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.

SourceResearch connectionFrom yearThrough year

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

At least 559 records · Page 31Linked to original sources

PubMed2000

Differential requirements of the MAP kinase and PI3 kinase signaling pathways in Src- versus insulin and IGF-1 receptors-induced growth and transformation of rat intestinal epithelial cells.

There have been few studies on the specific signaling pathways involved in the transformation of epithelial cells by oncogenic protein tyrosine kinases. Here we investigate the requirement of MAP (MAPK) and phosphatidylinositol 3- (PI3K) kinases in the transformation of rat intestinal epithelial (RIE) cells by oncogenic forms of insulin receptor (gag-IR), insulin-like growth factor-1 receptor (gag-IGFR), and v-Src. MAPK is not significantly activated in cells transformed by gag-IR and gag-IGFR but is activated in v-Src transformed cells. Treatment with PD98059, a MEK inhibitor, at concentrations where MAPK activity was reduced below the basal level showed that MAPK is partially required for the monolayer growth of parental and transformed RIE cells. However, MAPK is not essential for the focus forming ability of the three oncogene-transformed cells. It is also not necessary for the colony forming ability of gag-IR- and gag-IGFR-, but is partially required for v-Src-transformed cells. PI3K is significantly activated in all three oncogene transformed RIE cells. LY294002, a PI3K inhibitor, potently inhibited monolayer growth of all three oncogene-transformed cells. However, at concentrations of LY294002 where activated forms of Akt, a downstream component of the PI3K pathway, were undetectable, colony and focus forming abilities of the v-Src-RIE cells were only slightly affected whereas those of gag-IR/IGFR-RIE cells were greatly inhibited. These results were confirmed using a different pharmacological inhibitor, wortmannin, and a dominant negative form of PI3K, Ap85. Similarly, rapamycin, known to inhibit p70S6 kinase, a downstream component of the PI3K-Akt pathway, also inhibited gag-IR/IGFR-induced, but not v-Src-induced, focus and colony formation. We conclude that the MAPK and PI3K signaling pathways are differentially required for transformation of RIE cells by oncogenic IR and IGFR versus Src and the pattern of requirements is different from that of fibroblast transformation.

Androstadienes↗

PubMed2001

cAMP signaling selectively influences Ras effectors pathways.

Thyrotropin (TSH) stimulates survival and growth of thyroid cells via a seven transmembrane G protein-coupled receptor. TSH elevates the intracellular cyclic AMP (cAMP) levels activating protein kinase A (PKA). Recent evidence indicates that p21 Ras is required for TSH-induced mitogenesis, but the molecular mechanism(s) is not known. Here we report that Ras p21 activity is necessary for the Go- G1 transition in TSH induced cycle and that the downstream effector of Ras upon TSH signaling is p85-p110 PI3K. We show that PI3K inhibitors block TSH-induced DNA synthesis, cAMP-PKA stimulate the formation of the complex PI3K-p21 Ras and reduce the complex Ras-Raf1 in thyroid and other cells types. Moreover, PKA phosphorylates immunoprecipitated p85 and PKA phosphorylation of cell extracts significantly stimulates the formation of the complex PI3K-Ras. We suggest that PKA phosphorylates p85 and stabilizes the complex p110-p85, enhancing the interaction PI3K and p21 Ras. Simultaneously, cAMP inhibits Raf-1-ERK signaling by decreasing Raf1 availability to Ras. Under these circumstances PI3K signaling is favored. These results indicate that PI3K is an important mediator of Ras effects in cAMP-induced proliferation and illustrates how cAMP can selectively influence Ras effector pathways.

Cell Cycle↗

PubMed2006

A selective inhibitor of the p110delta isoform of PI 3-kinase inhibits AML cell proliferation and survival and increases the cytotoxic effects of VP16.

Current therapy for acute myeloid leukaemia (AML) is suboptimal with a high incidence of relapse. There is strong evidence that constitutive phosphoinositide 3-kinase (PI3K) activity plays a significant role in the pathophysiology of AML. PI3K products are derived from the activity of a number of PI3K catalytic isoforms (class I, II and III) but the relative contribution of these enzymes in AML remains unknown. As non-isoform-selective inhibitors of PI3K such as LY294002 may produce unwanted toxicity to normal tissues, we have investigated the role of the leukocyte-restricted p110delta PI3K isoform in 14 cases of AML. p110delta was detected in all cases whereas the expression levels of the other class I PI3Ks varied more widely, and were often undetectable. The p110delta-selective compound IC87114 inhibited constitutive phosphorylation of the PI3K target Akt/PKB and reduced cell number to a mean of 66+/-5% (range 14-88%). In eight cases, the combination of IC87114 and VP16 (a topoisomerase II inhibitor) was synergistic in reducing viable cell number, and was associated with a reduction in constitutive NF-kappaB activity. IC87114 did not have direct adverse effects or enhance the activity of VP16 on the proliferation and survival of normal haemopoietic progenitors. Overall, our results identify the p110delta isoform as a potential therapeutic target in AML and support a clinical approach to use isoform-selective over broad-spectrum PI3K inhibitors.

Adenine↗

PubMed2002

Hepatocyte growth factor activates phosphoinositide 3-kinase C2 beta in renal brush-border plasma membranes.

Upon stimulation of renal cortical slices with hepatocyte growth factor (HGF), inositol lipid metabolism was studied in basal-lateral plasma membranes (BLM) and brush-border plasma membranes (BBM). Whereas in BLM rapid increases in 1,2-diacylglycerol, PtdIns(3,4,5)P(3) and PtdIns(3,4)P(2) were observed, suggesting that in BLM HGF activates both phospholipase C (PLC) and phosphoinositide 3-kinase (PI3K), in BBM only HGF-induced transient accumulation of PtdIns3P was seen, which was temporarily delayed from signalling events in BLM and could be blocked by the PtdIns-specific-PLC inhibitor ET-18-OCH(3) and the calpain inhibitor calpeptin, suggesting that 3-kinase activation in BBM lies downstream of PLC activation in BLM and is a calpain-mediated event. Moreover, the increase in immunoprecipitable PI3K-C2 beta activity, which is sensitive to wortmannin (10 nM) and shows strong preference for PtdIns over PtdIns4P as a substrate, was observed only in BBM upon stimulation of renal cortical slices with HGF and could be mimicked by the Ca(2+) ionophore A23187 and blocked by the cell-penetrant Ca(2+) chelator BAPTA-AM [1,2-bis-(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid tetrakis(acetoxymethyl ester)]. On Western blots PI3K-C2 beta revealed a single immunoreactive band of 180 kDa in BLM and BBM, while after stimulation with HGF a gel shift of 18 kDa was noticed only in BBM, suggesting that the observed enzyme activation is achieved by proteolysis. When BBM were subjected to short-term (15 min) exposure to mu-calpain, a similar gel shift together with an increase in PI3K-C2 beta activity was observed, when compared with the BBM harvested after HGF stimulation. The above-mentioned gel shift and increase in PI3K-C2 beta activity could be prevented by the calpain inhibitor calpeptin. The data presented in this report show that in renal cells there is a spatial separation of the inositol lipid signalling system between BLM and BBM, and that HGF causes activation of PLC and PI3K primarily in BLM, which leads to calpain-mediated activation of PI3K-C2 beta in BBM with a concomitant increase in PtdIns3P.

Animals↗

PubMed2003

Class III phosphoinositide 3-kinase--Beclin1 complex mediates the amino acid-dependent regulation of autophagy in C2C12 myotubes.

Increased proteolysis contributes to muscle atrophy that prevails in many diseases. Elucidating the signalling pathways responsible for this activation is of obvious clinical importance. Autophagy is a ubiquitous degradation process, induced by amino acid starvation, that delivers cytoplasmic components to lysosomes. Starvation markedly stimulates autophagy in myotubes, and the present studies investigate the mechanisms of this regulation. In C(2)C(12) myotubes incubated with serum growth factors, amino acid starvation stimulated autophagic proteolysis independently of p38 and p42/p44 mitogen-activated protein kinases, but in a PI3K (phosphoinositide 3-kinase)-dependent manner. Starvation, however, did not alter activities of class I and class II PI3Ks, and was not sufficient to affect major signalling proteins downstream from class I PI3K (glycogen synthase kinase, Akt/protein kinase B and protein S6). In contrast, starvation increased class III PI3K activity in whole-myotube extracts. In fact, this increase was most pronounced for a population of class III PI3K that coimmunoprecipitated with Beclin1/Apg6 protein, a major determinant in the initiation of autophagy. Stimulation of proteolysis was reproduced by feeding myotubes with synthetic dipalmitoyl-PtdIns3 P, the class III PI3K product. Conversely, protein transfection of anti-class III PI3K inhibitory antibody into starved myotubes inverted the induction of proteolysis. Therefore, independently of class I PI3K/Akt, protein S6 and mitogen-activated protein kinase pathways, amino acid starvation stimulates proteolysis in myotubes by regulating class III PI3K-Beclin1 autophagic complexes.

Amino Acids↗

PubMed2004

Towards an understanding of isoform specificity in phosphoinositide 3-kinase signalling in lymphocytes.

The PI3K (phosphoinositide 3-kinase) signalling pathway promotes proliferation and transformation in many cell types. This is particularly well illustrated by studies of primary lymphocytes and their leukaemic counterparts. PI3K activation is required for proliferation of T cells and B cells, and certain oncogenes cause leukaemia in part by promoting PI3K signalling. Genetic manipulation of this pathway, together with biochemical studies in primary lymphocytes, has begun to shed light on the molecular mechanisms by which PI3K contributes to the proliferation of normal and transformed lymphocytes. In particular, targeted gene disruption in mice has allowed the identification of specific isoforms of PI3K that are required for distinct cellular responses. Continued investigation of isoform specificity in PI3K signalling, as well as the characterization of critical downstream targets of PI3K signalling, may reveal strategies for the therapeutic control of immune responses and leukaemia.

Animals↗

PubMed2001

RAFTK/Pyk2 involvement in platelet activation is mediated by phosphoinositide 3-kinase.

Platelet activation by different agonists initiates a signalling cascade involving the phosphorylation of several protein kinases, which control key regulatory events. Previously, we demonstrated that the related adhesion focal tyrosine kinase (RAFTK, Pyk2) was involved in an early phase of platelet activation, independent of integrin and glycoprotein IIb-IIIa activation. In this study, we demonstrate that RAFTK is co-immunoprecipitated with phosphoinositide 3-kinase (PI3K) upon platelet activation, and that thrombin, ADP and collagen induced the phosphorylation of both PI3K and RAFTK. A low dose of thrombin (0.015 U/ml) induced RAFTK phosphorylation and platelet aggregation in a PI3K activity-dependent manner, whereas a high dose of thrombin (0.1 U/ml) induced these events in a PI3K activity-independent manner. ADP and collagen also induced RAFTK phosphorylation and platelet aggregation in a PI3K activity-dependent manner, similar to that of the low-dose thrombin. Furthermore, protein tyrosine phosphatase activity was associated with RAFTK in response to platelet activation, and was found to be that of protein tyrosine phosphatase-2 (SHP-2). The association of SHP-2 with RAFTK was PI3K-dependent and was increased upon RAFTK phosphorylation. Taken together, our results strongly suggest that the involvement of RAFTK in platelet activation is mediated via the PI3K pathway.

Adenosine Diphosphate↗

PubMed2005

Modulation of epithelial neoplasia and lymphoid hyperplasia in PTEN+/- mice by the p85 regulatory subunits of phosphoinositide 3-kinase.

Mice with heterozygous deletion of the PTEN tumor suppressor gene develop a range of epithelial neoplasia as well as lymphoid hyperplasia. Previous studies suggest that PTEN suppresses tumor formation by acting as a phosphoinositide phosphatase to limit signaling by phosphoinositide 3-kinase (PI3K). Here, we examined the effect of deleting various regulatory subunits of PI3K (p85alpha and p85beta) on epithelial neoplasia and lymphoid hyperplasia in PTEN+/- mice. Interestingly, we found the loss of one p85alpha allele with or without the loss of p85beta led to increased incidence of intestinal polyps. Signaling downstream of PI3K was enhanced in the PTEN+/-p85alpha+/-p85beta-/- polyps, as judged by an increased fraction of both cells with cytoplasmic staining of the transcription factor FKHR and cells with positive staining for the proliferation marker Ki-67. In contrast, the incidence of prostate intraepithelial neoplasia was not significantly altered in PTEN+/- mice heterozygous for p85alpha or null for p85beta, whereas the fraction of proliferating cells in prostate intraepithelial neoplasia was reduced in mice lacking p85beta. Finally, there was no significant change in T lymphocyte hyperplasia in the PTEN+/- mice with various p85 deletions, although anti-CD3-stimulated AKT activation was somewhat reduced in the p85alpha+/- background. These results indicate that decreasing the levels of different p85 regulatory subunits can result in enhanced PI3K signaling in some tissues and decreased PI3K signaling in others, supporting the model that, although p85 proteins are essential for class I(A) PI3K signaling, they can function as inhibitors of PI3K signaling in some tissues and thus suppress tumor formation.

Animals↗

PubMed1998

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.

Apoptosis↗

PubMed1998

A novel class II phosphoinositide 3-kinase predominantly expressed in the liver and its enhanced expression during liver regeneration.

We report herein the cloning and characterization of a novel class II phosphoinositide 3-kinase, termed PI3K-IIgamma, from the cDNA library of regenerating rat liver. This cDNA encodes a protein of 1505 amino acids with a calculated molecular mass of 170,972 Da. The amino acid sequence of PI3K-IIgamma is highly similar to those of class II PI 3-kinases, including murine Cpk-m/p170 and human HsC2-PI3K. It contains a C2 domain at the C terminus but no recognizable protein motifs at its N terminus. PI3K-IIgamma displays a restricted substrate specificity for PtdIns and PtdIns 4-P, but not for PtdIns 4,5-P2. By epitope tag immunocytochemistry, the immunoreactivity for PI3K-IIgamma is localized in the juxtanuclear Golgi region at high levels and also in the plasma and nuclear membranes at low levels. By Northern blot analysis and in situ hybridization histochemistry, PI3K-IIgamma mRNA expression is confined to the liver throughout the development with much higher expression in adult liver than in fetal liver. In addition, its expression increases during liver regeneration after partial hepatectomy with maximal expression after the growth period, suggesting that PI3K-IIgamma may function mainly in highly differentiated hepatic cells.

Adult↗

PubMed1998

Binding of a diphosphotyrosine-containing peptide that mimics activated platelet-derived growth factor receptor beta induces oligomerization of phosphatidylinositol 3-kinase.

Phosphatidylinositol 3-kinase (PI3K) is a heterodimeric enzyme comprising a p110 catalytic subunit and a p85 regulatory subunit. We have recently shown that the isolated p85 subunit exists as a dimer; therefore, we examined whether the heterodimeric enzyme was capable of further self-association. Size-exclusion chromatography demonstrated that PI3K was a 1:1 complex of p85 and p110 under native conditions. However, binding of a diphosphotyrosine-containing peptide that mimics an activated platelet-derived growth factor receptor beta induced an increase in the apparent molecular mass of PI3K. This increase was due to dimerization of PI3K and was dependent on PI3K concentration but not diphosphopeptide concentration. Dimer formation was also observed directly using fluorescence resonance energy transfer. Diphosphopeptide-induced activation of PI3K (Carpenter, C. L., Auger, K. R., Chanudhuri, M., Yoakim, M., Schaffhausen, B., Shoelson, S., and Cantley, L. C. (1993) J. Biol. Chem. 268, 9478-9483; Rordorf-Nikolic, T., Van Horn, D. J., Chen, D., White, M. F., and Backer, J. M. (1995) J. Biol. Chem. 270, 3662-3666) was not a direct result of dimerization and occurred only when phosphatidylinositol, and not phosphatidylinositol-4,5-diphosphate, was the phosphorylation substrate. Binding of the tandem SH2 domains of the p85 regulatory subunit to activated receptor tyrosine kinases therefore induces dimerization of PI3K, which may be an early step in inositol lipid-mediated signal transduction.

Amino Acid Sequence↗

PubMed2000

Platelet-derived growth factor-induced H(2)O(2) production requires the activation of phosphatidylinositol 3-kinase.

Autophosphorylation of the platelet-derived growth factor (PDGF) receptor triggers intracellular signaling cascades as a result of recruitment of Src homology 2 domain-containing enzymes, including phosphatidylinositol 3-kinase (PI3K), the GTPase-activating protein of Ras (GAP), the protein-tyrosine phosphatase SHP-2, and phospholipase C-gamma1 (PLC-gamma1), to specific phosphotyrosine residues. The roles of these various effectors in PDGF-induced generation of H(2)O(2) have now been investigated in HepG2 cells expressing various PDGF receptor mutants. These mutants included a kinase-deficient receptor and receptors in which various combinations of the tyrosine residues required for the binding of PI3K (Tyr(740) and Tyr(751)), GAP (Tyr(771)), SHP-2 (Tyr(1009)), or PLC-gamma1 (Tyr(1021)) were mutated to Phe. PDGF failed to increase H(2)O(2) production in cells expressing either the kinase-deficient mutant or a receptor in which the two Tyr residues required for the binding of PI3K were replaced by Phe. In contrast, PDGF-induced H(2)O(2) production in cells expressing a receptor in which the binding sites for GAP, SHP-2, and PLC-gamma1 were all mutated was slightly greater than that in cells expressing the wild-type receptor. Only the PI3K binding site was alone sufficient for PDGF-induced H(2)O(2) production. The effect of PDGF on H(2)O(2) generation was blocked by the PI3K inhibitors LY294002 and wortmannin or by overexpression of a dominant negative mutant of Rac1. These results suggest that a product of PI3K is required for PDGF-induced production of H(2)O(2) in nonphagocytic cells, and that Rac1 mediates signaling between the PI3K product and the putative NADPH oxidase.

Binding Sites↗

PubMed2000

Differential protein phosphorylation in 3T3-L1 adipocytes in response to insulin versus platelet-derived growth factor. No evidence for a phosphatidylinositide 3-kinase-independent pathway in insulin signaling.

Insulin regulates glucose metabolism in adipocytes via a phosphatidylinositide 3-kinase (PI3K)-dependent pathway that appears to involve protein phosphorylation. However, the generation of phosphoinositides is not sufficient for insulin action, and it has been suggested that insulin regulation of glucose metabolism may involve both PI3K-dependent and -independent pathways, the latter being insulin specific. To test this hypothesis, we have designed a phosphoprotein screen to study insulin-specific phosphoproteins that may be either downstream or in parallel to PI3K. Nineteen insulin-regulated phosphospots were detected in the cytosol and high speed pellet fractions, only six of which were significantly regulated by platelet-derived growth factor. Importantly, almost all (92%) of the insulin-specific phosphoproteins identified using this approach were sensitive to the PI3K inhibitor wortmannin. Thus, we obtained no evidence for an insulin-specific, PI3K-independent signaling pathway. A large proportion (62%) of the insulin-specific phosphoproteins were enriched in the same high speed pellet fraction to which PI3K was recruited in response to insulin. Thus, our data suggest that insulin specifically stimulates the phosphorylation of a novel subset of downstream targets and this may in part be because of the unique localization of PI3K in response to insulin in adipocytes.

3T3 Cells↗

PubMed2001

Tyrosine phosphorylation of p85 relieves its inhibitory activity on phosphatidylinositol 3-kinase.

Under resting conditions, the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI3K) serves to both stabilize and inactivate the p110 catalytic subunit. The inhibitory activity of p85 is relieved by occupancy of the NH(2)-terminal SH2 domain of p85 by phosphorylated tyrosine. Src family kinases phosphorylate tyrosine 688 in p85, a process that we have shown to be reversed by the activity of the p85-associated SH2 domain-containing phosphatase SHP1. We demonstrate that phosphorylation of the downstream PI3K target Akt is increased in cells lacking SHP1, implicating phosphorylation of p85 in the regulation of PI3K activity. Furthermore, the in vitro specific activity of PI3K associated with tyrosine- phosphorylated p85 is higher than that associated with nonphosphorylated p85. Expression of wild-type p85 inhibits PI3K enzyme activity as indicated by PI3K- dependent Akt phosphorylation. The inhibitory activity of p85 is accentuated by mutation of tyrosine 688 to alanine and reversed by mutation of tyrosine 688 to aspartic acid, changes that block and mimic tyrosine phosphorylation, respectively Strikingly, mutation of tyrosine 688 to aspartic acid completely reverses the inhibitory activity of p85 on cell viability and activation of the downstream targets Akt and NFkappaB, indicative of the physiological relevance of p85 phosphorylation. Tyrosine phosphorylation of Tyr(688) or mutation of tyrosine 688 to aspartic acid is sufficient to allow binding to the NH(2)-terminal SH2 domain of p85. Thus an intramolecular interaction between phosphorylated Tyr(688) and the NH(2)-terminal SH2 domain of p85 can relieve the inhibitory activity of p85 on p110. Taken together, the data indicate that phosphorylation of Tyr(688) in p85 leads to a novel mechanism of PI3K regulation.

Animals↗

PubMed2001

Inhibitory mechanisms of tea polyphenols on the ultraviolet B-activated phosphatidylinositol 3-kinase-dependent pathway.

In this study, we investigated the effect of tea polyphenols, (-)-epigallocatechin-3-gallate or theaflavins, on UVB-induced phosphatidylinositol 3-kinase (PI3K) activation in mouse epidermal JB6 Cl 41 cells. Pretreatment of cells with these polyphenols inhibited UVB-induced PI3K activation. Furthermore, UVB-induced activation of Akt and ribosomal p70 S6 kinase (p70 S6-K), PI3K downstream effectors, were also attenuated by the polyphenols. In addition to LY294002, a PI3K inhibitor, pretreatment with a specific mitogen-activated protein/extracellular signal-regulated protein kinases (Erks) kinase 1 inhibitor, U0126, or a specific p38 kinase inhibitor, SB202190, blocked UVB-induced activation of both Akt and p70 S6-K. Pretreatment with LY294002 restrained UVB-induced phosphorylation of Erks, suggesting that in UVB signaling, the Erk pathway is mediated by PI3K. Moreover, pretreatment with rapamycin, an inhibitor of p70 S6-K, inhibited UVB-induced activation of p70 S6-K, but UVB-induced activation of Akt did not change. Interestingly, UVB-induced p70 S6-K activation was directly blocked by the addition of (-)-epigallocatechin-3-gallate or theaflavins, whereas these polyphenols showed only a weak inhibition on UVB-induced Akt activation. Because PI3K is an important factor in carcinogenesis, the inhibitory effect of these polyphenols on activation of PI3K and its downstream effects may further explain the anti-tumor promotion action of these tea constituents.

Animals↗

PubMed2001

Phosphatidylinositol 3-kinase controls human intestinal epithelial cell differentiation by promoting adherens junction assembly and p38 MAPK activation.

The signaling pathways mediating human intestinal epithelial cell differentiation remain largely undefined. Phosphatidylinositol 3-kinase (PI3K) is an important modulator of extracellular signals, including those elicited by E-cadherin-mediated cell-cell adhesion, which plays an important role in maintenance of the structural and functional integrity of epithelia. In this study, we analyzed the involvement of PI3K in the differentiation of human intestinal epithelial cells. We showed that inhibition of PI3K signaling in Caco-2/15 cells repressed sucrase-isomaltase and villin protein expression. Morphological differentiation of enterocyte-like features in Caco-2/15 cells such as epithelial cell polarity and brush-border formation were strongly attenuated by PI3K inhibition. Immunofluorescence and immunoprecipitation experiments revealed that PI3K was recruited to and activated by E-cadherin-mediated cell-cell contacts in confluent Caco-2/15 cells, and this activation appears to be essential for the integrity of adherens junctions and association with the cytoskeleton. We provide evidence that the assembly of calcium-dependent adherens junctions led to a rapid and remarkable increase in the state of activation of Akt and p38 MAPK pathways and that this increase was blocked in the presence of anti-E-cadherin antibodies and PI3K inhibitor. Therefore, our results indicate that PI3K promotes assembly of adherens junctions, which, in turn, control p38 MAPK activation and enterocyte differentiation.

Adherens Junctions↗

PubMed2002

Phosphoinositide 3-kinase activity is required for retinoic acid-induced expression and activation of the tissue transglutaminase.

Tissue transglutaminase (TGase) is a dual function enzyme that couples an ability to bind GTP with transamidation activity. Retinoic acid (RA) consistently induces TGase expression and activation, and it was recently shown that increased TGase expression protected cells from apoptosis. To better understand how RA regulates TGase, we considered whether RA employed pro-survival signaling pathways to mediate TGase expression and activation. It was found that RA stimulation of NIH3T3 cells activated ERK and phosphoinositide 3-kinase (PI3K); however, only PI3K activation was necessary for RA-induced TGase expression. The overexpression of a constitutively active form of PI3K did not induce TGase expression, indicating that PI3K signaling was necessary but not sufficient for TGase expression. The exposure of cells expressing exogenous TGase to the PI3K inhibitor, LY294002, reduced the ability of TGase to be photoaffinity-labeled with [alpha-(32)P]GTP, providing evidence that PI3K regulates the GTP binding activity of TGase as well as its expression. Moreover, cell viability assays showed that incubation of RA-treated cells with LY294002 together with the TGase inhibitor, monodansylcadaverine (MDC), converted RA from a differentiation factor to an apoptotic stimulus. These findings demonstrate that PI3K activity is required for the RA-stimulated expression and GTP binding activity of TGase, thereby linking the up-regulation of TGase with a well established cell survival factor.

3T3 Cells↗

PubMed2002

Phosphatidylinositol 3-kinase activity negatively regulates stability of cyclooxygenase 2 mRNA.

Human alveolar macrophages have both lipopolysaccharide (LPS)-induced and constitutive phosphatidylinositol 3-kinase (PI3K) activity. We observed that blocking PI3K activity increased release of prostaglandin E2 after LPS exposure, and increasing PI3K activity (interleukin-13) decreased release of prostaglandin E2 after LPS exposure. This was not because of an effect of PI3K on phospholipase 2 activity. PI3K inhibition resulted in an increase in cyclooxygenase 2 (COX2) protein, mRNA, and mRNA stability. PI3K negatively regulated activation of the p38 pathway (p38, MKK3/6, and MAPKAP2), and an active p38 was necessary for COX2 production. The data suggest that PI3K inhibition of p38 modulates COX2 expression via destabilization of LPS-induced COX2 mRNA.

Animals↗