Mechanism by which cAMP activates PI3-kinase and increases bile acid secretion in WIF-B9 cells

Tatehiro Kagawa, Lyuba Varticovski, Yoshimichi Sai, and Irwin M. Arias

Department of Physiology, Tufts University School of Medicine, Boston, Massachusetts 02111


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Previous studies in rat bile canalicular membrane vesicles and WIF-B9 cells revealed that cAMP-induced trafficking of ATP-binding cassette (ABC) transporters to the canalicular membrane and their activation require phosphoinositide 3-kinase (PI3-K) products. In the present studies, canalicular secretion of fluorescein isothiocyanate-glycocholate in WIF-B9 cells was increased by cAMP and a decapeptide that enhances PI3-K activity; these effects were inhibited by wortmannin. To determine the mechanism(s) whereby cAMP activates PI3-K, we examined signal transduction pathways in WIF-B9 and COS-7 cells. cAMP activated PI3-K in both cell lines in a phosphotyrosine-independent manner. PI3-K activity increased in association with p110beta in both cell lines. The effect of cAMP was KT-5720 sensitive, suggesting involvement of protein kinase A. Expression of a dominant-negative beta -adrenergic receptor kinase COOH terminus (beta -ARKct), which blocks Gbeta gamma signaling, decreased PI3-K activation in both cell lines. cAMP increased GTP-bound Ras in COS-7 but not WIF-B9 cells. Expression of dominant-negative Ras abolished cAMP-mediated PI3-K, which suggests that the effect is downstream of Ras and Gbeta gamma . These data indicate that cAMP activates PI3-K in a cell type-specific manner and provide insight regarding mechanisms of PI3-K activation required for bile acid secretion.

bile secretion; heterotrimeric G protein; Gbeta gamma ; protein kinase A; Ras


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

CAMP REGULATES critical biological functions through activation of several signaling pathways (44). In addition to activation of adenylyl cyclase isoforms and protein kinase A (PKA), cAMP-activated signaling pathways include A-kinase anchor proteins, phosphodiesterases, cAMP regulatory element-binding protein (CREB), CREB-binding proteins (CBP/p300) (39), and cAMP-regulated guanine nucleotide exchange factors (cAMP-GEFs), which activate small GTPases such as Rap1 and Rap2 (5, 22). Our previous studies (11, 31, 32, 42) suggest cross talk between cAMP and phosphoinositide 3-kinase (PI3-K) in regulation of bile acid secretion across the canalicular membrane. The mechanism is unknown and is the long-range goal of our studies.

Administration of dibutyryl cAMP (DBcAMP) to rats increases biliary secretion of taurocholate, which results from increased trafficking of bsep, the canalicular ATP-dependent bile acid transporter, from intracellular sites to the canalicular membrane (11, 24). The concentration of bsep in the canalicular membrane increased approximately threefold, concomitant with a similar increase in PI3-K activity and ATP-dependent transport of taurocholate (21). Administration of wortmannin in vivo or after addition to canalicular membrane vesicles inhibited each response to DBcAMP; however, addition of phosphoinositide products of PI3-K but not of phosphatidylinositol (PI)4-P, the enzyme's substrate, to canalicular membrane vesicles restored bsep activity in the presence of wortmannin (21). In studies using a polarized rat hepatoma-human fibroblast hybrid cell line (WIF-B9), administration of a decapeptide (PI3-K peptide; Refs. 15, 18), which enters cells and activates PI3-K, increased canalicular bile acid secretion in a wortmannin-inhibitable manner (32, 42, 53). These previously published studies in rat liver, canalicular membrane vesicles, and WIF-B9 cells suggest interactions between cAMP and PI3-K signal transduction pathways as related to bsep trafficking and activity in the canalicular membrane. Studies in other systems suggest similar cross talk. For example, wortmannin inhibits cAMP activation of Akt (2, 3, 13, 28, 54, 56) in a cell type-specific manner, as well as serum- and glycocorticoid-induced protein kinase (Sgk) (35).

The mechanism(s) responsible for cAMP and PI3-K cross talk have not been identified in any system and were the objective of the present studies. WIF-B9 and COS-7 cells were chosen for study on the basis of the following rationale. Preliminary studies in freshly isolated rat hepatocytes confirmed that cAMP increased PI3-K activity (54); however, primary cultures lack cellular polarity, which prevents correlation with bsep function or trafficking. Hepatocytes that retain tight junctions during preparation (so-called hepatocyte doublets) were not studied because they retain a hemi-canaliculus from adjacent cells that complicates investigation of canalicular membrane trafficking (24). Furthermore, transient transfection in primary hepatocytes is unpredictable for biochemical studies. Because transport studies in WIF-B9 cells yielded results similar to those observed in rat liver (42, 53), we chose to study the relation between cAMP administration and PI3-K activation in polarized WIF-B9 cells and in nonpolarized COS-7 cells as a control.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. DBcAMP, wortmannin, pertussis toxin (PTX), phosphatidylserine (PS), PI, PI 3,4-bisphosphate (PI 3,4-P2), and glycocholate were purchased from Sigma (St. Louis, MO). KT-5720, PP2, and forskolin were obtained from Calbiochem (San Diego, CA).

Cell culture. COS-7 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and antibiotics (Life Technologies, Rockville, MD). WIF-B9 cells, an immortalized cell line obtained from a rat hepatoma cell line-human fibroblast fusion that forms functional bile canaliculus (20, 45), were grown in F-12 medium (Life Technologies) supplemented with 5% FBS. Jurkat cells were cultured in RPMI 1640 (BioWhittaker, Walkersville, MD) with 10% FBS. All cells were cultured at 37°C in a humidified atmosphere with 5% (COS-7 and Jurkat) or 7% (WIF-B9) CO2. Cells were starved in 1% bovine serum albumin (Sigma) for 40 h for COS-7 and 16 h for WIF-B9 cells and treated with DBcAMP or forskolin for the indicated time period. When indicated, cells were incubated with inhibitors KT-5720 for 30 min, PTX overnight, or PP2 for 1 h before treatment with DBcAMP.

Expression of dominant-negative constructs. COS-7 cells were transfected with RasN17 (gift of Dr. L. A. Feig, Tufts University, Boston, MA) by using Lipofectamine Plus (Life Technologies) according to the manufacturer's instructions and cultured for 24 h. Cells were induced to quiescence and subjected to treatment with DBcAMP. For expression of dominant-negative Gbeta gamma , cells were infected with adenovirus vector alone or adenovirus expressing the beta -adrenergic receptor kinase COOH terminus (beta -ARKct) (gift of Dr. R. J. Lefkowitz, Duke University Medical Center, Durham, NC) at 100 multiplicities of infection (MOI) (7, 26). Medium was replaced with complete medium 2 h after infection. Cells were starved after 24 h and subjected to the experiments as indicated. The expression of beta -ARKct was tested by immunoblotting with a beta -ARK antiserum that recognizes the COOH terminus of beta -ARK2 (gift of Dr. R. J. Lefkowitz).

PI3-K activity assay. Cells were washed with ice-cold phosphate-buffered saline (PBS) three times and lysed in lysis buffer (50 mM HEPES, pH 7.5, 0.5 mM EGTA, 5 mM sodium orthovanadate, 10% glycerol, and 1% NP-40) with protease inhibitors (0.5 mM PMSF, 5 µg/ml pepstatin A, 10 µg/ml aprotinin, and 10 µg/ml leupeptin). Cell lysates were incubated on ice for 5 min and spun at 4°C for 10 min at 12,000 g to remove insoluble fractions. The supernatant was frozen immediately in aliquots by immersion in liquid nitrogen and stored at -80°C until being used. PI3-K activity assay was performed in immunoprecipitates by using 200 µg of protein from cell lysates after incubation with anti-p85 antibody (no. 06-497; Upstate Biotechnology, Lake Placid, NY) or 350 µg of protein incubated with anti-phosphotyrosine (anti-P-tyr) antibody (4G10; Upstate Biotechnology) or anti-p110alpha , -beta , or -gamma antibodies (H-201, H-198, and H-199, respectively; Santa Cruz Biotechnology, Santa Cruz, CA) at 4°C for 2 h followed by incubation with 30 µl of 50% suspension of protein A beads (Sigma) for 1 h. After the beads were washed once with lysis buffer and three times with PBS, they were sedimented by brief centrifugation and PI3-K activity was quantified as previously described (32, 50). Briefly, the reaction was performed in a total volume of 50 µl containing 150 µM ATP, 25 mM MOPS (pH 7.0), 5 mM MgCl2, 1 mM EGTA, and 0.2 mg/ml sonicated lipids: PS-PI-PI 4,5-bisphosphate (PI 4,5-P2) (1:1:1 vol/vol/vol) in sonication buffer (25 mM MOPS, pH 7.0, 1 mM EGTA) with 25 µCi of [gamma -32P]ATP per assay (NEN, Boston, MA). After incubation at 37°C for 20 min, the reaction was stopped with 90 µl of methanol-1 N HCl (1:1 vol/vol) and lipids were extracted twice in 100 µl of chloroform. The organic layer was combined, dried under nitrogen gas flow, and analyzed by TLC (Whatman, Clifton, NJ). Polyphosphoinositides were resolved in water-acetic acid-n-propanol (34:1:65 vol/vol/vol), and 32P label was detected by autoradiography. TLC spots corresponding to PI 3,4,5-trisphosphate (PIP3) products were scraped and eluted in scintillation liquid, and 32P incorporation into PIP3 was quantified by liquid scintillation counting. To compare the results obtained from different experiments, whole cell lysates from HEK-293 cells were diluted 1,000-fold (50) and used as control (designated as 1 unit of PI3-K activity).

Immunoblotting. Twenty micrograms of cell lysates obtained as described in PI3-K activity assay were separated by SDS-PAGE, transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA), and probed with antibodies to Akt and phospho-Akt (pAkt) (Cell Signaling Technology, Beverly, MA), total ERK1/2 (Upstate Biotechnology), and phospho-ERK1/2 (pERK1/2, Santa Cruz Biotechnology). The membranes were probed with appropriate horseradish peroxidase-coupled secondary antibodies (Cell Signaling) followed by enhanced chemiluminescence (ECL) reaction (NEN, Boston, MA). The densitometric analysis was performed with the NIH Image program, and the relative intensity was calculated relative to total protein content. Normal rabbit IgG (Santa Cruz Biotechnology) was used as a negative control.

Ras-GTP assay. GTP-bound Ras was quantified as described previously (12). Briefly, the Ras-binding domain of Raf was expressed as a glutathione S-transferase (GST)-fusion protein in Escherichia coli and affinity purified on glutathione Sepharose 4B beads (Amersham Pharmacia Biotech, Piscataway, NJ). Ten micrograms of immobilized fusion protein were incubated with four hundred micrograms of whole cell lysates for 2 h at 4°C. The beads were washed once with lysis buffer and three times with PBS. After addition of 30 µl of sample buffer, the beads were heated to 95°C for 5 min and centrifuged for 2 min at 12,000 g in a microcentrifuge column (Bio-Rad Laboratories, Hercules, CA) to remove the beads. The flow-through was separated by SDS-PAGE followed by immunoblotting with anti-Ras antibodies (Upstate Biotechnology). To confirm that an equal amount of total Ras protein was present in each sample, cell lysates were also immunoblotted with anti-Ras antibodies.

Bile acid transport in WIF-B9 cells. With a previously described technique (42), monolayers of WIF-B9 cells were grown on glass coverslips, mounted in a Dvorak-Stotler culture chamber that was thermostabilized at 37°C, and promptly viewed by confocal laser scanning microscopy. Cells were perfused with modified F-12 supplemented with 5% FBS and 12 mM HEPES (pH 7.0) with a perfusion pump (model 351; Sege Instruments, Cambridge, MA) at 0.3 ml/min. Images were obtained with an Odyssey XL confocal system (Noran Instruments, Middletown, WI) and an inverted Nikon Diaphot microscope equipped with a Nikon ×60 oil-immersion Planchromat lens (NA 1.4). After a baseline fluorescent image was acquired, the chamber was perfused for 20 min with a second solution containing 0.5 µM glycocholic acid conjugated with fluorescein isothiocyanate (FITC-GC), which exhibits the requisite structural features of conventional bile acids and is a substrate for the bile acid transporter (16, 25). Excitation was at 488 nm with a krypton-argon laser. The microscope was calibrated daily by adjusting the gain and offset to obtain constant values for an intensity calibration curve. Linearity of the signal as a function of the concentration of the fluorescent probe was verified. Quantification was performed with Inter Vision computer software by measuring fluorescence intensity in the perinuclear cytoplasmic and canalicular regions, which were delimited by boundaries drawn on the first image of the series. Similar studies were performed in WIF-B9 cells that were preincubated for 10 min at 37°C with DBcAMP (300 µM) or rhodamine-linked PI3-K peptide (5, 20 µM) (15, 18, 42) and washed twice with PBS before perfusion with FITC-GC. Cells were also incubated in wortmannin (100 nM) for 10 min at 37°C before addition of DBcAMP, PI3-K peptide, or FITC-GC. Control cells for experiments using wortmannin were exposed to DMSO (0.01%).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

cAMP and PI3-K peptide increased FITC-GC secretion in a wortmannin-inhibitable manner in WIF-B9 cells. As shown in Table 1, cAMP and PI3-K peptide significantly increased FITC-GC secretion into bile canaliculi of WIF-B9 cells 2.0- and 2.1-fold, respectively; both responses were inhibited by prior incubation with wortmannin (100 nM). In a previous study (42), canalicular secretion of FITC-GC by WIF-B9 cells was time dependent, saturable, stimulated by preincubation of cells with taurocholate (100 µM) or PI3-K peptide (5 µM), and decreased by preincubation with wortmannin (100 nM). The present study demonstrated that, like taurocholate, cAMP and PI3-K peptide significantly increased FITC-GC secretion in WIF-B9 cells. The stimulatory effects of cAMP and PI3-K peptide were both inhibited by wortmannin (100 nM), which suggests that cAMP may enhance bile acid secretion through activation of PI3-K. These studies provided the impetus to identify the biochemical mechanism(s) whereby cAMP and PI3-K interact with one another as regards bile acid secretion.

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Canalicular secretion of FITC-GC in WIF-B9 cells and effects of cAMP, PI3-K peptide, and wortmannin

cAMP activated PI3-K in a time- and dose-dependent manner in COS-7 and WIF-B9 cell lines. PI3-K activity was quantified before and after stimulation with the cell-permeant cAMP analog DBcAMP. Within 15 min, cAMP activated p85-associated PI3-K in both cell lines (Fig. 1, A and C for COS-7 and B and D for WIF-B9 cells). Growth factors such as PDGF produced maximal activation within 10 min (50); in contrast, the maximal response (2.0- to 2.5-fold) to cAMP was observed at 60 min; p85-associated PI3-K activation returned to basal levels after 4 h in both cell lines. Stimulation with forskolin (50 µM, 30 min) also activated PI3-K by 2.8 ± 0.5- and 2.3 ± 0.2 (mean ± SE)-fold in COS-7 and WIF-B9 cells, respectively (Fig. 1E for COS-7, Fig. 1F for WIF-B9 cells). The increase in association with p85 was not smaller than that observed by growth factors; EGF (10 ng/ml, 15 min) in COS-7 and insulin (10 ng/ml, 15 min) in WIF-B9 induced 2.4 ± 0.5- and 2.6 ± 0.4 (mean ± SE)-fold increase, respectively. The cAMP effect on PI3-K was dose dependent (Fig. 1,G and H). The calculated ED50 was 67.0 and 56.2 µM in COS-7 and WIF-B9 cells, respectively. The increase in PI3-K activity resulted from modification in intrinsic catalytic activity, because the amount of immunoreactive p110 subunits associated with p85 did not vary after cAMP stimulation (data not shown). In whole lysates, PI3-K activity increased 1.5-fold in both cell lines within 1 h (data not shown).


View larger version (30K):
[in this window]
[in a new window]
 
Fig. 1.   cAMP activates phosphoinositide 3-kinase (PI3-K) in a time- and dose-dependent manner. COS-7 (A, C, E, and G) and WIF-B9 (B, D, F, and H) cells were induced to quiescence and treated with cAMP (100 µM) for indicated times (A-D); with cAMP (100 µM) for 60 min, forskolin (50 µM) for 30 min, EGF (10 ng/ml) for 15 min, and insulin (10 ng/ml) for 15 min (E and F); or with cAMP with different concentrations of cAMP for 60 min (G and H). Whole cell lysates (200 µg) were immunoprecipitated with anti-p85 antibodies followed by PI3-K assay, and radioactivity incorporated into the phosphatidylinositol (PI) 3,4,5-trisphosphate (PIP3) was quantified. Representative autoradiographs (A for COS-7 and B for WIF-B9 cells) are shown. Data (C-H) were obtained from 3-8 independent experiments; each value represents the mean ± SE. *P < 0.05 vs. basal levels (repeated-measure 1-way ANOVA).

cAMP-mediated PI3-K activation was independent of phosphotyrosine. cAMP stimulation did not generate tyrosine-phosphorylated proteins, in contrast to the effect of EGF and insulin in COS-7 and WIF-B9 cells, respectively (Fig. 2A). Phosphotyrosine-associated PI3-K activity did not change after cAMP stimulation at any time period, in contrast to the effect of EGF and insulin, which induced robust activation in COS-7 and WIF-B9 cells (Fig. 2B). These observations indicate that cAMP-mediated PI3-K activation is phosphotyrosine independent. The rapidity of PI3-K activation by cAMP suggests that transcription is unaffected. Therefore, on the basis of evidence from other pathways that involve cAMP, we investigated the role of PKA, Gbeta gamma , and Ras in cAMP-mediated activation of PI3-K in COS-7 and WIF-B9 cells.


View larger version (63K):
[in this window]
[in a new window]
 
Fig. 2.   cAMP activates PI3-K in a phosphotyrosine-independent manner. A: immunoblot of tyrosine-phosphorylated proteins. COS-7 or WIF-B9 cells were stimulated with EGF (10 ng/ml) or insulin (10 ng/ml) for 15 min or with cAMP (100 µM) for indicated times. Whole cell lysates (20 µg) were separated by SDS-PAGE and probed with anti-phosphotyrosine (anti-P-tyr) antibodies. B: effect of cAMP on anti-P-tyr-associated PI3-K activity. PI3-K activity was determined in immunoprecipitates from 400 µg of protein with anti-P-tyr antibodies. Data represent means ± SE from 3 independent experiments.

cAMP activated Akt and ERK1/2 in COS-7 cells. To determine the effect of cAMP on downstream targets of PI3-K, we quantified activation of Akt by determining phosphorylation of Akt on Ser473 (Fig. 3, A and C). In COS-7 cells, Akt phosphorylation was detected 15-30 min after addition of cAMP. The effect of cAMP on Akt phosphorylation was significant [relative intensity at 15 min (mean ± SE): 2.3 ± 0.3; P < 0.05 vs. basal level], although substantially smaller than the response to EGF (9.0-fold). This effect was wortmannin inhibitable (Fig. 3A), suggesting that cAMP-mediated Akt activation, although modest and transient, is PI3-K dependent. In contrast, Akt activation was not detectable in WIF-B9 cells at any time (Fig. 3C). EGF also failed to activate Akt, possibly because WIF-B9 cells lack the EGF receptor. WIF-B9 cells possess a functional insulin receptor, and Akt was activated by insulin (13.7-fold). Similar events were observed in ERK1/2 activation by cAMP, namely, cAMP transiently activated ERK1/2 by 2.5-fold in COS-7 cells (Fig. 3B), consistent with a previous report (9). There was no activation of ERK1/2 in WIF-B9 cells (Fig. 3D). Insulin also activated ERK1/2 in WIF-B9 cells. Higher concentrations of cAMP (up to 1 mM) did not activate Akt or ERK1/2 in WIF-B9 cells (data not shown).


View larger version (37K):
[in this window]
[in a new window]
 
Fig. 3.   cAMP activates Akt and ERK1/2 in COS-7 but not WIF-B9 cells. Cell lysates (20 µg) from COS-7 (A and B) and WIF-B9 cells (C and D) were subjected to SDS-PAGE and analyzed by immunoblotting with anti-phospho-Akt (pAkt), anti-Akt (A and C), anti-phospho-ERK1/2 (pERK1/2) or anti-ERK1/2 antibodies (B and D). EGF (10 ng/ml) or insulin (10 ng/ml)-treated cells were used as controls. Wortmannin (WM, 100 nM) was added for 30 min before the addition of cAMP (A). Relative intensity represents the mean ratio of intensity of phosphorylated proteins relative to total protein from 6 independent experiments. *P < 0.05 vs. basal level (paired t-test).

p110 Catalytic subunit-specific PI3-K activity and expression of each subunit. To determine why cAMP did not activate Akt in WIF-B9 cells despite cAMP activation of p85-associated PI3-K, we characterized expression of PI3-K catalytic subunits by measuring catalytic subunit-associated PI3-K activity and protein expression by Western blotting. cAMP increased p110alpha -associated PI3-K activity in COS-7 cells (3-fold) and in WIF-B9 cells (2.5-fold) (Fig. 4A). p110beta -associated PI3-K activity increased 1.7-fold in COS-7 cells, whereas basal and cAMP-stimulated activities in immunoprecipitates were substantially lower in WIF-B9 cells. PI3-K activity associated with p110gamma was undetectable in both cell lines even in the presence of cAMP. These results are not due to an inhibitory effect of anti-p110gamma antibodies on PI3-K because p110gamma -associated PI3-K activity was detected in Jurkat cells. Immunoblotting revealed p110alpha expression in cell lysates and p85 immunoprecipitates in COS-7 and WIF-B9 cells (Fig. 4B). In contrast, p110beta was expressed only in COS-7 cells in concordance with the activity assay data described above. No protein was detected in immunoprecipitates with normal rabbit IgG, which confirmed specificity of the results. Expression of p110gamma was detected only in Jurkat cells, an acute T cell leukemia cell line, confirming that expression of p110gamma , originally cloned from a bone marrow cDNA library (49), is relatively restricted to hematopoietic cells. The class 1A PI3-K catalytic subunit, p110delta , was not tested because its expression is restricted to leukocytes (52). These results reveal that COS-7 cells express p110alpha and p110beta , whereas WIF-B9 cells only express p110alpha .


View larger version (34K):
[in this window]
[in a new window]
 
Fig. 4.   Activation and expression of individual PI3-K catalytic subunits in COS-7 and WIF-B9 cells. A: cells were incubated in the absence or presence of cAMP (100 µM) for 15 min. PI3-K activity was determined in cell lysates after immunoprecipitation with antibodies to p110alpha , -beta , or -gamma . PI3-K activity in whole cell lysates from HEK-293 was used as a standard as indicated in MATERIALS AND METHODS. Each value represents the mean ± SE from 3 independent experiments. B: expression of each catalytic subunit was determined by immunoblotting. Total cell lysates (20 µg) or anti-p85 immunoprecipitates from 200 µg of protein were separated by SDS-PAGE, blotted onto polyvinylidene difluoride (PVDF) membranes, and probed with antibodies to p110alpha , -beta , or -gamma . Extracts from Jurkat cells were used as a control for p110gamma antibodies.

cAMP-mediated PI3-K activation was sensitive to an inhibitor of PKA, KT-5720. KT-5720, an inhibitor of PKA, did not affect basal PI3-K activity but decreased cAMP-mediated PI3-K activity by 74% in COS-7 and 79% in WIF-B9 cells (Fig. 5A). Addition of PKA catalytic subunit to PI3-K in vitro did not change PI3-K activity (data not shown). Src kinase can be activated by cAMP and bind to and activate PI3-K (14). We found that cAMP induces activation of Src in COS-7 cells (data not shown). However, PP2, a Src inhibitor, had no effect on PI3-K activity (Fig. 5B). Because several reports indicate cross talk between Gs and Gi signaling (4), we also investigated the involvement of Gi signaling with PTX. PTX also failed to block cAMP-mediated PI3-K activation (Fig. 5B), suggesting that neither Src nor Gi signaling is involved.


View larger version (44K):
[in this window]
[in a new window]
 
Fig. 5.   cAMP-induced PI3-K activation is KT-5720 sensitive. COS-7 and WIF-B9 cells were treated with KT-5720 (10 µM) for 30 min (A) or pertussis toxin (PTX; 100 ng/ml) overnight or PP2 (10 µg/ml) for 1 h (B) before stimulation with cAMP (100 µM) for 15 min. Whole cell lysates (200 µg) were immunoprecipitated with anti-p85 antibodies, and PI3-K activity was assayed as in Fig. 1. Each value represents the mean ± SE from 3-6 independent experiments. *P < 0.05 vs. cAMP alone (paired t-test).

Role of Ras in cAMP-mediated PI3-K activation. Ras induces PI3-K activation by direct binding to the p110 catalytic subunits (41) in a phosphotyrosine-independent manner. Addition of cAMP to COS-7 cells increased GTP-bound Ras in 15-30 min [2.2 ± 0.4 (mean ± SE)-fold increase at 30 min; P < 0.05 compared with basal level; Fig. 6A]. To determine whether Ras activation in COS-7 cells is upstream of PI3-K, we determined the effect of wortmannin on cAMP-induced Ras activation. Wortmannin did not affect cAMP-mediated Ras activation (Fig. 6B). In WIF-B9 cells, cAMP did not activate Ras; however, in the presence of cAMP, wortmannin reduced Ras activation to below control levels (Fig. 6B). These data suggest that, in COS-7 cells, Ras participates in PI3-K activation and is either upstream or independent. In contrast, cAMP-mediated increase in PI3-K activity in WIF-B9 cells does not involve activation of Ras.


View larger version (39K):
[in this window]
[in a new window]
 
Fig. 6.   cAMP activates PI3-K in a Ras-dependent manner in COS-7 cells. Cells were stimulated with cAMP (100 µM) for indicated times, and 400 µg of cell lysates were incubated with glutathione agarose bound to the Ras-binding domain of Raf as described in MATERIALS AND METHODS. The GTP-bound Ras was detected by anti-Ras antibodies (A, top; active Ras). To confirm that an equal amount of Ras was present in each sample, 20 µg of each cell lysate were immunoblotted with anti-Ras antibodies (A, bottom; total Ras). B: cells were treated with wortmannin (100 nM) for 30 min before stimulation with cAMP (100 µM) for 30 min. Activated Ras was detected as in A. C: COS-7 cells were transfected with RasN17 and stimulated with cAMP (100 µM) for 30 min or with EGF (10 ng/ml) for 15 min. Two hundred micrograms of whole cell lysates were immunoprecipitated with anti-p85 antibodies, and PI3-K activity was determined. Each value represents the mean ± SE obtained from 3 independent experiments. *P < 0.05 vs. cAMP alone (paired t-test). Whole cell lysates (20 µg) were also immunoblotted with anti-Ras antibodies to confirm the expression of Ras (C, right).

To determine whether cAMP-induced PI3-K activation is dependent of Ras, we transfected COS-7 cells with dominant-negative Ras (Ras17N). Figure 6C, right, shows overexpression of immunoreactive Ras in transfected cells. Transfection of Ras17N did not affect EGF-mediated PI3-K activation, which is consistent with recruitment of p85 to tyrosine-phosphorylated proteins in EGF-mediated activation of PI3-K (Ref. 55; Fig. 6C, left). In contrast, transfection of Ras17N abolished cAMP-mediated PI3-K activation, suggesting that Ras is upstream of PI3-K after stimulation with cAMP in COS-7 cells. Similar experiments could not be performed in WIF-B9 cells because, as we (42) and others have observed, conventional techniques for transient transfection of WIF-B9 cells produce insufficient yield for biochemical studies.

Gbeta gamma involvement in cAMP-mediated PI3-K activation. Gbeta gamma is reported to stimulate PI3-K in many cell types (27, 47, 49, 51). We used an adenoviral vector that expresses beta -ARKct to inhibit signaling through Gbeta gamma . beta -ARKct specifically binds to and sequesters Gbeta gamma complexes (7). Cells were infected with the adenovirus, and the effect of cAMP on PI3-K activation was quantified. The expression of beta -ARKct was confirmed by Western blotting (Fig. 7A, top). Whereas control adenovirus infection had no effect, beta -ARKct infection attenuated cAMP-mediated PI3-K activation by 68% and 70% in COS-7 and WIF-B9 cells, respectively (Fig. 7A, bottom), suggesting involvement of Gbeta gamma in this process. We also determined whether Gbeta gamma is required for cAMP-induced Ras activation in COS-7 cells (Fig. 7B). Expression of beta -ARKct blocked cAMP-mediated activation of Ras, whereas infection with control adenovirus did not affect Ras activation [fold increase: 1.8 ± 0.3 (mean ± SE) for cAMP + empty adenovirus and 0.9 ± 0.2 for cAMP + beta -ARKct; P < 0.05]. Similar results were observed with pERK1/2 [fold increase: 2.5 ± 0.5 (mean ± SE) for cAMP + empty adenovirus and 1.2 ± 0.4 for cAMP + beta -ARKct; P < 0.05; Fig. 7B], which provides a positive control for attenuation of Ras function in response to beta -ARKct. These observations suggest that Gbeta gamma is upstream of Ras in response to cAMP in COS-7 cells.


View larger version (37K):
[in this window]
[in a new window]
 
Fig. 7.   cAMP-induced PI3-K activation is Gbeta gamma dependent. A: COS-7 and WIF-B9 cells were infected with adenovirus expressing beta -adrenergic receptor kinase COOH terminus (beta -ARKct) (beta -ARKct AV) at 100 multiplicities of infection (MOI) and incubated for 2 days in complete medium. Empty adenovirus (empty AV) was used as a control. After starvation, cells were stimulated with cAMP (100 µM) for 15 min. PI3-K activity was assayed in whole cell lysates (200 µg) immunoprecipitated with anti-p85 antibodies. Each value represents the mean ± SE from 3 independent experiments. *P < 0.05 vs. cAMP alone (paired t-test). Whole cell lysates (20 µg) were immunoblotted with a beta -ARK antiserum that recognizes the carboxyl terminus of beta -ARK2 (top). B: activation of Ras and ERK1/2 is Gbeta gamma dependent in COS-7 cells. COS-7 cells infected with beta -ARKct AV were stimulated with cAMP (100 µM) for 30 min, and GTP-bound Ras and ERK1/2 phosphorylation were determined. To confirm an equal amount of total Ras in each sample, 20 µg of total cell lysates were immunoblotted with anti-Ras antibodies. Similar studies were performed with antibodies to ERK1/2 or pERK1/2. The blot shown is representative of 3 independent experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

cAMP activated class IA PI3-K in a time- and dose-dependent manner in COS-7 and WIF-B9 cells. There have been few studies determining PI3-K activity in response to cAMP. PI3-K activation was reported in FRTL-5, a rat thyroid follicular cell line (33), and PC12, a pheochromocytoma cell line (37). In our study, increased PI3-K activity was detected at 15 min, remained elevated for 2 h, and declined at 4 h. This time course of response differs from effects observed after addition of growth factors such as PDGF, in which PI3-K activity peaks at 10-15 min and returns to baseline within 1 h (6, 48, 50). On stimulation with growth factors, p85 subunit SH2 domains interact with specific phosphotyrosine motifs (46), resulting in activation of PI3-K activity (8). Tyrosine phosphorylation can be removed quickly by tyrosine phosphatases, which leads to dissociation of p85 from target proteins. In contrast to the response induced by growth factors, cAMP activation of p85-associated PI3-K was independent of phosphotyrosine in both cell lines.

Akt is a downstream target of PI3-K and, in some studies, cAMP-induced Akt activation was blocked by a PI3-K inhibitor, wortmannin (13, 37, 54). However, in 293-EBNA cells, forskolin activation of Akt was PI3-K independent (10), suggesting that Akt response to cAMP may differ by cell type. Although cAMP increased PI3-K activity in both COS-7 and WIF-B9 cells, Akt activation was detected only in COS-7 cells and was inhibited by nanomolar concentrations of wortmannin. In WIF-B9 cells, insulin but not cAMP activated Akt, which suggests that different pools of PI3-K are utilized. Because cAMP activates Akt in hepatocytes (28, 54, 56), WIF-B9 cells may lack intermediate molecules required for cAMP-mediated Akt activation in response to PI3-K. Additional observations suggest that PI3-K activation by cAMP may not be directly linked to Akt. In both cell lines, maximal PI3-K activation was observed at 60 min (Fig. 1, C and D) whereas maximal phosphorylation of Akt occurred at 15 min and declined to baseline levels by 60 min (Fig. 3, A and B).

To examine mechanisms of activation based on PI3-K catalytic subunits, we determined expression of p110 isoforms in COS-7 and WIF-B9 cells. In COS-7 cells p110alpha and p110beta catalytic subunits were expressed, whereas only p110alpha was detectable in WIF-B9 cells. These data suggest that activation of p110alpha is insufficient for transduction of some PI3-K downstream signals, such as Akt. Because p110beta was originally purified and cloned from rat liver and is widely expressed, the lack of p110beta expression in WIF-B9 was unexpected. Expression of p110beta may be lost during fusion or subsequent passages, similar to loss of p110gamma expression in colon cancer cell lines despite expression of this protein in colonic epithelium (43). Lysophosphatidic acid- and carbachol-induced Akt activation require p110beta , which suggests that p110beta expression may be essential for transducing cAMP signaling to Akt in WIF-B9 cells.

Gbeta gamma specifically activates p110gamma (47) and p110beta (19, 27, 30). Blocking downstream signaling from heterotrimeric G proteins by expression of beta -ARKct, which specifically binds to and sequesters Gbeta gamma complexes (7), impaired activation of PI3-K by cAMP in both cell lines. Because WIF-B9 cells express only p110alpha , it is likely that Gbeta gamma involvement is indirect. PI3-K activation was also sensitive to KT-5720, suggesting involvement of PKA. The molecular mechanism whereby PKA leads to release of Gbeta gamma is not well understood; a signal switch to Gi from Gs has been proposed in response to beta 2-adrenergic receptor stimulation (4). Because PTX did not block cAMP-induced PI3-K activation, other pathways probably exist. For example, reactive oxygen species can induce Gbeta gamma liberation without activating G protein-coupled receptors (34).

An additional mechanism for PI3-K activation is activation of Ras. GTP-bound Ras can directly bind to PI3-K catalytic subunits, resulting in conformational changes and increased catalytic activity (40). Ras can also be a downstream target of PI3-K (17, 55). cAMP induced an increase in GTP-bound Ras in COS-7 and melanocytes (1) but not in WIF-B9 cells. Activation of Ras in COS-7 cells was not blocked by wortmannin, and expression of the dominant-negative Ras construct (RasN17) abolished PI3-K activation by cAMP. These data suggest that Ras activation is upstream of PI3-K in COS-7 cells and is required for cAMP-mediated PI3-K activation. These observations are also consistent with activation of ERK1/2 by cAMP, which was not sensitive to wortmannin (data not shown). Several mechanisms have been proposed for Ras activation in response to cAMP. cAMP-GEFs can directly activate Rap1/2 (5, 22), and a cyclic nucleotide Ras-GEF, CDS-25Mm, which is phosphorylated by PKA, has also been described (36). However, expression of these GEFs has been found specifically in brain. Other, as yet unidentified, molecules may function as GEFs for Ras in response to cAMP in other cells. Ras and ERK1/2 activation were blocked by expression of beta -ARKct, which suggests that Gbeta gamma release is required for Ras activation in COS-7 cells. Although the molecular mechanisms are not well defined, Gbeta gamma release may activate Ras by initiating assembly of a multiprotein complex including beta -arrestin or c-Src in clathrin-coated pits (29, 38).

A recent report showed that cAMP had an opposite effect in COS cells that overexpress PI3-K and Akt (23). Under these conditions, cAMP blocked PI3-K and Akt activation. The difference between our data and these results may be due to the use of different clones of COS cells or to the fact that overexpression of these proteins leads to a different response.

In conclusion, cAMP activated PI3-K in COS-7 and WIF-B9 cells and the pathway leading to PI3-K activation was cell type specific. PKA and Gbeta gamma were required in both cell lines; however, the response in COS-7 cells required Ras activation whereas the response in WIF-B9 cells did not. Our study indicates that there are different pathways by which cAMP activates PI3-K. How these individual mechanisms relate to specific cellular responses, such as bile acid secretion, requires additional studies, which are in progress.


    ACKNOWLEDGEMENTS

This research was supported in part by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-54785 (to I. M. Arias) and 30-DK-34928 (Digestive Disease Center).


    FOOTNOTES

Address for reprint requests and other correspondence: I. M. Arias, Dept. of Physiology, Tufts Univ. School of Medicine, 136 Harrison Ave., Boston, MA 02111 (E-mail: irwin.arias{at}tufts.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

August 7, 2002;10.1152/ajpcell.00041.2002

Received 28 January 2002; accepted in final form 24 July 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Busca, R, Abbe P, Mantoux F, Aberdam E, Peyssonnaux C, Eychene A, Ortonne JP, and Ballotti R. Ras mediates the cAMP-dependent activation of extracellular signal-regulated kinases (ERKs) in melanocytes. EMBO J 19: 2900-2910, 2000[Abstract/Free Full Text].

2.   Cass, LA, Summers SA, Prendergast GV, Backer JM, Birnbaum MJ, and Meinkoth JL. Protein kinase A-dependent and -independent signaling pathways contribute to cyclic AMP-stimulated proliferation. Mol Cell Biol 19: 5882-5891, 1999[Abstract/Free Full Text].

3.   Crowder, RJ, and Freeman RS. The survival of sympathetic neurons promoted by potassium depolarization, but not by cyclic AMP, requires phosphatidylinositol 3-kinase and Akt. J Neurochem 73: 466-475, 1999[ISI][Medline].

4.   Daaka, Y, Luttrell LM, and Lefkowitz RJ. Switching of the coupling of the beta2-adrenergic receptor to different G proteins by protein kinase A. Nature 390: 88-91, 1997[ISI][Medline].

5.   De Rooij, J, Zwartkruis FJ, Verheijen MH, Cool RH, Nijman SM, Wittinghofer A, and Bos JL. Epac is a Rap1 guanine-nucleotide-exchange factor directly activated by cyclic AMP. Nature 396: 474-477, 1998[ISI][Medline].

6.   Domin, J, Dhand R, and Waterfield MD. Binding to the platelet-derived growth factor receptor transiently activates the p85alpha -p110alpha phosphoinositide 3-kinase complex in vivo. J Biol Chem 271: 21614-21621, 1996[Abstract/Free Full Text].

7.   Drazner, MH, Peppel KC, Dyer S, Grant AO, Koch WJ, and Lefkowitz RJ. Potentiation of beta-adrenergic signaling by adenoviral-mediated gene transfer in adult rabbit ventricular myocytes. J Clin Invest 99: 288-296, 1997[Abstract/Free Full Text].

8.   Fantl, WJ, Escobedo JA, Martin GA, Turck CW, del Rosario M, McCormick F, and Williams LT. Distinct phosphotyrosines on a growth factor receptor bind to specific molecules that mediate different signaling pathways. Cell 69: 413-423, 1992[ISI][Medline].

9.   Faure, M, Voyno-Yasenetskaya TA, and Bourne HR. cAMP and beta gamma subunits of heterotrimeric G proteins stimulate the mitogen-activated protein kinase pathway in COS-7 cells. J Biol Chem 269: 7851-7854, 1994[Abstract/Free Full Text].

10.   Filippa, N, Sable CL, Filloux C, Hemmings B, and Van Obberghen E. Mechanism of protein kinase B activation by cyclic AMP-dependent protein kinase. Mol Cell Biol 19: 4989-5000, 1999[Abstract/Free Full Text].

11.   Gatmaitan, ZC, Nies AT, and Arias IM. Regulation and translocation of ATP-dependent apical membrane proteins in rat liver. Am J Physiol Gastrointest Liver Physiol 272: G1041-G1049, 1997[Abstract/Free Full Text].

12.   Goi, T, Rusanescu G, Urano T, and Feig LA. Ral-specific guanine nucleotide exchange factor activity opposes other Ras effectors in PC12 cells by inhibiting neurite outgrowth. Mol Cell Biol 19: 1731-1741, 1999[Abstract/Free Full Text].

13.   Gonzalez-Robayna, IJ, Falender AE, Ochsner S, Firestone GL, and Richards JS. Follicle-stimulating hormone (FSH) stimulates phosphorylation and activation of protein kinase B (PKB/Akt) and serum and glucocorticoid-induced kinase (Sgk): evidence for A kinase-independent signaling by FSH in granulosa cells. Mol Endocrinol 14: 1283-1300, 2000[Abstract/Free Full Text].

14.   Haefner, B, Baxter R, Fincham VJ, Downes CP, and Frame MC. Cooperation of Src homology domains in the regulated binding of phosphatidylinositol 3-kinase. A role for the Src homology 2 domain. J Biol Chem 270: 7937-7943, 1995[Abstract/Free Full Text].

15.   Hartwig, JH, Kung S, Kovacsovics T, Janmey PA, Cantley LC, Stossel TP, and Toker A. D3 phosphoinositides and outside-in integrin signaling by glycoprotein IIb-IIIa mediate platelet actin assembly and filopodial extension induced by phorbol 12-myristate 13-acetate. J Biol Chem 271: 32986-32993, 1996[Abstract/Free Full Text].

16.   Holzinger, F, Schteingart CD, Ton-Nu HT, Cerre C, Steinbach JH, Yeh HZ, and Hofmann AF. Transport of fluorescent bile acids by the isolated perfused rat liver: kinetics, sequestration, and mobilization. Hepatology 28: 510-520, 1998[ISI][Medline].

17.   Hu, Q, Klippel A, Muslin AJ, Fantl WJ, and Williams LT. Ras-dependent induction of cellular responses by constitutively active phosphatidylinositol-3 kinase. Science 268: 100-102, 1995[ISI][Medline].

18.   Hubner, S, Couvillon AD, Kas JA, Bankaitis VA, Vegners R, Carpenter CL, and Janmey PA. Enhancement of phosphoinositide 3-kinase (PI 3-kinase) activity by membrane curvature and inositol-phospholipid-binding peptides. Eur J Biochem 258: 846-853, 1998[Abstract].

19.   Igarashi, J, Bernier SG, and Michel T. Sphingosine 1-phosphate and activation of endothelial nitric-oxide synthase. Differential regulation of Akt and MAP kinase pathways by EDG and bradykinin receptors in vascular endothelial cells. J Biol Chem 276: 12420-12426, 2001[Abstract/Free Full Text].

20.   Ihrke, G, Neufeld EB, Meads T, Shanks MR, Cassio D, Laurent M, Schroer TA, Pagano RE, and Hubbard AL. WIF-B cells: an in vitro model for studies of hepatocyte polarity. J Cell Biol 123: 1761-1775, 1993[Abstract].

21.   Kagawa, T, Misra S, Varticovski L, and Arias IM. The mechanisms whereby cAMP activates PI 3-kinase which regulates canalicular spgp (Abstract). Hepatology 32: 306A, 2000.

22.   Kawasaki, H, Springett GM, Mochizuki N, Toki S, Nakaya M, Matsuda M, Housman DE, and Graybiel AM. A family of cAMP-binding proteins that directly activate Rap1. Science 282: 2275-2279, 1998[Abstract/Free Full Text].

23.   Kim, S, Jee K, Kim D, Koh H, and Chung J. Cyclic AMP inhibits Akt activity by blocking the membrane localization of PDK1. J Biol Chem 276: 12864-12870, 2001[Abstract/Free Full Text].

24.   Kipp, H, Pichetshote N, and Arias IM. Transporters on demand: intrahepatic pools of canalicular ATP binding cassette transporters in rat liver. J Biol Chem 276: 7218-7224, 2001[Abstract/Free Full Text].

25.   Kitamura, T, Gatmaitan Z, and Arias IM. Serial quantitative image analysis and confocal microscopy of hepatic uptake, intracellular distribution and biliary secretion of a fluorescent bile acid analog in rat hepatocyte doublets. Hepatology 12: 1358-1364, 1990[ISI][Medline].

26.   Koch, WJ, Hawes BE, Inglese J, Luttrell LM, and Lefkowitz RJ. Cellular expression of the carboxyl terminus of a G protein-coupled receptor kinase attenuates G beta gamma-mediated signaling. J Biol Chem 269: 6193-6197, 1994[Abstract/Free Full Text].

27.   Kurosu, H, Maehama T, Okada T, Yamamoto T, Hoshino S, Fukui Y, Ui M, Hazeki O, and Katada T. Heterodimeric phosphoinositide 3-kinase consisting of p85 and p110beta is synergistically activated by the beta gamma subunits of G proteins and phosphotyrosyl peptide. J Biol Chem 272: 24252-24256, 1997[Abstract/Free Full Text].

28.   Li, J, Yang S, and Billiar TR. Cyclic nucleotides suppress tumor necrosis factor alpha -mediated apoptosis by inhibiting caspase activation and cytochrome c release in primary hepatocytes via a mechanism independent of Akt activation. J Biol Chem 275: 13026-13034, 2000[Abstract/Free Full Text].

29.   Luttrell, LM, Ferguson SS, Daaka Y, Miller WE, Maudsley S, Della Rocca GJ, Lin F, Kawakatsu H, Owada K, Luttrell DK, Caron MG, and Lefkowitz RJ. Beta-arrestin-dependent formation of beta2 adrenergic receptor-Src protein kinase complexes. Science 283: 655-661, 1999[Abstract/Free Full Text].

30.   Maier, U, Babich A, and Nurnberg B. Roles of non-catalytic subunits in Gbeta gamma -induced activation of class I phosphoinositide 3-kinase isoforms beta  and gamma . J Biol Chem 274: 29311-29317, 1999[Abstract/Free Full Text].

31.   Misra, S, Ujhazy P, Gatmaitan Z, Varticovski L, and Arias IM. The role of phosphoinositide 3-kinase in taurocholate-induced trafficking of ATP-dependent canalicular transporters in rat liver. J Biol Chem 273: 26638-26644, 1998[Abstract/Free Full Text].

32.   Misra, S, Ujhazy P, Varticovski L, and Arias IM. Phosphoinositide 3-kinase lipid products regulate ATP-dependent transport by sister of P-glycoprotein and multidrug resistance associated protein 2 in bile canalicular membrane vesicles. Proc Natl Acad Sci USA 96: 5814-5819, 1999[Abstract/Free Full Text].

33.   Nedachi, T, Akahori M, Ariga M, Sakamoto H, Suzuki N, Umesaki K, Hakuno F, and Takahashi SI. Tyrosine kinase and phosphatidylinositol 3-kinase activation are required for cyclic adenosine 3',5'-monophosphate-dependent potentiation of deoxyribonucleic acid synthesis induced by insulin-like growth factor-I in FRTL-5 cells. Endocrinology 141: 2429-2438, 2000[Abstract/Free Full Text].

34.   Nishida, M, Maruyama Y, Tanaka R, Kontani K, Nagao T, and Kurose H. G alpha(i) and G alpha(o) are target proteins of reactive oxygen species. Nature 408: 492-495, 2000[ISI][Medline].

35.   Perrotti, N, He RA, Phillips SA, Haft CR, and Taylor SI. Activation of serum- and glucocorticoid-induced protein kinase (Sgk) by cyclic AMP and insulin. J Biol Chem 276: 9406-9412, 2001[Abstract/Free Full Text].

36.   Pham, N, Cheglakov I, Koch CA, de Hoog CL, Moran MF, and Rotin D. The guanine nucleotide exchange factor CNrasGEF activates ras in response to cAMP and cGMP. Curr Biol 10: 555-558, 2000[ISI][Medline].

37.   Powers, JF, Misra S, Schelling K, Varticovski L, and Tischler AS. Mitogenic signaling by cyclic adenosine monophosphate in chromaffin cells involves phosphatidylinositol 3-kinase activation. J Cell Biochem Suppl 36: 89-98, 2001.

38.   Pumiglia, KM, LeVine H, Haske T, Habib T, Jove R, and Decker SJ. A direct interaction between G-protein beta gamma subunits and the Raf-1 protein kinase. J Biol Chem 270: 14251-14254, 1995[Abstract/Free Full Text].

39.   Richards, JS. New signaling pathways for hormones and cyclic adenosine 3',5'-monophosphate action in endocrine cells. Mol Endocrinol 15: 209-218, 2001[Abstract/Free Full Text].

40.   Rodriguez-Viciana, P, Warne PH, Dhand R, Vanhaesebroeck B, Gout I, Fry MJ, Waterfield MD, and Downward J. Phosphatidylinositol-3-OH kinase as a direct target of Ras. Nature 370: 527-532, 1994[ISI][Medline].

41.   Rodriguez-Viciana, P, Warne PH, Khwaja A, Marte BM, Pappin D, Das P, Waterfield MD, Ridley A, and Downward J. Role of phosphoinositide 3-OH kinase in cell transformation and control of the actin cytoskeleton by Ras. Cell 89: 457-467, 1997[ISI][Medline].

42.   Sai, Y, Nies AT, and Arias IM. Bile acid secretion and direct targeting of mdr1-green fluorescent protein from Golgi to the canalicular membrane in polarized WIF-B cells. J Cell Sci 112: 4535-4545, 1999[Abstract/Free Full Text].

43.   Sasaki, T, Irie-Sasaki J, Horie Y, Bachmaier K, Fata JE, Li M, Suzuki A, Bouchard D, Ho A, Redston M, Gallinger S, Khokha R, Mak TW, Hawkins PT, Stephens L, Scherer SW, Tsao M, and Penninger JM. Colorectal carcinomas in mice lacking the catalytic subunit of PI(3)Kgamma. Nature 406: 897-902, 2000[ISI][Medline].

44.   Servillo, G, Della Fazia MA, and Sassone-Corti P. Cyclic AMP signaling in the liver: coupling transcription to physiology and proliferation. In: The Liver: Biology and Pathobiology (4th ed.), edited by Arias IM, Boyer JL, Chisari FV, Fausto N, Schachter D, and Shafritz DA.. Philadelphia, PA: Lippincott Williams and Wilkins, 2001, p. 525-536.

45.   Shanks, MR, Cassio D, Lecoq O, and Hubbard AL. An improved polarized rat hepatoma hybrid cell line. Generation and comparison with its hepatoma relatives and hepatocytes in vivo. J Cell Sci 107: 813-825, 1994[Abstract/Free Full Text].

46.   Songyang, Z, Shoelson SE, McGlade J, Olivier P, Pawson T, Bustelo XR, Barbacid M, Sabe H, Hanafusa H, Yi T, and Cantley LC. Specific motifs recognized by the SH2 domains of Csk, 3BP2, fps/fes, GRB-2, HCP, SHC, Syk, and Vav. Mol Cell Biol 14: 2777-2785, 1994[Abstract].

47.   Stephens, L, Smrcka A, Cooke FT, Jackson TR, Sternweis PC, and Hawkins PT. A novel phosphoinositide 3 kinase activity in myeloid-derived cells is activated by G protein beta gamma subunits. Cell 77: 83-93, 1994[ISI][Medline].

48.   Stephens, LR, Jackson TR, and Hawkins PT. Agonist-stimulated synthesis of phosphatidylinositol(3,4,5)-trisphosphate: a new intracellular signalling system? Biochim Biophys Acta 1179: 27-75, 1993[ISI][Medline].

49.   Stoyanov, B, Volinia S, Hanck T, Rubio I, Loubtchenkov M, Malek D, Stoyanova S, Vanhaesebroeck B, Dhand R, Nurnberg B, Gierschik P, Seedorf K, Hsuan JJ, Waterfield MD, and Wetzker R. Cloning and characterization of a G protein-activated human phosphoinositide-3 kinase. Science 269: 690-693, 1995[ISI][Medline].

50.   Susa, M, Keeler M, and Varticovski L. Platelet-derived growth factor activates membrane-associated phosphatidylinositol 3-kinase and mediates its translocation from the cytosol. Detection of enzyme activity in detergent-solubilized cell extracts. J Biol Chem 267: 22951-22956, 1992[Abstract/Free Full Text].

51.   Thomason, PA, James SR, Casey PJ, and Downes CP. A G-protein beta gamma-subunit-responsive phosphoinositide 3-kinase activity in human platelet cytosol. J Biol Chem 269: 16525-16528, 1994[Abstract/Free Full Text].

52.   Vanhaesebroeck, B, and Waterfield MD. Signaling by distinct classes of phosphoinositide 3-kinases. Exp Cell Res 253: 239-254, 1999[ISI][Medline].

53.   Wakabayashi, Y, and Arias IM. Microtubular-dependent bidirectional trafficking of BSEP in polarized WIF-B9 cells (Abstract). Hepatology 34: 258A, 2001.

54.   Webster, CR, and Anwer MS. Role of the PI3K/PKB signaling pathway in cAMP-mediated translocation of rat liver Ntcp. Am J Physiol Gastrointest Liver Physiol 277: G1165-G1172, 1999[Abstract/Free Full Text].

55.   Yart, A, Laffargue M, Mayeux P, Chretien S, Peres C, Tonks N, Roche S, Payrastre B, Chap H, and Raynal P. A critical role for phosphoinositide 3-kinase upstream of Gab1 and SHP2 in the activation of ras and mitogen-activated protein kinases by epidermal growth factor. J Biol Chem 276: 8856-8864, 2001[Abstract/Free Full Text].

56.   Zhao, AZ, Shinohara MM, Huang D, Shimizu M, Eldar-Finkelman H, Krebs EG, Beavo JA, and Bornfeldt KE. Leptin induces insulin-like signaling that antagonizes cAMP elevation by glucagon in hepatocytes. J Biol Chem 275: 11348-11354, 2000[Abstract/Free Full Text].


Am J Physiol Cell Physiol 283(6):C1655-C1666
0363-6143/02 $5.00 Copyright © 2002 the American Physiological Society