Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “PI3K”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 451 records · Page 25Linked to original sources
PubMed1998

Functional association of platelet endothelial cell adhesion molecule-1 and phosphoinositide 3-kinase in human neutrophils.

In this paper we show that the engagement of the platelet-endothelial cell adhesion molecule-1 (PECAM-1/CD31) up-regulates the adhesion of human neutrophils to the EA.hy926 endothelial cell line through a phosphoinositide 3-kinase (PI3K)-dependent pathway. Indeed, LY294002 and wortmannin prevented the effect of PECAM-1/CD31 cross-linking on cell adhesion, at concentrations known to inhibit PI3K without affecting other kinases. Both compounds blocked neutrophil binding to murine fibroblasts transfected with human ICAM-1, to purified ICAM-1 protein, or to fibronectin, suggesting that PECAM-1/CD31-mediated up-regulation of beta2 and beta1 integrin-mediated adhesion is PI3K-sensitive. We also provide evidence for the association of PECAM-1/CD31 to PI3K, because PI3K was detectable in neutrophil lysates after PECAM-1/CD31 cross-linking and immunoprecipitation. PECAM-1/CD31-dependent recruitment of PI3K was suggested by the finding that the serine/threonine kinase p70 S6 kinase (S6K), a signaling protein downstream of PI3K, is activated in neutrophils upon PECAM-1/CD31 cross-linking, based on the appearance of serine phosphorylation in S6K immunoprecipitates. In turn, S6K is not directly involved in the up-regulation of integrin function because rapamycin, which can inhibit S6K independent of PI3K, did not block PECAM-1/CD31-induced adhesion of neutrophils to beta1 and beta2 integrin substrates. In conclusion, PECAM-1/CD31 appears to be one of the molecules functionally coupled to PI3K, suggesting that this enzyme may represent a common pathway of integrin and adhesiveness regulation in leukocytes.

Cell Adhesion↗
PubMed2000

Distinct classes of phosphatidylinositol 3'-kinases are involved in signaling pathways that control macroautophagy in HT-29 cells.

3-Methyladenine which stops macroautophagy at the sequestration step in mammalian cells also inhibits the phosphoinositide 3-kinase (PI3K) activity raising the possibility that PI3K signaling controls the macroautophagic pathway (Blommaart, E. F. C., Krause, U., Schellens, J. P. M., Vreeling-Sindelárová, H., and Meijer, A. J. (1997) Eur. J. Biochem. 243, 240-246). The aim of this study was to identify PI3Ks involved in the control of macroautophagic sequestration in human colon cancer HT-29 cells. An increase of class I PI3K products (phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-triphosphate) caused by either feeding cells with synthetic lipids (dipalmitoyl phosphatidylinositol 3, 4-bisphosphate and dipalmitoyl phosphatidylinositol 3,4, 5-triphosphate) or by stimulating the enzymatic activity by interleukin-13 reduced macroautophagy. In contrast, an increase in the class III PI3K product (phosphatidylinositol 3-phosphate), either by feeding cells with a synthetic lipid or by overexpressing the p150 adaptor, stimulates macroautophagy. Transfection of a specific class III PI3K antisense oligonucleotide greatly inhibited the rate of macroautophagy. In accordance with a role of class III PI3K, wortmannin (an inhibitor of PI3Ks) inhibits macroautophagic sequestration and protein degradation in the low nanomolar range (IC(50) 5-15 nM). Further in vitro enzymatic assay showed that 3-methyladenine inhibits the class III PI3K activity. Dipalmitoyl phosphatidylinositol 3-phosphate supplementation or p150 overexpression rescued the macroautophagic pathway in HT-29 cells overexpressing a GTPase-deficient mutant of the Galpha(i3) protein suggesting that both class III PI3K and trimeric G(i3) protein signaling are required in the control macroautophagy in HT-29 cells. In conclusion, our results demonstrate that distinct classes of PI3K control the macroautophagic pathway in opposite directions. The roles of PI3Ks in macroautophagy are discussed in the context of membrane recycling.

Adenocarcinoma↗
PubMed2000

Novel role of phosphatidylinositol 3-kinase in CD28-mediated costimulation.

Ligation of the CD28 surface receptor provides a major costimulatory signal for full scale T cell activation. Despite extensive studies, the intracellular signaling pathways delivered by CD28 ligation are not fully understood. A particularly controversial matter is the role of phosphatidylinositol 3-kinase (PI3K) in CD28-mediated costimulation. It is known that the binding site for PI3K and Grb-2 lies nested within the YMNM motif of the CD28 cytoplasmic domain. To elucidate the role of PI3K during CD28-mediated interleukin-2 (IL-2) production, CD28 YMNM point and deletion mutants were expressed in Jurkat cells. We then measured IL-2 promoter activation after CD28 ligation. Our results showed that the Y189F mutant, which disrupts binding by PI3K, and the YMNM deletion mutant both demonstrated reduced but significant activity for IL-2 promoter activation. In contrast, the N191A mutant, which retains PI3K binding ability, resulted in a complete abrogation of activity, suggesting that PI3K mediates a negative effect upon transcriptional activation of the IL-2 gene. Consistent with this idea, we found that the addition of a PI3K pharmacological inhibitor augmented IL-2 promoter activity, whereas coexpression of a constitutively active form of PI3K reduced this activity. Taken together, these data indicate that PI3K, when associated with the YMNM motif, may act as a negative mediator in CD28-mediated IL-2 gene transcription.

Amino Acid Motifs↗
PubMed2000

Phosphatidylinositol 3-kinase activation and interaction with focal adhesion kinase in Escherichia coli K1 invasion of human brain microvascular endothelial cells.

Invasion of brain microvascular endothelial cells (BMEC) is a prerequisite for successful crossing of the blood-brain barrier by Escherichia coli K1. We have previously demonstrated the requirement of cytoskeletal rearrangements and activation of focal adhesion kinase (FAK) in E. coli K1 invasion of human BMEC (HBMEC). The current study investigated the role of phosphatidylinositol 3-kinase (PI3K) activation and PI3K interaction with FAK in E. coli invasion of HBMEC. PI3K inhibitor LY294002 blocked E. coli K1 invasion of HBMEC in a dose-dependent manner, whereas an inactive analogue LY303511 had no such effect. In HBMEC, E. coli K1 increased phosphorylation of Akt, a downstream effector of PI3K, which was completely blocked by LY294002. In contrast, non-invasive E. coli failed to activate PI3K. Overexpression of PI3K mutants Deltap85 and catalytically inactive p110 in HBMEC significantly inhibited both PI3K/Akt activation and E. coli K1 invasion of HBMEC. Stimulation of HBMEC with E. coli K1 increased PI3K association with FAK. Furthermore, PI3K/Akt activation was blocked in HBMEC-overexpressing FAK dominant-negative mutants (FRNK and Phe397FAK). These results demonstrated the involvement of PI3K signaling in E. coli K1 invasion of HBMEC and identified a novel role for PI3K interaction with FAK in the pathogenesis of E. coli meningitis.

Blood-Brain Barrier↗
PubMed2001

The hepatitis B virus-X protein activates a phosphatidylinositol 3-kinase-dependent survival signaling cascade.

The hepatitis B virus-X (HBx) protein is known as a multifunctional protein that not only coactivates transcription of viral and cellular genes but coordinates the balance between proliferation and programmed cell death, by inducing or blocking apoptosis. In this study the role of the HBx protein in activation of phosphatidylinositol 3-kinase (PI3K) was investigated as a possible cause of anti-apoptosis in liver cells. HBx relieved serum deprivation-induced and pro-apoptic stimuli-induced apoptosis in Chang liver (CHL) cells. Treatment with 1-d-3-deoxy-3-fluoro-myo-inositol, an antagonist to PI3K, which blocks the formation of 3'-phosphorylated phosphatidyl inositol in CHL cells transformed by HBx (CHL-X) but not normal Chang liver (CHL) cells, showed a marked loss of viability with evidence of apoptosis. Similarly, treatment with wortmannin, an inhibitor of PI3K, stimulated apoptosis in HBx-transformed CHL cells but not in normal cells, confirming that HBx blocks apoptosis through the PI3K pathway. The serine 47 threonine kinase, Akt, one of the downstream effectors of PI3K-dependent survival signaling was 2-fold higher in HBx-transformed CHL (CHL-X) cells than CHL cells. Phosphorylation of Akt at serine 473 and Bad at serine 136 were induced by HBx, which were specifically blocked by wortmannin and dominant negative mutants of Akt and Bad, respectively. We also demonstrated that HBx inhibits caspase 3 activity and HBx down-regulation of caspase 3 activity was blocked by the PI3K inhibitor. Regions required for PI3K phosphorylation on the HBx protein overlap with the known transactivation domains. HBx blocks apoptosis induced by serum withdrawal in CHL cells in a p53-independent manner. The results indicate that, unlike other DNA tumor viruses that block apoptosis by inactivating p53, the hepatitis B virus achieves protection from apoptotic death through a HBx-PI3K-Akt-Bad pathway and by inactivating caspase 3 activity that is at least partially p53-independent in liver cells. Moreover, these data suggest that modulation of the PI3K activity may represent a potential therapeutic strategy to counteract the occurrence of apoptosis in human hepatocellular carcinoma.

Apoptosis↗
PubMed2002

Requirement of phosphoinositide 3-kinase and Akt for interferon-beta-mediated induction of the beta-R1 (SCYB11) gene.

We previously reported (Rani, M. R., Asthagiri, A. R., Singh, A., Sizemore, N., Sathe, S. S., Li, X., DiDonato, J. D., Stark, G. R., and Ransohoff, R. M. (2001) J. Biol. Chem. 276, 44365-44368) that IFN-beta induction of beta-R1 in fibrosarcoma cells required transcription factors ISGF-3 and NF-kappa B. IFN-beta treatment did not augment the abundance of NF-kappa B, but led to phosphorylation of the NF-kappa B subunit p65 and induced a nuclear activity capable of phosphorylating a p65-GST fusion construct in the carboxy-terminal transactivation domain (TAD), residues 354-551. We now present evidence for the involvement of phosphoinositide 3-kinase (PI3K) in this pathway. Pretreatment of HT1080-derived fibrosarcoma cells with pharmacological inhibitors of PI3K (wortmannin or LY294002) selectively inhibited IFN-beta-induced beta-R1 mRNA accumulation in a dose-dependent manner. In stably transfected cell lines, bovine p85, the regulatory subunit of PI3K, functioned as a dominant-negative inhibitor of interferon (IFN) signaling via PI3K and selectively suppressed IFN-beta-mediated induction of beta-R1. Overexpression of PTEN (phosphatase and tensin homologue mutated on chromosome ten), an antagonist of PI3K, blocked induction of a beta-R1 promoter-reporter construct. Studies with PTEN mutants suggested that the lipid kinase activity of PI3K was essential for IFN-beta-induced transcription of beta-R1. Consistent with this finding, a dominant-negative mutant of the serine-threonine kinase Akt, a downstream effector of PI3K, selectively blocked IFN-beta-mediated induction of the beta-R1 promoter reporter. Furthermore, IFN-beta-mediated phosphorylation of GST-p65 was blocked by pretreatment with LY294002. These data suggest that IFN-beta acts through PI3K to enhance the transactivation competence of NF-kappa B complexes through phosphorylation of p65 within the TAD. The results provide novel insight into the role of PI3K in the transcriptional response to IFN-beta.

Animals↗
PubMed2003

Phosphoinositide 3-kinase activation regulates cell division time by coordinated control of cell mass and cell cycle progression rate.

Cells must increase their mass in coordination with cell cycle progression to ensure that their size and macromolecular composition remain constant for any given proliferation rate. To this end, growth factors activate early signaling cascades that simultaneously promote cell mass increase and induce cell cycle entry. Nonetheless, the mechanism that controls the concerted regulation of cell growth and cell cycle entry in mammals remains unknown. The phosphatidylinositol 3-kinase (PI3K)/protein kinase B pathway regulates cell cycle entry by inactivating forkhead transcription factors and promoting cyclin D synthesis. PI3K/protein kinase B-derived signals also affect activation of p70 S6 kinase and the mammalian target of rapamycin, enzymes involved in cell growth control. We previously showed that enhancement of PI3K activation accelerates cell cycle entry, whereas reduction of PI3K activation retarded this process. Here we examined whether expression of different PI3K mutants affects cell growth during cell division. We show that diminishing or enhancing the magnitude of PI3K activation in a transient manner reduces or increases, respectively, the protein synthesis rate. Alteration of cell growth and cell cycle entry by PI3K forms appears to be concerted, because it results in lengthening or shortening of cell division time without altering cell size. In support of a central role for PI3K in growth control, expression of a deregulated, constitutive active PI3K mutant affects p70 S6 kinase and mammalian target of rapamycin activities and increases cell size. Together, the results show that transient PI3K activation regulates cell growth and cell cycle in a coordinated manner, which in turn controls cell division time.

3T3 Cells↗
PubMed2003

The phosphatidylinositol 3-kinase/Akt pathway enhances Smad3-stimulated mesangial cell collagen I expression in response to transforming growth factor-beta1.

Transforming growth factor (TGF)-beta has been associated with renal glomerular matrix accumulation. We previously showed that Smad3 promotes COL1A2 gene activation by TGF-beta1 in human glomerular mesangial cells. Here, we report that the PI3K/Akt pathway also plays a role in TGF-beta1-increased collagen I expression. TGF-beta1 stimulates the activity of phosphoinositide-dependent kinase (PDK)-1, a downstream target of PI3K, starting at 1 min. Akt, a kinase downstream of PDK-1, is phosphorylated and concentrates in the membrane fraction within 5 min of TGF-beta1 treatment. The PI3K inhibitor LY294002 decreases TGF-beta1-stimulated alpha1(I) and alpha2(I) collagen mRNA expression. Similarly, LY294002 or an Akt dominant negative construct blocks TGF-beta1 induction of COL1A2 promoter activity. However, PI3K stimulation alone is not sufficient to increase collagen I expression, since neither a constitutively active p110 PI3K construct nor PDGF, which induces Akt phosphorylation, is able to stimulate COL1A2 promoter activity or mRNA expression, respectively. LY294002 inhibits stimulation of COL1A2 promoter activity by Smad3. In a Gal4-LUC assay system, blockade of the PI3K pathway significantly decreases TGF-beta1-induced transcriptional activity of Gal4-Smad3. Activity of SBE-LUC, a Smad3/4-responsive construct, is stimulated by over-expression of Smad3 or Smad3D, in which the three C-terminal serine phospho-acceptor residues are mutated. This induction is blocked by LY294002, suggesting that inhibition of the PI3K pathway decreases Smad3 transcriptional activity independently of C-terminal serine phosphorylation. However, TGF-beta1-induced total serine phosphorylation of Smad3 is decreased by LY294002, suggesting that Smad3 is phosphorylated by the PI3K pathway at serine residues other than the direct TGF-beta receptor I target site. Thus, although the PI3K-PDK1-Akt pathway alone is insufficient to stimulate COL1A2 gene transcription, its activation by TGF-beta1 enhances Smad3 transcriptional activity leading to increased collagen I expression in human mesangial cells. This cross-talk between the Smad and PI3K pathways likely contributes to TGF-beta1 induction of glomerular scarring.

Cells, Cultured↗
PubMed2004

Direct activation of the epithelial Na(+) channel by phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 3,4-bisphosphate produced by phosphoinositide 3-OH kinase.

The phospholipid phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)) is accepted to be a direct modulator of ion channel activity. The products of phosphoinositide 3-OH kinase (PI3K), PtdIns(3,4)P(2) and phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)), in contrast, are not. We report here activation of the epithelial Na(+) channel (ENaC) reconstituted in Chinese hamster ovary cells by PI3K. Insulin-like growth factor-I also activated reconstituted ENaC and increased Na(+) reabsorption across renal A6 epithelial cell monolayers via PI3K. Neither IGF-I nor PI3K affected the levels of ENaC in the plasma membrane. The effects of PI3K and IGF-I on ENaC activity paralleled changes in the plasma membrane levels of the PI3K product phospholipids, PtdIns(3,4)P(2)/PtdIns(3,4,5)P(3), as measured by evanescent field fluorescence microscopy. Both PtdIns(3,4)P(2) and PtdIns(3,4,5)P(3) activated ENaC in excised patches. Activation of ENaC by PI3K and its phospholipid products corresponded to changes in channel open probability. We conclude that PI3K directly modulates ENaC activity via PtdIns(3,4)P(2) and PtdIns(3,4,5)P(3). This represents a novel transduction pathway whereby growth factors, such as IGF-I, rapidly modulate target proteins independent of signaling elicited by kinases downstream of PI3K.

Animals↗
PubMed2004

Phosphatidylinositol 3-kinase and frabin mediate Cryptosporidium parvum cellular invasion via activation of Cdc42.

Cryptosporidium parvum invades target epithelia via a mechanism that involves host cell actin reorganization. We previously demonstrated that C. parvum activates the Cdc42/neural Wiskott-Aldrich syndrome protein network in host cells resulting in actin remodeling at the host cell-parasite interface, thus facilitating C. parvum cellular invasion. Here, we tested the role of phosphatidylinositol 3-kinase (PI3K) and frabin, a guanine nucleotide exchange factor specific for Cdc42 in the activation of Cdc42 during C. parvum infection of biliary epithelial cells. We found that C. parvum infection of cultured human biliary epithelial cells induced the accumulation of PI3K at the host cell-parasite interface and resulted in the activation of PI3K in infected cells. Frabin also was recruited to the host cell-parasite interface, a process inhibited by two PI3K inhibitors, wortmannin and LY294002. The cellular expression of either a dominant negative mutant of PI3K (PI3K-Deltap85) or functionally deficient mutants of frabin inhibited C. parvum-induced Cdc42 accumulation at the host cell-parasite interface. Moreover, LY294002 abolished C. parvum-induced Cdc42 activation in infected cells. Inhibition of PI3K by cellular overexpression of PI3K-Deltap85 or by wortmannin or LY294002, as well as inhibition of frabin by various functionally deficient mutants, decreased C. parvum-induced actin accumulation and inhibited C. parvum cellular invasion. In contrast, the overexpression of the p85 subunit of PI3K promoted C. parvum invasion. Our data suggest that an important component of the complex process of C. parvum invasion of target epithelia results from the ability of the organism to trigger host cell PI3K/frabin signaling to activate the Cdc42 pathway, resulting in host cell actin remodeling at the host cell-parasite interface.

Actins↗
PubMed2005

Class II phosphoinositide 3-kinase defines a novel signaling pathway in cell migration.

The lipid products of phosphoinositide 3-kinase (PI3K) are involved in many cellular responses such as proliferation, migration, and survival. Disregulation of PI3K-activated pathways is implicated in different diseases including cancer and diabetes. Among the three classes of PI3Ks, class I is the best characterized, whereas class II has received increasing attention only recently and the precise role of these isoforms is unclear. Similarly, the role of phosphatidylinositol-3-phosphate (PtdIns-3-P) as an intracellular second messenger is only just beginning to be appreciated. Here, we show that lysophosphatidic acid (LPA) stimulates the production of PtdIns-3-P through activation of a class II PI3K (PI3K-C2beta). Both PtdIns-3-P and PI3K-C2beta are involved in LPA-mediated cell migration. This study is the first identification of PtdIns-3-P and PI3K-C2beta as downstream effectors in LPA signaling and demonstration of an intracellular role for a class II PI3K. Defining this novel PI3K-C2beta-PtdIns-3-P signaling pathway may help clarify the process of cell migration and may shed new light on PI3K-mediated intracellular events.

Animals↗
PubMed2000

Increased phosphoinositide 3-kinase activity induces a lymphoproliferative disorder and contributes to tumor generation in vivo.

Alterations in the cell division:cell death ratio induce multiple autoimmune and transformation processes. Phosphoinositide 3-kinase (PI3K) controls cell division and cell death in vitro, but its effect on the function of the cellular immune system and on tumor formation in mammals is poorly characterized. Here we show that transgenic mice expressing in T lymphocytes an active form of PI3K derived from a thymic lymphoma, p65(PI3K), developed an infiltrating lymphoproliferative disorder and autoimmune renal disease with an increased number of T lymphocytes exhibiting a memory phenotype and reduced apoptosis. This pathology was strikingly similar to that described in mice exhibiting heterozygous loss of the tumor suppressor PTEN, a lipid and protein phosphatase. We show that overexpression of PTEN selectively blocks p65(PI3K)-induced 3T3 fibroblast transformation. Moreover, the early development of T cell lymphomas in p65(PI3K) Tg p53(-/-) mice indicated that PI3K contributes to tumor development. These observations demonstrate that constitutive activation of PI3K extends T cell survival in vivo, affects T cell homeostasis, and contributes to tumor generation, supporting a model in which selective increases in one type of PTEN substrate, the PI3K-derived lipid products, induce tumorigenesis. PI3K thus emerges as a potential target in autoimmune disease and cancer therapy.

Animals↗
PubMed1997

Differential effects of constitutively active phosphatidylinositol 3-kinase on glucose transport, glycogen synthase activity, and DNA synthesis in 3T3-L1 adipocytes.

Phosphatidylinositol 3-kinase (PI3K) activation is necessary for many insulin-induced metabolic and mitogenic responses. However, it is unclear whether PI3K activation is sufficient for any of these effects. To address this question we increased PI3K activity in differentiated 3T3-L1 adipocytes by adenovirus-mediated expression of both the inter-SH2 region of the regulatory p85 subunit of PI3K (iSH2) and the catalytic p110 alpha subunit (p110). Coexpression resulted in PI3K activity that exceeded insulin-stimulated activity by two- to fivefold in cytosol, total membranes, and the low density microsome (LDM) fraction, the site of greatest insulin stimulation. While insulin increased glucose transport 15-fold, coexpression of iSH2-p110 increased transport (5.2-) +/- 0.7-fold with a parallel increase in GLUT4 translocation to the plasma membrane. Constitutive activation of PI3K had no effect on maximally insulin-stimulated glucose transport. Neither basal nor insulin-stimulated activity of glycogen synthase or mitogen-activated protein kinase was altered by iSH2-p110 coexpression. DNA synthesis was increased twofold by insulin in control 3T3-L1 adipocytes transduced with beta-galactosidase-encoding recombinant adenovirus, while iSH2-p110 coexpression increased DNA synthesis fivefold. These data indicate that (i) increased PI3K activity is sufficient to activate some but not all metabolic responses to insulin, (ii) activation of PI3K to levels exceeding the effect of insulin in adipocyte LDM results in only a partial stimulation of glucose transport, and (iii) increased PI3K activity in the absence of growth factor or oncoprotein stimulation is a potent stimulus of DNA synthesis.

3T3 Cells↗
PubMed1999

Activation of phosphatidylinositol 3-kinase in response to interleukin-1 leads to phosphorylation and activation of the NF-kappaB p65/RelA subunit.

The work of Reddy et al. (S. A. Reddy, J. A. Huang, and W. S. Liao, J. Biol. Chem. 272:29167-29173, 1997) reveals that phosphatidylinositol 3-kinase (PI3K) plays a role in transducing a signal from the occupied interleukin-1 (IL-1) receptor to nuclear factor kappaB (NF-kappaB), but the underlying mechanism remains to be determined. We have found that IL-1 stimulates interaction of the IL-1 receptor accessory protein with the p85 regulatory subunit of PI3K, leading to the activation of the p110 catalytic subunit. Specific PI3K inhibitors strongly inhibit both PI3K activation and NF-kappaB-dependent gene expression but have no effect on the IL-1-stimulated degradation of IkappaBalpha, the nuclear translocation of NF-kappaB, or the ability of NF-kappaB to bind to DNA. In contrast, PI3K inhibitors block the IL-1-stimulated phosphorylation of NF-kappaB itself, especially the p65/RelA subunit. Furthermore, by using a fusion protein containing the p65/RelA transactivation domain, we found that overexpression of the p110 catalytic subunit of PI3K induces p65/RelA-mediated transactivation and that the specific PI3K inhibitor LY294,002 represses this process. Additionally, the expression of a constitutively activated form of either p110 or the PI3K-activated protein kinase Akt also induces p65/RelA-mediated transactivation. Therefore, IL-1 stimulates the PI3K-dependent phosphorylation and transactivation of NF-kappaB, a process quite distinct from the liberation of NF-kappaB from its cytoplasmic inhibitor IkappaB.

Androstadienes↗
PubMed2003

Modulation of Rab5 and Rab7 recruitment to phagosomes by phosphatidylinositol 3-kinase.

Phagosomal biogenesis is central for microbial killing and antigen presentation by leukocytes. However, the molecular mechanisms governing phagosome maturation are poorly understood. We analyzed the role and site of action of phosphatidylinositol 3-kinases (PI3K) and of Rab GTPases in maturation using both professional and engineered phagocytes. Rab5, which is recruited rapidly and transiently to the phagosome, was found to be essential for the recruitment of Rab7 and for progression to phagolysosomes. Similarly, functional PI3K is required for successful maturation. Remarkably, inhibition of PI3K did not preclude Rab5 recruitment to phagosomes but instead enhanced and prolonged it. Moreover, in the presence of PI3K inhibitors Rab5 was found to be active, as deduced from measurements of early endosome antigen 1 binding and by photobleaching recovery determinations. Though their ability to fuse with late endosomes and lysosomes was virtually eliminated by wortmannin, phagosomes nevertheless recruited a sizable amount of Rab7. Moreover, Rab7 recruited to phagosomes in the presence of PI3K antagonists retained the ability to bind its effector, Rab7-interacting lysosomal protein, suggesting that it is functionally active. These findings imply that (i) dissociation of Rab5 from phagosomes requires products of PI3K, (ii) PI3K-dependent effectors of Rab5 are not essential for the recruitment of Rab7 by phagosomes, and (iii) recruitment and activation of Rab7 are insufficient to induce fusion of phagosomes with late endosomes and lysosomes. Accordingly, transfection of constitutively active Rab7 did not bypass the block of phagolysosome formation exerted by wortmannin. We propose that Rab5 activates both PI3K-dependent and PI3K-independent effectors that act in parallel to promote phagosome maturation.

Adaptor Proteins, Signal Transducing↗
PubMed2003

SAM68: a downstream target of angiotensin II signaling in vascular smooth muscle cells in genetic hypertension.

We investigated whether phosphatidylinositol 3-kinase (PI3K) and 68-kDa Src associated during mitosis (SAM68) are involved in angiotensin II (ANG II) growth signaling in vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rats (SHR). PI3K activity was assessed by measuring the phosphorylation of the regulatory subunit p85alpha and kinase activity of the catalytic 110-kDa subunit of PI3K. The PI3K-SAM68 interaction was assessed by coimmunoprecipitation, and SAM68 activity was evaluated by poly(U) binding. SAM68 expression was manipulated by SAM68 antisense oligonucleotide transfection. VSMC growth was evaluated by measuring [3H]leucine and [3H]thymidine incorporation as indexes of protein and DNA synthesis, respectively. ANG II increased the phosphorylation of p85alpha and kinase activity of the 110-kDa PI3K subunit in VSMCs from SHR and transiently increased p85alpha-SAM68 association. In Wistar-Kyoto (WKY) rat cells, ANG II increased SAM68 phosphorylation without influencing poly(U) binding. In SHR, ANG II did not influence SAM68 phosphorylation but increased SAM68 binding to poly(U). ANG II stimulated phosphoinositol phosphate synthesis by PI3K in SAM68 immunoprecipitates in both groups, with significantly enhanced effects in SHR. Inhibition of PI3K, using the selective inhibitor LY-294002, and downregulation of SAM68, by antisense oligonucleotides, significantly decreased ANG II-stimulated incorporation of [3H]leucine and [3H]thymidine in VSMCs, showing the functional significance of PI3K and SAM68. Our data demonstrate that PI3K and SAM68 are involved in ANG II signaling and that SAM68 is differentially regulated in VSMCs from SHR. These processes may contribute to the enhanced ANG II signaling and altered VSMC growth in SHR.

Angiotensin II↗
PubMed2006

Competitive displacement of phosphoinositide 3-kinase from beta-adrenergic receptor kinase-1 improves postinfarction adverse myocardial remodeling.

Adverse remodeling after myocardial infarction (MI) determines the progression of heart failure. Failing hearts are characterized by downregulation of beta-adrenergic receptor (beta-AR) signaling in part because of increased beta-AR kinase 1 activity. Our previous studies have shown that overexpression of the phosphoinositide kinase (PIK) domain of phosphoinositide 3-kinase (PI3K), prevents beta-AR downregulation and enhances adrenergic agonist responsiveness by inhibiting the targeting of PI3K to the beta-AR complex. To investigate whether preventing beta-AR downregulation in the heart ameliorates cardiac function post-MI, transgenic mice with cardiac-specific overexpression of the PIK domain peptide (TgPIK) underwent left coronary artery ligation and were subsequently followed by serial echocardiography at 4, 8, 12, 16, and 20 wk. Despite having similar infarction sizes, TgPIK mice showed better systolic function, less cardiac dilatation, and improved hemodynamic response to dobutamine compared with littermate controls after MI. To test that displacement of PI3K from the beta-AR complex, but not the total loss of PI3K-gamma, is critical for amelioration of cardiac function, mice lacking the PI3K-gamma (PI3K-gamma-KO) underwent MI, and their cardiac function was assessed 20 wk post-MI. Serial echocardiographic measurements showed severe reduction in contractile performance in PI3K-gamma-KO compared with TgPIK mice. Furthermore, significant beta-AR downregulation and desensitization were only seen in infarcted wild-type and PI3K-gamma-KO mice and not in TgPIK mice. Together, these results demonstrate that adverse remodeling of the ventricle after MI can be attenuated by a strategy that prevents recruitment of PI3K to the plasma membrane and restores normal beta-AR function.

Animals↗
PubMed2005

Inhibition of phosphatidylinositol 3-kinase stimulates activity of the small-conductance K channel in the CCD.

We used Western blotting to examine the expression of phosphatidylinositol 3-kinase (PI3K) in the renal cortex and outer medulla and employed the patch-clamp technique to study the effect of PI3K on the ROMK-like small-conductance K (SK) channels in the cortical collecting duct (CCD). Low K intake increased the expression of the 110-kDa alpha-subunit (p110alpha) of PI3K compared with rats on a normal-K diet. Because low K intake increases superoxide levels (2), the possibility that increases in superoxide anions may be responsible for the effect of low K intake on the expression of PI3K is supported by finding that addition of H(2)O(2) stimulates the expression of p110alpha in M1 cells. Inhibition of PI3K with either wortmannin or LY-294002 significantly increased channel activity in the CCD from rats on a K-deficient (KD) diet or on a normal-K diet. The stimulatory effect of wortmannin on ROMK channel activity cannot be mimicked by inhibition of phospholipase C with U-73122. This suggests that the effect of inhibiting PI3K was not the result of increasing the phosphatidylinositol 4,5-bisphosphate level. Moreover, application of the exogenous phosphatidylinositol 3,4,5-trisphosphate analog had no effect on channel activity in excised patches. Because low K intake has been shown to increase the activity of protein tyrosine kinase (PTK), we explored the role of the interaction between PTK and PI3K in the regulation of the SK channel activity. Inhibition of PTK increased SK channel activity in the CCD from rats on a KD diet. However, addition of wortmannin did not further increase ROMK channel activity. Also, the effect of wortmannin was abolished by treatment of CCD with phalloidin. We conclude that PI3K is involved in mediating the effect of low K intake on ROMK channel activity in the CCD and that the effect of PI3K on SK channels requires the involvement of PTK and the cytoskeleton.

Animals↗