAR?-driven energy metabolism. These results reveal that PPAR? activation elicits a distinct metabolic and metabolomic profile in tumors that is in part related to PDK1 and AKT signaling.

| S-EPMC3020974 | biostudies-literature

Regulation of Hippo pathway by mitogenic growth factors via phosphoinositide 3-kinase and phosphoinositide-dependent kinase-1.

Project description:The Hippo signaling pathway inhibits cell growth and regulates organ size through a kinase cascade that leads to the phosphorylation and nuclear exclusion of the growth-promoting transcriptional coactivator Yes-associated protein (YAP)/Yorkie. It mediates contact inhibition of cell growth downstream of cadherin adhesion molecules and other cell surface proteins. Contact inhibition is often antagonized by mitogenic growth factor signaling. We report an important mechanism for this antagonism, inhibition of Hippo pathway signaling by mitogenic growth factors. EGF treatment of immortalized mammary cells triggers the rapid translocation of YAP into the nucleus along with YAP dephosphorylation, both of which depend on Lats, the terminal kinase in the Hippo pathway. A small-molecule inhibitor screen of downstream effector pathways shows that EGF receptor inhibits the Hippo pathway through activation of PI3-kinase (PI3K) and phosphoinositide-dependent kinase (PDK1), but independent of AKT activity. The PI3K-PDK1 pathway also mediates YAP nuclear translocation downstream of lysophosphatidic acid and serum as a result of constitutive oncogenic activation of PI3K. PDK1 associates with the core Hippo pathway-kinase complex through the scaffold protein Salvador. The entire Hippo core complex dissociates in response to EGF signaling in a PI3K-PDK1-dependent manner, leading to inactivation of Lats, dephosphorylation of YAP, and YAP nuclear accumulation and transcriptional activation of its target gene, CTGF. These findings show that an important activity of mitogenic signaling pathways is to inactivate the growth-inhibitory Hippo pathway and provide a mechanism for antagonism between contact inhibition and growth factor action.

| S-EPMC3574943 | biostudies-literature

RAS and RHO families of GTPases directly regulate distinct phosphoinositide 3-kinase isoforms.

Project description:RAS proteins are important direct activators of p110α, p110γ, and p110δ type I phosphoinositide 3-kinases (PI3Ks), interacting via an amino-terminal RAS-binding domain (RBD). Here, we investigate the regulation of the ubiquitous p110β isoform of PI3K, implicated in G-protein-coupled receptor (GPCR) signaling, PTEN-loss-driven cancers, and thrombocyte function. Unexpectedly, RAS is unable to interact with p110β, but instead RAC1 and CDC42 from the RHO subfamily of small GTPases bind and activate p110β via its RBD. In fibroblasts, GPCRs couple to PI3K through Dock180/Elmo1-mediated RAC activation and subsequent interaction with p110β. Cells from mice carrying mutations in the p110β RBD show reduced PI3K activity and defective chemotaxis, and these mice are resistant to experimental lung fibrosis. These findings revise our understanding of the regulation of type I PI3K by showing that both RAS and RHO family GTPases directly regulate distinct ubiquitous PI3K isoforms and that RAC activates p110β downstream of GPCRs.

| S-EPMC3690480 | biostudies-literature

Ras is an indispensable coregulator of the class IB phosphoinositide 3-kinase p87/p110gamma.

Project description:Class I(B) phosphoinositide 3-kinase gamma (PI3Kgamma) elicits various immunologic and cardiovascular responses; however, the molecular basis for this signal heterogeneity is unclear. PI3Kgamma consists of a catalytic p110gamma and a regulatory p87(PIKAP) (p87, also p84) or p101 subunit. Hitherto p87 and p101 are generally assumed to exhibit redundant functions in receptor-induced and G protein betagamma (Gbetagamma)-mediated PI3Kgamma regulation. Here we investigated the molecular mechanism for receptor-dependent p87/p110gamma activation. By analyzing GFP-tagged proteins expressed in HEK293 cells, PI3Kgamma-complemented bone marrow-derived mast cells (BMMCs) from p110gamma(-/-) mice, and purified recombinant proteins reconstituted to lipid vesicles, we elucidated a novel pathway of p87-dependent, G protein-coupled receptor (GPCR)-induced PI3Kgamma activation. Although p101 strongly interacted with Gbetagamma, thereby mediating PI3Kgamma membrane recruitment and stimulation, p87 exhibited only a weak interaction, resulting in modest kinase activation and lack of membrane recruitment. Surprisingly, Ras-GTP substituted the missing Gbetagamma-dependent membrane recruitment of p87/p110gamma by direct interaction with p110gamma, suggesting the indispensability of Ras for activation of p87/p110gamma. Consequently, interference with Ras signaling indeed selectively blocked p87/p110gamma, but not p101/p110gamma, kinase activity in HEK293 and BMMC cells, revealing an important crosstalk between monomeric and trimeric G proteins for p87/p110gamma activation. Our data display distinct signaling requirements of p87 and p101, conferring signaling specificity to PI3Kgamma that could open up new possibilities for therapeutic intervention.

| S-EPMC2787109 | biostudies-literature

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