Chemistry and Physics of Lipids 183 (2014) 68–76

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Interaction of protein phosphatase inhibitors with membrane lipids assessed by surface plasmon resonance based binding technique Bálint Bécsi a,b , Andrea Kiss a , Ferenc ErdÅdi a,b, * a b

Department of Medical Chemistry, Faculty of Medicine, University of Debrecen, H-4032 Debrecen, Hungary MTA-DE Cell Biology and Signaling Research Group, Faculty of Medicine, University of Debrecen, Debrecen, Hungary

A R T I C L E I N F O

A B S T R A C T

Article history: Received 14 January 2014 Received in revised form 25 April 2014 Accepted 28 May 2014 Available online 2 June 2014

The interaction of okadaic acid (OA), tautomycin (TM), microcystin-LR (MC-LR), cantharidin (CA), epigallocatechin-gallate (EGCG) and cyclosporin A (CsA), inhibitors of protein phosphatases, with liposome covered surfaces prepared from the lipid extracts of bovine brain, heart and liver was investigated by surface plasmon resonance (SPR) based binding technique. The SPR sensorgrams indicated reversible association or partial intercalation of the inhibitors with liposomes at 20  C or 37  C, respectively. Distinct lipid composition specificities were reflected in different saturation values of inhibitor binding in a decreasing order of liver > heart >> brain lipids. Assaying the effect of OA, TM, MCLR, CA and EGCG on the activity of protein phosphatases in neuroblastoma B50, cardiomyoblast H9C2 and hepatocarcinoma HepG2 cells implied that the cell type specific association of phosphatase inhibitors with membrane lipids may influence their inhibitory potencies exerted on intact cells. ã 2014 Elsevier Ireland Ltd. All rights reserved.

Keywords: Protein phosphatase inhibitors Protein phosphatase-1 (PP1) and -2A (PP2A) Surface plasmon resonance (SPR) Lipid immobilization L1 sensor chip

1. Introduction Reversible phosphorylation of proteins on serine (Ser), threonine (Thr) and tyrosine (Tyr) residues is a major regulatory device in many cellular processes (Cohen, 2002). More than 90% of protein phosphorylation occurs at Ser/Thr residues, therefore, identification of the interconverting enzymes, the Ser/Thr specific protein kinases and phosphatases, involved in specific cellular events, have received widespread attention for the past few decades. Initially, the protein kinases were assumed as major targets of regulatory interventions, while the protein phosphatases were thought of as the necessary “housekeeping” enzymes to ensure the reversibility of phosphorylation (Brautigan, 2013). It has become clear, however, that the protein phosphatases are also regulated by a wide variety of mechanisms (Bollen et al., 2010; Hartshorne et al., 2004) and they are important mediators of the strength and duration of the cellular signals (Heinrich et al., 2002). It is also well established that three types of Ser/Thr specific phosphatases, protein phosphatase-1 (PP1), -2A (PP2A) and -2B (PP2B) are responsible for the vast majority of dephosphorylation of phosphoSer/Thr residues in proteins (Mansuy and Shenolikar, 2006).

* Corresponding author at: Department of Medical Chemistry, Faculty of Medicine, University of Debrecen, Egyetem tér 1, H-4032 Debrecen, Hungary. Tel.: +36 52 412345; fax: +36 52 412566. E-mail address: [email protected] (F. ErdÅdi). http://dx.doi.org/10.1016/j.chemphyslip.2014.05.009 0009-3084/ ã 2014 Elsevier Ireland Ltd. All rights reserved.

The discovery of specific, membrane permeable inhibitors of PP1, PP2A and PP2B, and their application to cells permitted to assess the physiological functions of these enzymes (Honkanen and Golden, 2002). Okadaic acid (OA) was the first PP1 and PP2A inhibitory toxin identified (Takai et al., 1987), followed by calyculin-A (CLA) (Ishihara et al., 1989) and tautomycin (TM) (MacKintosh and Klumpp, 1990). OA, CLA and TM are ineffective on PP2B, or inhibit its activity only at much higher concentrations than they do PP1 or PP2A. In contrast, cyclosporin A (CsA) forming a complex with cyclophilin in cells specifically inhibits PP2B, but does not influence directly PP1 or PP2A (Ke and Huai, 2004; Rusnak and Mertz, 2000). Later, microcystin-LR (MC-LR), a hepatotoxic cyclic heptapeptide produced by cyanobacteria (MacKintosh et al., 1990) as well as cantharidin (CA) and its derivatives were also identified as inhibitors of PP1 and PP2A (Li and Casida, 1992). The latter compounds also exerted cytotoxic effects not only on hepatic tissue but other cells, too (ErdÅdi et al., 1995). Recently, polyphenolic compounds such as penta-O-galloyl-b-D-glucose (PGG) as well as epigallocatechin-gallate (EGCG) and its derivatives were shown to inhibit PP1 with preference compared to PP2A (Kiss et al., 2013). The structural backgrounds for the protein phosphatase–inhibitor complexes have been uncovered in several cases and these data may initiate the design of drugs for type specific regulation of these enzymes (Zhang et al., 2013). While the inhibitory potency and selectivity of the phosphatase inhibitory molecules to the different types of enzymes are quite well characterized in vitro, their action, effective concentrations

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and targeted enzymes in the cells are less known. Favre et al. (Favre et al., 1997) characterized the effects of OA, CLA and TM on the phosphatase activity of different cell lines and concluded that OA had relative specificity toward inhibition of PP2A, while TM preferentially blocked PP1 in cells, whereas CLA suppressed the activity of both enzymes. In addition, the extent of inhibition exerted by these molecules was dependent upon the duration of their incubation with cells. It is assumed that the permeation of the inhibitors through the cell membrane could be an important factor in the development of the inhibitory effect, and these processes may be dependent upon the cell types with cell membranes of different lipid constituents. The present study was undertaken to characterize the associationdissociation features of distinct phosphatase inhibitory molecules with lipid vesicles derived from different tissues by surface plasmon resonance (SPR) based binding technique. The liposomes prepared from the lipid mixtures representing the compositions characteristic for brain, heart and liver tissues were immobilized on L1 sensor chip and binding of inhibitors were recorded as sensorgrams. It is seen that the association of inhibitors with liposomes differs depending on the temperature and the tissue specific composition and ratio of lipids implying possible differences in the membrane permeation of these molecules in various tissues and cells. Consistent with these findings, in phosphatase activity assays the inhibitory potencies of these molecules proved to be different in neuroblastoma B50, cardiomyoblast H9C2 and hepatocarcinoma HepG2 cell lines. 2. Materials and methods 2.1. Materials Chemicals and vendors were as follows: [g-32P]ATP was from Hungarian Isotope Institute (Budapest, Hungary); L-glutamine, DMEM and fetal bovine serum were from PAA Laboratories (Pasching, Austria); MC-LR isolated and purified as described (Máthé et al., 2009) was a gift from C. Máthé (Department of Botany, University of Debrecen); EGCG, OA, TM, BSA and Chaps were from Sigma–Aldrich (St. Louis, MO, USA); CA was from LC Services (Woburn, MA, USA); Complete Mini Protease Inhibitor Cocktail was from Roche (Penzberg, Germany); Sensor Chip L1 was from Biacore AB (Uppsala, Sweden); Bovine brain, heart and liver total lipid extracts were from Avanti Polar Lipids (Alabaster, Alabama, USA). All other chemicals used were of the highest purity commercially available. 2.2. Liposome preparation The composition of brain, heart and liver total lipid extracts are shown in Table 1 as given by the description of the commercial source, Avanti Polar Lipids. Lipids were prepared in the same way, regardless of their compositions using the methodology described previously (Abdiche and Myszka, 2004). Briefly, lipid extract powders were hydrated at room temperature in running buffer (50 mM Hepes, 150 mM NaCl, pH 7.4) used in the interaction analysis. Lipid suspensions (3 mg/ml) were subjected to four cycles of freezing ( 80  C), thawing (20  C), and vortexing (5 s) to ensure thorough agitation prior to extrusion through a polycarbonate filter of defined pore diameter (100 nm) using an Avanti Mini-Extruder kit. This involved sandwiching a membrane between two syringes, loading the crude lipid suspension into one syringe, and passing it many times (a minimum of 15) through the membrane. The extruded product, containing uniformly sized micelles was always unloaded from the opposing syringe to avoid contamination present in the original sample. The prepared liposomes were diluted in running buffer to 1 mg/ml.

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Table 1 The composition of the total lipid extracts of brain, heart and liver. Component

(%/wt.)

Brain total lipid extract Phosphatidylethanolamine Phosphatidylserine Phosphatidylcholine Phosphatidic acid Phosphatidylinositol Other

16.7 10.6 9.6 2.8 1.6 58.7

Heart total lipid extract Phosphatidylethanolamine Phosphatidylinositol Cardiolipin Phosphatidylcholine Phosphatidic acid Neutral lipid Other

6.8 2.5 2.3 5.4 1.1 49.8 32.1

Liver total lipid extract Cholesterol Phosphatidylethanolamine Phosphatidylinositol Phosphatidylcholine Lysophosphatidylinositol Others, including neutral lipids

7.0 22.0 8.0 42.0 1.0 20.0

2.3. Capture of liposomes on the L1 sensor chip The L1 sensor chip surfaces were washed with three consecutive one minute pulses of 20 mM Chaps at a flow rate of 100 ml/min, followed by the rinsing routine Extraclean. Liposomes were captured across isolated flow cells around saturation to approximately the same immobilization level (7500 RU) at a flow rate of 2 ml/min. The reference (lipid free) and the three lipid surfaces were blocked with 0.1 mg/ml BSA at a flow rate of 5 ml/min for 15 min. BSA binds in a high amount to the L1 chip surface composed of dextran matrix modified with lipophilic moiety (Cho et al., 2004; Erb et al., 2000). The flow rate then was switched to 100 ml/min and the surfaces were washed with running buffer for three minutes. 2.4. SPR measurements The interactions were investigated at 20 and 37  C using Biacore 3000 instrument equipped with an L1 sensor chip. The protein phosphatase inhibitors were diluted in 10 mM Hepes (pH 7.4) plus 150 mM NaCl to give a concentration series. The Kinject injection type was used and 120 s durations were chosen for both the association and dissociation phases. Inhibitor samples were dispensed into single-used snap-capped vials and injected across the three different lipid and the lipid-free (control) surfaces in a single step. In case of all inhibitors the injections were repeated with increasing sample concentration in successions. The application of these successive injections was possible since the inhibitors dissociated completely from the surfaces during the dissociation phase. After each binding series, the sensor surfaces were regenerated to the original matrix by injecting 4:6 v/v% isopropanol/50 mM NaOH at a flow rate of 100 ml/min followed by washing with 20 mM Chaps. The sensor surfaces were recoated with fresh liposome solutions for the next binding series. 2.5. Cell cultures, incubation with inhibitors and assay of phosphatase activity B50 (neuroblastoma), H9C2 (cardiomyoblast) and HepG2 (hepatocarcinoma) cell lines were obtained from the European Collection of Cell Cultures, and cultured according to the supplier's

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recommendations at 37  C. Prior to treatments, cells were incubated in serum-free medium for 16 h. To investigate the influence of the inhibitors on phosphatase activity, cells were treated with various concentrations of OA, TM, MC-LR, CA and EGCG for 60 min in serum-free media and lysates were prepared for phosphatase assays as described previously (Favre et al., 1997). Briefly, cells were washed with phosphate-buffered saline (PBS) followed by 0.1 M Tris–HCl (pH 7.6), 150 mM NaCl (TBS) containing 0.1 mM EDTA, and then collected in 75 ml ice-cold TBS containing 0.1 mM EDTA supplemented with 0.5% protease inhibitor cocktail and 50 mM 2-mercaptoethanol (extraction buffer). Cells were frozen in liquid nitrogen, then thawed, sonicated and the lysates were clarified by centrifugation at 16,000 g for 10 min. Minimally diluted (6–15-fold final dilution in the assays) supernatants were assayed with 1 mM 32P-labelled 20 kDa light chain of turkey gizzard myosin (32P-MLC20) (ErdÅdi et al., 1995) at 30  C in 20 mM Tris–HCl (pH 7.4) and 0.1% 2-mercaptoethanol. The reaction was initiated by addition of the substrate. After one minute incubation the reaction was terminated by the addition of 200 ml 10% TCA and 200 ml 6 mg/ ml BSA. The precipitated proteins were collected by centrifugation and the released 32Pi was determined from the supernatant (370 ml) in a scintillation counter.

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In SPR experiments the reference response collected across the reference surface was subtracted from all sample responses and then the sensorgrams were analyzed using BIAevaluation software (Biacore AB, Uppsala, Sweden). The apparent dissociation constants (KD(app)) for the binding of phosphatase inhibitors to liposomes were determined from the concentration dependence of the RUmax values analyzing the data using GraphPad software of nonlinear regression for one phase exponential association. Data were derived from at least three independent experiments and

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2.6. Data analysis

Heart lipid Response unit (RU)

Response unit (RU)

Okadaic acid

Brain lipid 15

To examine the total phosphatase activity of B50, H9C2 and HepG2 cells, cell cultures grown to 80–90% confluency were washed with PBS and incubated with trypsin/EDTA mixture until the cells were detached from the surface of cell culture dishes, and then medium was added. Cells were suspended thoroughly and counted in Bürker chamber. Then the cells were collected by centrifugation, washed with PBS and lysed in extraction buffer at 107 cell/ml. Phosphatase activity of the lysates was measured at 225–450-fold dilution in the absence or presence of 2 nM or 1 mM OA as described above.

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Fig. 1. Binding of phosphatase inhibitory toxins to lipids immobilized on L1 sensor chip at 20  C. The liposomes prepared from brain, heart and liver total lipid extracts were immobilized on the surface of L1 sensor chip, then the reference and the three lipid surfaces were blocked with 0.1 mg/ml BSA solution. The interactions between lipid surfaces and OA (upper panel), TM (middle panel) and MC-LR (lower panel) were assayed by injecting the inhibitors over the lipid surfaces at different concentrations for 120 s, then the dissociation phase was recorded changing the inhibitor solution to running buffer for another 120 s. The sensorgrams are representatives of three experiments with similar results.

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given as mean  SEM. Unpaired t-test was used to analyze the results with 95% confidence intervals. 3. Results and discussion 3.1. Interaction of phosphatase inhibitors with liposomes assessed by SPR About the same experimental condition was adjusted at all interaction analysis: typically the immobilization level of liposomes was set to 7500  200 RU, the measurements were carried out at 20  C, the same flow rate and buffer was used, thus the parameters of different phosphatase inhibitor–liposome interactions could be compared. Fig. 1 shows the sensorgrams obtained with the injections of the most commonly used phosphatase inhibitors (OA, TM and MC-LR) on the various liposome covered surfaces. It is seen that the extent of binding of OA was independent of the toxin concentrations in the assayed range. The use of lower OA concentrations was constrained by the detection limit of SPR method for the binding of low molecular mass molecules. In contrast, the binding to liposomes appeared to be concentration dependent in case of TM or MC-LR. Fig. 2 shows liposome binding of CA, EGCG and CsA, inhibitors known to show preference for the inhibition of PP2A (Li and Casida, 1992), PP1

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(Kiss et al., 2013) and PP2B (Rusnak and Mertz, 2000), respectively. These inhibitors exhibit also limited (CA and CsA) or complete (EGCG) concentration dependence of binding to liposomes. Previous studies (Abdiche and Myszka, 2004) established that interactions of drugs with liposome may generally fall into three categories such as nonbinding, transient binding or stable complexes which dissociate slowly. As seen on Figs. 1 and 2, the sensorgrams of inhibitor–liposome interactions exhibit quite steep dissociation phases suggesting that phosphatase inhibitors could be regarded as transient, reversible binding molecules to liposomes. The dissociation of EGCG molecules from the heart or liver liposomes is not complete at all concentrations, however, this might be due to the tendency of stacking of this molecule on the binding surface as suggested in earlier studies (Kiss et al., 2013; Wroblewski et al., 2001). Fig. 3 shows the binding of inhibitors to liposomes obtained by plotting the actual RUmax values from the sensorgrams against the inhibitor concentration (solid lines). It is noteworthy that all inhibitors followed the order of liver > heart > brain lipids with respect to the extent of binding to the liposomes indicated by the different saturation values of inhibitor binding (Fig. 3). It appears that the association of the inhibitors with brain lipids is significantly lower than that of liver or heart lipids in all cases.

Heart lipid Response unit (RU)

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Cantharidin

Brain lipid 30

71

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Fig. 2. Binding of small molecule PP1/PP2A inhibitors (CA and EGCG) and the PP2B inhibitor (CsA) to lipids immobilized on L1 sensor chip at 20  C. The interactions between lipid surfaces and CA (upper panel), EGCG (middle panel) and CsA (lower panel) were assayed by injecting the inhibitors over the lipid surfaces under the same conditions described in the legend to Fig. 1.

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40

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Fig. 3. Concentration dependent binding of the phosphatase inhibitors to the liposomes of different tissues. The RUmax values of the sensorgrams were plotted against the concentration of the inhibitors (solid lines). Dotted lines represent nonlinear regression curve fitting for one phase exponential association.

These data imply that the association of the inhibitors with the cell membranes may involve certain tissue specific features due to the presence of different lipids in distinct ratios. In Fig. 3 the dotted lines exhibit nonlinear fitting of the binding curves for one phase exponential association to derive apparent dissociation constants (KD(app)) for the interactions. Table 2 displays the KD(app) values for the inhibitor–liposome interactions derived from the concentration dependent RUmax values in cases it was applicable. It is apparent that the KD(app) values for a given inhibitor vary only slightly when compared for the different liposomes (see Table 2), the differences are less than 5-fold even in case of the most marked variation of KD(app) values for the interaction of TM with the various liposomes. These data imply that the distinct inhibitor binding capacities of the liposomes may determine the effectiveness of the inhibitor on different tissues, rather than the otherwise subtle differences in the binding affinities. Most of the drug–lipid interaction studies by SPR have been carried out at 20–25  C (Abdiche and Myszka, 2004; Lombardi et al., 2009a), whereas these interactions occur in cellular systems at physiological temperature of 37  C. Therefore, we decided to assess the interaction of phosphatase inhibitors with liposomes at 37  C. The extent of liposome immobilization on L1 chip at 37  C Table 2 Apparent dissociation constants (KD(app)) for the liposome–phosphatase inhibitor interactions determined from the concentration dependence of RUmax values at 20  C. *

Okadaic acid Tautomycin Microcystin-LR Cantharidin EGCG Cyclosporin A

KD(app), mM

Brain lipid

Heart lipid

Liver lipid

n.d. 3.82  0.71 7.33  1.50 0.37  0.07 185.2  16.25 n.d.

n.d. 2.04  0.13 5.11  0.51 0.12  0.04 111.8  5.25 n.d.

0.03  0.01 0.86  0.04 3.16  0.05 0.21  0.03 110.2  19.20 0.05  0.01

n.d.: not determined. * Apparent KD values with the standard deviations for the fits.

was approximately at the same level (7500–8000 RU) as at 20  C, but the stability of the surface was slightly weaker, since a subtle “leaking” (less than 5% of the total immobilized liposome in 30 min) was observed (data are not shown) during the washing period. Nevertheless, binding studies with phosphatase inhibitors could be completed, but due to the slight instability of the surface the sensorgrams obtained at 37  C were “noisy” and not as regular as at 20  C. However, even these sensorgrams were suitable to draw qualitative conclusions for the temperature dependence of the interactions (Fig. 4). In Fig. 4A binding of MC-LR to brain, heart and liver liposome surfaces are shown as representative sensorgrams of association of an inhibitor molecule with the three different lipid surfaces. There are several distinctive features of the binding curves of MC-LR–liposome interactions at 37  C (Fig. 4A) compared to that of 20  C (Fig. 1, lower panel): the concentration dependence of the association kinetics and the equilibrium RUmax values almost disappeared at 37  C, and the dissociation of the inhibitors from the liposomes became significantly slower. Fig. 4B illustrates that similar changes to that of MC-LR in the binding characteristics of OA, TM and CsA were observed at 37  C exemplified with their interactions with liver liposomes. In contrast, CA and EGCG binding to liposomes did not differ profoundly at these two temperatures. These data suggest that the mechanism of interaction of some inhibitors with liposomes is different at higher temperature: MC-LR, OA, TM and CsA are partially retained in the liposomes and this is reflected in their lower dissociation rates. It is speculated that the higher fluidity of the liposomes at 37  C might allow partial intercalation and retention of the relatively larger inhibitor molecules (MC-LR, OA, TM and CsA) in the lipid bilayer. 3.2. Effect of the inhibitors on the phosphatase activity of B50, H9C2 and HepG2 cells The different association of phosphatase inhibitors with membrane lipids may also have influence on their inhibitory

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Fig. 4. Binding of phosphatase inhibitory toxins to lipids immobilized on L1 sensor chip at 37  C. (A) The concentration dependent binding of MC-LR to liposomes prepared from brain (left), heart (middle) and liver (right) lipid extract. (B) Binding of OA, TM, CA, EGCG and CsA to liver lipids. Assay conditions were the same as described in Fig. 1 except that the temperature was set at 37  C.

potencies exerted on the phosphatase activity of different cell lines. It was assumed that the different lipid compositions of brain, heart and liver may also be reflected in cell lines of similar tissue origins. First, we estimated and compared the amount and distribution of PP1 and PP2A in B50, H9C2 and HepG2 cells (normalized for 106 cells of each cell line) using 32P-MLC20 as substrate which is known to be dephosphorylated by these two types of phosphatase in cell lysates. OA in 2 nM was applied in the phosphatase assays to differentiate between PP1 and PP2A activity in diluted lysates: the phosphatase activity inhibited by 2 nM OA was considered as PP2A (Fig. 5). Phosphatase activity was also determined in the presence of 1 mM OA which is supposed to inhibit entirely both PP1 and PP2A. Fig. 5A shows that the amount of total phosphatase activity followed the order of H9C2 > B50 > HepG2 cells for the same number of cells. It is also to note that phosphatase activity was not completely diminished even in the presence of 1 mM OA (Fig. 5A), however, this PP1/PP2A independent activity represented less than 10% of the total phosphatase activity in each cell line (Fig. 5B). In B50 cells the PP2A activity

exceeded PP1 activity, whereas the opposite (PP1 over PP2A activity) was observed in H9C2 and HepG2 cells. Then, the inhibitors (OA, TM, MC-LR, CA and EGCG) were incubated for 60 min with B50, H9C2 and HepG2 cells and the phosphatase activity in the cell lysates was determined using 32P-MLC20 as substrate. Fig. 6 depicts that the inhibitors had differential effects on the phosphatase activity of distinct cell lines. The phosphatase activity of B50 cells (Fig. 1, upper panel) was in some cases slightly, but less inhibited by OA and MC-LR. TM diminished the phosphatase activity of H9C2 cells almost completely, while in B50 and HepG2 cells only partial inhibition was observed. Interestingly, CA was without effect (except for H9C2), while EGCG inhibited the phosphatase activity of all cell lines significantly, but to a similarly moderate extent (20–30%). These data imply distinctive inhibitory features of these molecules depending upon the cell types. Although the membrane–lipid compositions of B50, H9C2 and HepG2 cells are not known, the inhibitory potencies of the phosphatase inhibitory drugs in most cases are lower for the neuroblastoma cell type than that of the cardiomyoblast and

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Fig. 5. Comparison of the total phosphatase activities and the distribution of PP1 and PP2A in B50, H9C2 and HepG2 cells. (A) Phosphatase activity of the lysates of B50, H9C2 and HepG2 cells (106 cells) was determined in the absence and in the presence of 2 nM or 1 mM OA using 32P-MLC20 as substrate as described in Section 2. (B) Distribution of PP1 and PP2A activities in B50, H9C2 and HepG2 cells.

hepatocarcinoma cell lines. These differences are reminiscent of the distinct association features and binding extents of phosphatase inhibitors to liposomes of brain, heart and liver lipids. Moreover, there appears to be no direct correlation between the apparent dissociation constants and the inhibitory potencies of the inhibitors (see Table 2 and Fig. 6). For the classical inhibitors, OA has the highest affinity (KD(app)  0.03 mM) to the liposomes, however, TM with lower affinity (KD(app)  0.8–3.8 mM) inhibits phosphatase activity in the cells more potently. Similarly, CA has relatively high affinity for liposomes, even though its slight inhibitory influence could be observed on H9C2 cells only. These data indicate that besides the strengths of association of inhibitors with lipids other factors may also be involved in the phosphatase inhibitory effectiveness in cells. These factors may include the differential interaction of the inhibitors with the liposomes at physiological temperature indicating retention of the inhibitors in the lipid bilayer. It is presumed that the rate determining step for the accumulation of the inhibitors within the cells may be the release of the inhibitors from the membrane toward the intracellular space. In accord with the above assumptions it was previously shown that cells need to be incubated with OA for at least 2 h to achieve effective inhibition and the development of TM inhibition was found to be even slower (Favre et al., 1997) at micromolar concentrations of both inhibitors. Our results confirmed the first observation in part, since OA exerted slight, but

*

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0.1 1.5 2.5 OA

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10 100 500µM EGCG

Fig. 6. Differential effects of protein phosphatase inhibitors on the activity of PP1 and PP2A in B50, H9C2 and HepG2 cells. Cells were treated with OA, TM, MC-LR, CA and EGCG for 60 min at the concentrations indicated in the figure. After incubation in the absence or in the presence of these drugs, cells were lysed and phosphatase activity was determined with 32P-MLC20 substrate. The phosphatase activity in the absence of the inhibitors was taken as 100%. The values represent the average  SEM of 3–5 independent experiments. Significance of changes compared to controls is indicated as follows: n.s., non-significant; *p < 0.05, **p < 0.01 and ***p < 0.001.

significant phosphatase inhibition (during 1 h incubation) even at 0.1 mM, which was more prevalent at 1.5–2.5 mM, but the increase in the extent of inhibition (1.8–2.5-fold) was not proportional with the increase (15–25-fold) in the inhibitor concentration. In contrast, we found that TM inhibited cellular PP1 and PP2A more readily than that was suggested earlier (Favre et al., 1997) and even 1 mM TM diminished almost completely the phosphatase activity in H9C2 cells, whereas in B50 and HepG2 cells it was less effective. MC-LR, a potent hepatotoxin, is known to enter only hepatocytes and its primary transport through the cell membranes to the intracellular space occurs via the bile acid/organic anion transporter(s) of these cells (Runnegar et al., 1995). Our present data suggest that MC-LR inhibits the phosphatase activity in all cell lines in a similar fashion at relatively high concentrations, although

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HepG2 cells would be expected to be more sensitive to MC-LR inhibition of phosphatase activity based on the facts described above. However, the lack of differences in the inhibition of the HepG cells compared to the other cell lines is not surprising since it has been demonstrated (Boyer et al., 1993) that the bile acid/ organic anion transporter(s) is not present in HepG2 cells. The above data imply that MC-LR may be inhibitory on the cellular phosphatases in cells which lack bile acid/organic anion transporters at higher toxin concentrations via interaction and permeation of cell membranes. In accord with this assumption MC-LR also has an inhibitory effect on the phosphatase activity of plant cells (Máthé et al., 2009) where the above transport mechanism does not operate either. 4. Conclusion Study of the binding of proteins (Ferguson et al., 2005) or drugs (Abdiche and Myszka, 2004; Kim et al., 2004; Lombardi et al., 2009a,b) to liposomes derived from tissue lipid extracts or reconstituted from different lipids by SPR technique is often applied to model interactions of these molecules with lipid membranes. These experiments may also provide clues for the kinetics of membrane permeation of the drugs, since the association phase of the sensorgrams reflects the interaction of these molecules with the membrane at the extracellular side, while the dissociation phase is characteristic for the reversibility of the interactions and fast dissociation may contribute to reaching the drug's intracellular targets readily. The protein phosphatases are enzymes that are considered as future drug targets with clinical applications (Honkanen and Golden, 2002; Zhang et al., 2013). We investigated the drug–ipid interaction features of classical and toxic (OA, TM, MC-LR and CA), non-toxic (CsA) as well as recently recognized (EGCG) inhibitors of protein phosphatases by SPR on the first time. The major conclusions of this study are as follows: (i) SPR is suitable for the interaction analysis of phosphatase

inhibitors with membrane lipids; however, the technique may have certain limitations with respect to determination of the full binding concentration range due to the low SPR responses at lower concentration of these low molecular mass analytes. (ii) Some of the phosphatase inhibitors exhibit different affinity towards the liposomes formed from total lipid extracts of brain, heart and liver tissues. It could be assumed that the different composition and ratio of lipids in the distinct liposomes are responsible for the differential binding of phosphatase inhibitors. The phosphatase inhibitor–liposome interactions are reversible and the dissociation kinetics exhibit only slight liposome dependence at 20  C. Therefore, the differences in lipid association of the inhibitors may primarily due to distinct association kinetics. However, the inhibitors interact with liposomes at 37  C in a different manner compared to that of 20  C. The differences in association kinetics apparently disappear and similar saturation binding values are observed at distinct concentrations. Moreover, the dissociation of the inhibitors becomes profoundly slower implicating their retention in the liposomes. Therefore, we hypothesize a mechanism suggesting that the inhibitors (OA, TM, MC-LR) may first bind at the membrane surface, then become intercalated in the bilayer and dissociates slowly towards the intracellular space in agreement with the need of relatively long incubation period to achieve effective inhibition (Favre et al., 1997). CA and EGCG might follow different routes with much lower permeation presumably due to their partially hydrophilic nature and to the lack of their effective intercalation in the membrane. In addition, the lower inhibitory

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effectiveness of EGCG might also due to its partial selectivity for PP1 and to binding to targets different from phosphatases (Kiss et al., 2013). On the other hand, the weak inhibitory potency of CA on cellular phosphatases and its low extent of permeation through cell membranes were also observed in our previous study (ErdÅdi et al., 1995). The above data direct attention to the importance of assaying biochemical interactions at physiological temperature to obtain data for drawing physiologically relevant conclusions. (iii) The capacity of the liposomes to bind inhibitors also varies and brain lipids exhibit the lowest extent of binding compared to heart or liver lipids. The differential binding of inhibitors to the membrane lipids appears to be reflected in different extent of phosphatase inhibition of distinct cell types. In cells the lipid constituent dependent saturation of cell membranes with inhibitors occurs even at relatively low inhibitor concentration. Therefore, the dissociation of the membrane-retained inhibitor may determine the rate of accumulation of the inhibitors in the cells. The operation of such a mechanism is supported by the data of phosphatase inhibition by the inhibitors in intact cells as a 10–40-fold increase in the applied inhibitor concentration resulted in only a moderate or not even significant decrease of the phosphatase activity. In summary, it is demonstrated that SPR studies of the interaction of phosphatase inhibitors with lipids extend the tools of investigating the mechanisms of action of these drugs and together with phosphatase assays may provide novel clues to assess their cell type specific effectiveness. Conflict of interest The authors declare that there are no conflicts of interest. Transparency document The Transparency document associated with this article can be found in the online version. Acknowledgments This work was supported by the following grants: TÁMOP4.2.2/B-10/-1-2010-0024, TÁMOP-4.2.2.A-11/1/KONV-2012-0025 and TÁMOP 4.2.4. A/2-11-1-2012-0001 ‘National Excellence Program’ supported by the European Union and the State of Hungary, co-financed by the European Social Fund, OTKA K109249 from the Hungarian Science Research Fund; Bridging Fund 2012 from the University of Debrecen Faculty of Medicine Research Fund. The authors are indebted to Mrs. Ágnes Németh for excellent technical assistance. References Cohen, P., 2002. The origins of protein phosphorylation. Nat. Cell Biol. 4, E127–E130. Brautigan, D.L., 2013. Protein Ser/Thr phosphatases–the ugly ducklings of cell signalling. FEBS J. 280, 324–345. Hartshorne, D.J., Ito, M., ErdÅdi, F., 2004. Role of protein phosphatase type 1 in contractile functions: myosin phosphatase. J. Biol. Chem. 279, 37211–37214. Bollen, M., Peti, W., Ragusa, M.J., Beullens, M., 2010. The extended PP1 toolkit: designed to create specificity. Trends Biochem. Sci. 35, 450–458. Heinrich, R., Neel, B.G., Rapoport, T.A., 2002. Mathematical models of protein kinase signal transduction. Mol. Cell. 9, 957–970. Mansuy, I.M., Shenolikar, S., 2006. Protein serine/threonine phosphatases in neuronal plasticity and disorders of learning and memory. Trends Neurosci. 29, 679–686. Honkanen, R.E., Golden, T., 2002. Regulators of serine/threonine protein phosphatases at the dawn of a clinical era? Curr. Med. Chem. 9, 2055–2075. Takai, A., Bialojan, C., Troschka, M., Ruegg, J.C., 1987. Smooth muscle myosin phosphatase inhibition and force enhancement by black sponge toxin. FEBS Lett. 217, 81–84.

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