Ciprofibrate stimulates protein kinase C-dependent phosphorylation of an 85 kDa protein in rat Fao hepatic derived cells

Biochimie 82 (2000) 749−753 © 2000 Société française de biochimie et biologie moléculaire / Éditions scientifiques et médicales Elsevier SAS. All rights reserved. S0300908400011573/FLA

Ciprofibrate stimulates protein kinase C-dependent phosphorylation of an 85 kDa protein in rat Fao hepatic derived cells Patricia Passilly-Degrace*, Brigitte Jannin, Daniel Boscoboinik**, Kiyoto Motojima***, Norbert Latruffe**** Laboratoire de Biologie Moléculaire et Cellulaire, Université de Bourgogne, Faculté des Sciences Gabriel, 6, boulevard Gabriel, 21000 Dijon, France (Received 21 January 2000; accepted 21 April 2000) Abstract — The effect of ciprofibrate on early events of signal transduction was previously studied in Fao cells. Protein kinase C (PKC) assays performed on permeabilized cells showed a more than two-fold increase in PKC activity in cells treated for 24 h with 500 µM ciprofibrate. To show the subsequent effect of this increase on protein phosphorylation, the in vitro phosphorylation on particulate fractions obtained from Fao cells was studied. Among several modifications, the phosphorylation of protein(s) with an apparent molecular mass of 85 kDa was investigated. This modification appeared in the first 24 h of treatment with 500 µM ciprofibrate. It was shown to occur on Ser/Thr residue(s). It was calcium but not calmodulin-dependent. The phosphorylation level of this/these protein(s) was reduced with kinase inhibitors and especially with 300 nM GF-109203X, a specific inhibitor of PKC. All these results suggest that the phosphorylation of the 85 kDa protein(s) is due to a PKC or to another Ser/Thr kinase activated via a PKC pathway. A possible biochemical candidate for 85 kDa protein seems to be the β isoform of phosphatidylinositol 3-kinase regulatory subunit. © 2000 Société française de biochimie et biologie moléculaire / Éditions scientifiques et médicales Elsevier SAS Fao cell line / ciprofibrate / protein phosphorylation / protein kinase C / signal transduction

1. Introduction Peroxisome proliferators and especially hypolipidaemic drugs such as ciprofibrate are known to be hepatocarcinogens in rodents. In spite of their structural diversity, peroxisome proliferators are believed to act through a common mechanism. The transcription of several genes is enhanced in the presence of these compounds and involves a PPAR [2, 3]. Although peroxisome proliferator compounds may directly activate the nuclearlocated PPAR [4], it is also possible that these drugs act via a signal-transduction pathway. We investigated the effects of ciprofibrate on signal transduction in a well-differentiated rat hepatoma cell line, Fao, known to respond to peroxisome proliferators [5]. The analysis of protein phosphorylation in intact cells with [32P] orthophosphate showed that ciprofibrate stimulates the phosphorylation level of several proteins [1]. The aim of the present study was to show the effect of ciprofibrate on PKC activation in Fao whole cells and to * Present adress: Université de Strasbourg I, Illkirch, France. ** Present adress: University of Bern, Switzerland. *** Present adress: Toho University, Chiba, Japan. **** Correspondence and reprints: [email protected] Abbreviations: DMEM, Dulbecco’s modified Eagle’s medium; DMSO, dimethylsulfoxide; PI3-K, phosphatidylinositol 3-kinase; PKC, protein kinase C; PPAR, peroxisome proliferator-activated receptor

search for targets for this enzyme by in vitro phosphorylation on subcellular fractions obtained from Fao cultured cells. 2. Materials and methods 2.1. Chemicals Ciprofibrate {2-[4-(2,2-dichlorocyclopropyl)phenoxy]2-methylpropanoic acid} was a gift from Sterling Winthrop (Dijon, France). Protease inhibitors (PMSF, leupeptin, aprotinin and pepstatin A) were purchased from Sigma Chemical Co. (L’Isle d’Abeau Chesnes, France). Streptolysin-O, GF 109203X (bisindolylmaleimide I) and Gö 6976 [12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13methyl-5-oxo-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole] were purchased from Alexis L.C. Laboratories (San Diego, USA). PKC substrate (peptide derived from glycogen synthase) was purchased from Bachem (Bubendorf, Switzerland). [γ-32P]ATP was obtained from Amersham (Little Chaffont, UK). Ham F12 medium, DMEM and fetal calf serum were obtained from Gibco-BRL (Life Technologies, France). 2.2. Cells The Fao cell line was routinely cultured in Ham F12: DMEM (1:1, by vol.) medium supplemented with 5%

750 (v/v) fetal calf serum at 37 °C in a humidified atmosphere of 10% CO2/90% air as previously described [1]. Medium was supplemented with 125 IU/mL specillin G and 125 µg/mL streptomycin. For cell treatment, peroxisome proliferators were dissolved in DMSO and added to the media at the appropriate concentration. Control cells received solvent at the same final concentration (0.1%). 2.3. PKC assays in permeabilized cells Cells were grown in 25 cm2 flasks (Falcon) and at 90% confluence were trypsinated and collected by centrifugation (100 g for 10 min). The cell pellet was washed once with PBS, once with an intracellular buffer (5.16 mM MgCl2, 94 mM KCl, 12.5 mM Hepes pH 7.4, 12.5 mM EGTA and 8.17 mM CaCl2), resuspended in this buffer and aliquoted in 185 µL portions (approximately 5 × 105 cells) for the PKC assays. The peptide substrate was added to the final concentration of 66 µM without or with 100 nM Gö 6976, a specific inhibitor of classical PKC isoforms. The permeabilization of Fao cells, first monitored by the lactate dehydrogenase release, was achieved with 2.8 UI/mL streptolysin O for 7 min in ice, given that in these conditions 85% of the lactate dehydrogenase activity was recovered in the medium. Mixtures were incubated for 7 min on ice and for 3 min at 37 °C just before the addition of labeled ATP. Assays were started by adding 20 µL of [γ-32P] ATP (approximately 50 cpm/pmol) to a final concentration of 250 µM of ATP and reaction mixture was incubated at 37 °C for 10 min. Reactions were stopped by addition of 80 µL of trichloroacetic acid 25% (v/v) in 2 M acetic acid. After 10 min on ice, 80 µL aliquots were spotted onto P81 ion exchange chromatographic paper (Whatman International) within 4.5 cm × 4.5 cm pre-drawn squares. The paper was then washed four times for 10 min with 30% (v/v) acetic acid containing 1% (w/v) phosphoric acid and once with ethanol, dried, and the radioactivity of the squares determined using a liquid scintillation analyzer. The difference between total phosphorylation without Gö 6976 and non-specific phosphorylation with Gö 6976 allowed the determination of the specific activity of PKC. 2.4. Cell fractionation After treatment with ciprofibrate, Fao cells were scrapped, disrupted with a Potter homogenizer and then submitted to cell fractionation according to the protocol described by Motojima et al. [6]. The pellet recovered after the step of centrifugation to 20 000 g, named ‘P1 fraction’ is a particulate fraction which was found to be enriched in mitochondria, lysosomes, peroxisomes and plasma membranes as monitored by the measurement of the specific activities of marker enzymes, glutamate dehy-

Passilly-Degrace et al. drogenase, acid phosphatase, catalase and phosphodiesterase respectively. Protein concentration was determined according to the method of Bradford [7]. The fractions were stored at –80 °C until use. 2.5. In vitro phosphorylation In vitro phosphorylation assay was carried out with 18 µg of P1 fraction proteins in a standard phosphorylation buffer containing 30 mM Tris HCl (pH 7.5), 10 mM MgCl2, 1 mM CaCl2, 0.1 mM dithiothreitol, 0.1% Triton X-100 and phosphatase inhibitors (5 mM sodium fluoride, 3 mM sodium pyrophosphate, 10 µM sodium orthovanadate and 5 mM β-glycerophosphate). The reactions were started by the addition of 1 µM [γ-32P]ATP (approximately 50 cpm/pmol). They were carried out at 30 °C for 2, 5 or 10 min and terminated by the addition of 2 vol of SDS-PAGE sample buffer followed by boiling for 5 min. In some experiments, fractions were incubated 5 or 10 min in ice with protein kinase inhibitors (staurosporine, H-7 or GF-109203X) or calmodulin and/or EGTA before in vitro phosphorylation reactions. Phosphorylated proteins were then separated by SDS-gel electrophoresis using a 10% (w/v) polyacrylamide gel. The 32P-labeled gels were fixed with 40% (v/v) ethanol and 3.7% (v/v) formaldehyde for 30 min and dried. In some experiments, fixed gels were treated with either 7% (v/v) acetic acid at 50 °C for 30 min or 1 M NaOH at 50 °C for 1 h. Autoradiograms were realized using an XO-Mat film from Kodak and scanned using a Shimadzu densitometer (model CS 9000). 3. Results 3.1. In vivo PKC-dependent phosphorylation 3.1.1. PKC activity is increased in Fao cells after treatment with ciprofibrate

We performed several assays of PKC activity on Fao cells untreated or treated with 500 µM ciprofibrate for different times. The permeabilized cells were incubated with [γ-32P]ATP and a peptidic substrate specific for PKC, as described in Materials and methods. The results of a representative experiment are shown in figure 1. The specific activities for control cells and with the substrate used were around 10 pmol per minute and per mg of proteins. No modification of this PKC activity was observed in cells treated for 4 h, whereas increases in PKC activity were found in cells treated for 24 h or 48 h. After 24 h of treatment with 500 µM ciprofibrate, PKC activity was increased 2.3-fold. 3.2. In vitro protein phosphorylation 3.2.1. Ciprofibrate especially increases the level of phosphorylation of an 85 kDa protein

Particulate fractions prepared from Fao cells, untreated or treated for different times with 500 µM ciprofibrate (not

The effect of ciprofibrate on early events of signal transduction

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Figure 1. PKC activity in Fao cells treated with ciprofibrate. Fao cells were treated for 24 h with 500 µM ciprofibrate (cipro) or with solvent alone in control cultures (control). After harvest, PKC assays were performed on intact cells according to the protocol described in Materials and methods. The specific activities, in picomol of phosphate incorporated/min/mg protein, are the mean ± S.E.M. of the values obtained from two different cell dishes, each one assayed in duplicate.

cytotoxic at this concentration upon 48 h [5]) were assayed for the in vitro phosphorylation as described in Materials and methods. Amounts and phosphorylation level of many proteins were not affected by ciprofibrate treatment. However, a protein with an apparent molecular mass of 85 kDa showed a two-fold increase in its phosphorylation level, as estimated by scanning the autoradiograms, upon 24 h or 48 h treatment of cells with ciprofibrate (figure 2). No changes were observed in samples that were treated for 4 h or 12 h with 500 µM ciprofibrate (results not shown). The observation of the gels after Coomassie blue staining showed that the intensity of the band at 85 kDa was similar between control and treated cells (not reported here), indicating that the increase in labeled 85 kDa electrophoretic band is due to an increase of its phosphorylation level rather than an increase in the synthesis of this protein. The increase in the phosphorylation of 85 kDa band was also observed with other peroxisome proliferators, such as Wy-14,643, 2-EHA (2-ethylhexanoic acid) and ibuprofen (data not shown). To determine what is the phosphate acceptor amino acid, phosphoproteins were analyzed on three parallel

Figure 2. Effects of ciprofibrate on the phosphorylation level of 85 kDa protein(s) in the Fao cell line. Fao cells were treated with 500 µM ciprofibrate for 48 h (cipro) or cultured in medium containing solvent alone, i.e., 0.1% DMSO (control) and then harvested and submitted to cell fractionation as described in Materials and methods. Proteins of particulate fractions were submitted to in vitro phosphorylation by [γ-32P]ATP, separated by SDS-PAGE and autoradiographied. This figure represents a typical experiment among several identical ones.

gels, in neutral, acid or alkaline conditions, as described by Passilly et al. [1]. The radioactive 85 kDa band was not affected by acid treatment of the gel whereas it disappeared after alkali treatment (data not shown), indicating that the phosphorylated amino acids is (are) serine and/or threonine, knowing that phosphohistidine is labile in acid and that phosphotyrosine is resistant in both acid and alkali.

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3.2.2. The in vitro phosphorylation of 85 kDa protein(s) requires Ca2+ but not calmodulin 2+

To investigate the involvement of Ca and/or calmodulin-dependent kinases, particulate fractions were incubated at 4 °C with or without 1 µM calmodulin and with or without 5 mM EGTA and submitted to in vitro phosphorylation. Phosphoproteins were separated by SDS-PAGE and autoradiographied (not shown). The radioactive band at 85 kDa disappeared when the samples were preincubated with EGTA. This result shows that 85 kDa protein phosphorylation requires Ca2+. Conversely, in the presence of Ca2+, the radioactive band was the same with or without calmodulin, suggesting that the kinase implicated in 85 kDa protein phosphorylation is not calmodulin-dependent. 3.2.3. The in vitro phosphorylation level of 85 kDa band decreases in the presence of PKC inhibitors

Particulate fractions prepared from untreated or ciprofibrate treated cells were preincubated at 4 °C with several PKC inhibitors (staurosporine, H-7 or GF-109203X) and then submitted to an in vitro phosphorylation assay. SDS-PAGE analysis of the phosphoproteins showed that the 85 kDa band phosphorylation was halved with 5 nM staurosporine or 12.5 µM H-7 and almost completely abolished in presence of 250 nM staurosporine or 120 µM H-7 (data not shown). To confirm the involvement of PKC, a strong specific inhibitor of this kinase, GF109203X, was used. We observed that phosphorylation of 85 kDa band was completely abolished with 300 nM of this inhibitor (not shown).

Moreover, we have shown that, in the Fao cell line, PPARα is a phosphoprotein and that ciprofibrate increases the level of its phosphorylation [12]. Therefore, this modification of the phosphorylation status of PPARα suggests interplay between signal transduction pathways and transcriptional regulatory pathways altered by ciprofibrate. Our PKC assays in whole treated cells showed a two-fold increase in the PKC activity of cells treated for 24 h with ciprofibrate, using a specific substrate. This increase in PKC activity can be associated with an increase in cell proliferation and indeed we have shown by flow cytometry an increase of the proportion of proliferative cells in ciprofibrate treated cells [13]. Moreover, activated PKC has been shown to induce expression of c-myc and c-fos proto-oncogenes [14]. Therefore, the increased expression of these oncogenes after a peroxisome proliferator treatment, already described in our laboratory [15], may be correlated to this increase in PKC activity.

4. Discussion

As already described by in vivo phosphorylation [1], the in vitro phosphorylation experiments on membrane fraction proteins prepared from treated Fao cells showed that ciprofibrate modifies the phosphorylation status of several proteins. It especially increases the phosphorylation of a protein of 85 kDa and this effect is found again by treatment with other peroxisome proliferators. The involvement of Ser/Thr residues, the absolute requirement of Ca2+ and the suppression of phosphorylation by GF109203X, strongly suggest that the kinase implicated in the increased phosphorylation of the protein of 85 kDa belong to the PKC family.

The activation of many kinases by peroxisome proliferators was already reported (for review see [6]), for instance an induction by bezafibrate of the phosphorylation of a nuclear protein by an AMPc dependent protein kinase, an increase in the phosphorylation of elongation factor 2 (substrate of a calcium/calmodulin dependent kinase) in rat liver by peroxisome proliferators or a stimulation of a protein histidine kinase by clofibrate in rat liver. The involvement of PKCs was first demonstrated in in vitro assays by Bronfman et al. [8] who showed that the activity of rat brain PKC was increased by acyl-CoA thioesters of peroxisome proliferators. The effect of peroxisome proliferators in intact cells was studied by Bieri et al. [9] who showed that a staurosporine analogue, a potent and specific inhibitor of PKCs, suppresses the induction of DNA synthesis by nafenopin. In isolated rat hepatocytes, Orellana et al. [10] found an increase in the phosphorylation of epidermal growth factor receptor, a PKC substrate, after treatment with ciprofibrate. Bojes and Thurman [11] showed an increase in PKC activity in liver of rats treated with various peroxisome proliferators.

Two hypotheses may be proposed for the biochemical function of this target protein for PKC found in Fao cells. In view of the coincidence of the apparent molecular mass, this protein may be a MARCKS (myristoylated alanine-rich C kinase substrate) protein, preferential and major cellular substrate for PKC, as suggested in [6] by Motojima et al. or may be the p85 regulatory subunit of phosphatidylinositide 3-kinase (PI3-K). Under effect of insulin, this last protein is translocated to intracellular membrane compartment. Both isoforms α and β were detected by immunoblotting in Fao cells [16]. Immunodetection of subunit α on our Fao cells extracts showed that the amount of this protein was not modified by ciprofibrate (Jennifer Rieusset, personal communication). Moreover, Reif et al. [17] showed that the stimulation of PKC leads to a rapid increase in phosphorylation of p85 β subunit of PI3-K on threonine residues. Therefore, these similarities in apparent molecular mass, localization and PKC-dependent phosphorylation are good arguments in favor of the correspondence of our protein of 85 kDa with p85 β subunit of PI3-K. This hypothesis is under investigation.

The effect of ciprofibrate on early events of signal transduction Since the phosphorylation of 85 kDa protein is linked to the activation of PKC, the increase in 85 kDa labeled band in cell extracts may be a useful indicator of PKC activation. Acknowledgments This work has been supported by grants from the ‘Ligue Bourguignonne contre le Cancer’ and the ‘Conseil Régional de Bourgogne’. We thank Jennifer Rieusset (INSERM U449, Lyon) for immunodetection of p85 subunit of phosphatidylinositol 3-kinase.

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