cAMP-dependent protein kinase and protein kinase C ... - Europe PMC

The EMBO Journal vol. 10 no. 1 pp. 1 17 - 122, 1991

Okadaic acid induction of the urokinase-type plasminogen activator gene occurs independently of cAMP-dependent protein kinase and protein kinase C and is sensitive to protein synthesis inhibition Yoshikuni Nagamine and Annemarie Ziegler Friedrich Miescher Institute, PO Box 2543. CH 4002 Basel, Switzerland Communicated by B.Groner

Urokinase-type plasminogen activator (uPA) gene expression in LLC-PK, cells is induced by activation of cAMP-dependent protein kinase (cAMP-PK) or protein kinase C (PK-C). To determine whether protein phosphatases can also modulate uPA gene expression, we tested okadaic acid, a potent speciflc inhibitor of protein phosphatases 1 and 2A, in the presence and absence of cAMP-PK and PK-C activators. Okadaic acid by itself induced uPA mRNA accumulation. This induction was strongly attenuated by the inhibition of protein synthesis. In contrast, the inhibition of protein synthesis enhanced induction by 8-bromo-cAMP and only delayed induction by 12-0-tetradecanoylphorbol-13-acetate (TPA). In addition, down-regulation of PK-C by chronic treatment with TPA did not abrogate the okadaic acid-dependent induction. These results provide evidence for a novel signal transduction pathway leading to gene regulation that involves protein phosphorylation but is independent of both cAMP-PK and PK-C. Key words: gene expression/okadaic acid/plasminogen activator/protein phosphatase/signal transduction

Introduction Protein phosphorylation is thought to play a key role in many biological processes such as glycolysis (Cohen, 1983), the cell cycle (Hunt, 1989) and the coupling of cell surface receptors to intracellular regulation (Yarden and Ullrich, 1988; Witters, 1990). In accordance with this, various types of protein kinases have been isolated and the genes encoding them cloned, and novel protein kinases are still being reported. Since the finding of the hormonal activation of cAMP-dependent protein kinase (cAMP-PK) (Sutherland, 1972), studies of the regulation of a given biological process through protein phosphorylation have centred around the characterization of a protein kinase involved in the process. More recently, a concept is emerging that the regulation is a given biological process by protein phosphorylation of through a dynamic equilibrium between the phosphorylation and dephosphorylation of proteins important in regulation (Cohen and Cohen, 1989). According to this view, it is conceivable that modulation of protein phosphatase activity may reverse the effect of protein kinase activity on a given biological process. Several lines of evidence indicate that protein kinase activity is essential for the induction of gene transcription during activation by the cAMP-dependent and TPAdependent signalling pathways (Boney et al., 1983; Degen Oxford University Press

et al., 1985; Grove et al., 1987; Nakagawa et al., 1988; Riabowol et al., 1988). However, very little is known about the role of protein phosphatases in gene regulation. The activity of protein kinase is modulated by a variety of natural and pharmacological agents. cAMP-PK can be activated by peptide hormones (Schramm and Selinger, 1984), cholera toxin (Cassel and Pfeuffer, 1978) or forskolin (Seamon et al., 1981). Protein kinase C (PK-C) can be activated by a number of hormones (Berridge and Irvine, 1984; Nishizuka, 1986) or tumor-promoting phorbol esters (Castagna et al., 1982). On the other hand, the modulation of protein phosphatase activities has not been easily attainable. However, the recent finding that okadaic acid is a potent inhibitor of protein phosphatase 1 and 2A (Bialojan and Takai, 1988), has enabled us to investigate this possibility. Okadaic acid is a polyether fatty acid first isolated from the marine sponges Halichondria okadaii and Halichondria melanodocia (Tachibana et al., 1981). In cellfree extracts, it inhibits protein phosphatase 2A at low concentrations (-0.1 nM) and protein phosphatase 1 at high concentrations (- 10 nM) (MacKintosh and Cohen, 1989). To test the possibility that the state of protein phosphorylation and consequently the regulation of gene expression are the net result of phosphorylation and dephosphorylation, we examined the effects of okadaic acid on urokinase-type plasminogen activator (uPA) gene regulation in LLC-PKI cells. In LLC-PK1 cells (Hull et al., 1976), a cell line derived from porcine renal proximal tubule epithelia, the uPA gene is induced independently by cAMP-PK and PK-C (Nagamine et al., 1983; Degen et al., 1985; Nakagawa et al., 1988; von der Ahe et al., 1990). Both kinases phosphorylate proteins at a serine or threonine residue in specific sequences (Kempt et al., 1977; Edelman et al., 1987; Nishizuka, 1986; Woodgett et al., 1986) and many phosphoproteins phosphorylated by these kinases can be dephosphorylated, at least in vitro, by protein phosphatase 1 and 2A (Cohen, 1989; Cohen and Cohen, 1989), which are the targets of okadaic acid. In this paper we show that okadaic acid induces uPA mRNA induction independently of cAMP-PK and PK-C.

Results Induction of uPA mRNA by okadaic acid In our initial experiments we looked at the effects of okadaic acid on uPA gene expression. We measured the steady-state levels of uPA mRNA because earlier studies had shown that uPA mRNA levels reflect uPA gene transcription (Degen et al., 1985; Andrus et al., 1988; Ziegler et al., 1990). Our working hypothesis was that okadaic acid would affect a pathway involving cAMP-PK or PK-C. Therefore, we measured uPA mRNA levels in cells treated with different concentrations of okadaic acid in the presence or absence of suboptimal concentrations of 8-bromo-cAMP (Br-cAMP;

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Y.Nagamine and A.Ziegler

0.1 mM) and 12-O-tetradecanoylphorbol-13-acetate (TPA; 1 ng/tnl). If okadaic acid-sensitive phosphatases are responsible for the dephosphorylation of a factor involved in uPA gene regulation that is phosphorylated by cAMPPK or PK-C, then okadaic acid treatment should enhance the uPA mRNA accumulation induced by either or both activators. Furthermore, if the basal level of protein kinase activity is very low, then the effect of okadaic acid will be seen only when cells are treated with inducers. The effect of okadaic acid on uPA mRNA levels in total RNA prepared 2 and 7 h after treatment of cells is shown in Figure 1. Okadaic acid induced uPA mRNA accumulation at the highest concentration tested at both time points in the absence of Br-cAMP and TPA. This implies that a significant level of phosphorylation of a factor that is involved in uPA gene regulation occurs constitutively: this factor is normally dephosphorylated by the action of okadaic acid-sensitive protein phosphatase(s) (lane 6). Treatment of cells with Br-cAMP and TPA also induced uPA mRNA. Okadaic acid potentiated the effect of both agonists, but only at the highest concentration used (lanes 12 and 18). When the levels of uPA mRNA at 2 h were compared, the effect of okadaic acid on Br-cAMP- and TPA-dependent uPA mRNA accumulation was more than additive. The levels of uPA mRNA induced by okadaic acid, Br-cAMP and TPA were 60, 170 and 30 c.p.m. (upper panel: lanes 6, 7 and 13) respectively, whereas those induced by Br-cAMP plus okadaic acid and TPA plus okadaic acid were 400 and 220 c.p.m. (upper panel: lanes 12 and 18) respectively. This suggests a functional interaction between an okadaic acidsensitive protein phosphatase and cAMP-PK- and PK-Cdependent signalling pathways. Although okadaic aciddependent induction became more prominent with longer incubation, the synergistic effect of okadaic acid was less obvious (lower panel). In a more detailed second experiment, we measured the time course of uPA mRNA accumulation following treatment of cells with combinations of okadaic acid, Br-cAMP and TPA. To obtain comparable levels of mRNA accumulation, the concentrations of Br-cAMP and TPA were increased to 0.3 mM and 30 ng/ml respectively. Okadaic acid gave a different time course of uPA mRNA accumulation compared

to Br-cAMP and TPA (Figure 2). uPA mRNA accumulation reached maximum at 4-6 h after okadaic acid treatment, while the maximum was 2 h for both Br-cAMP and TPA, suggesting that the mechanisms underlying okadaic aciddependent uPA mRNA induction are qualitatively different from that of Br-cAMP- and TPA-dependent induction. When okadaic acid was combined with Br-cAMP or TPA, the level of uPA mRNA was higher than the sum of each induction at every time point, but the pattern of uPA mRNA accumulation was similar to that obtained with okadaic acid alone; the optimal level of induction was attained after 4-6 h of induction and declined thereafter. The synergistic effect of okadaic acid was clearly observed at 1 and 2 h after treatment when no induction by okadaic acid alone could be detected. Therefore, it appears that okadaic acid affects uPA gene expression independently of as well as dependently on Br-cAMP- and TPA-dependent signalling pathways. Effect of protein synthesis inhibition on uPA mRNA induction As previous experiments suggested the presence of a distinct signalling pathway for okadaic acid-dependent uPA mRNA induction, we performed the following experiment that would possibly differentiate between those pathways involving okadaic acid-sensitive phosphatase, cAMP-PK and PK-C. Because okadaic acid-induced uPA mRNA accumulation was slower than accumulation induced by Br-cAMP or TPA, we considered the possibility that the okadaic acid-dependent uPA mRNA induction required protein synthesis. To test this possibility, we treated the cells with okadaic acid in the presence or absence of cycloheximide, an inhibitor of eukaryotic protein synthesis. Previously, we have shown that inhibition of protein synthesis enhances uPA mRNA accumulation induced by calcitonin, a peptide hormone coupled to adenylate cyclase, and delays but does not reduce the magnitude of the TPA-dependent induction (Altus et al., 1987). The results show that cycloheximide markedly inhibited the okadaic acid-induced accumulation of uPA mRNA (Figure 3). Six hours after treatment, 90% of uPA mRNA accumulation was suppressed by cycloheximide

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Fig. 1. Effect of okadaic acid on uPA mRNA accumulation in LLC-PKI cells. Cells were treated with indicated concentrations of okadaic acid (OA) for 1 h (lanes 1-18) and further incubated for 2 h (upper panel) or 7 h (lower panel) in the absence (lanes 1-6) or presence of 0.1 mM Br-cAMP (lanes 7-12) or 1 ng/ml TPA (lanes 13-18). In lane 1, cells were without any treatment. Total RNA was prepared and analyzed by Northern blot hybridization. RNA was hybridized simultaneously with uPA (1 106 c.p.m./ml) and actin (1 x 105 c.p.m./ml) probes. In cultures of all lanes except lane 19, 0.1% dimethyl fluoride solvent of okadaic acid, was present. 118

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Fig. 2. Time course of uPA mRNA accumulation. Cells were treated with different reagents and incubated for the time shown in the figure. Total RNA was then prepared and analyzed by Northern blot hybridization using the radioactive uPA probe. After hybridization areas corresponding to uPA mRNA in the filter were excised and counted in a scintillation counter. Cells were treated with: 125 nM

okadaic acid (x); 0.3 mM Br-cAMP (0); 30 ng/ml TPA (A); 125 nM okadaic acid plus 0.3 mM Br-cAMP (0); 125 nM okadaic acid plus 30 ng/ml TPA (LI).

Okadaic acid-dependent uPA gene regulation

(from 370 to 39 c.p.m.). On the other hand, as expected, Br-cAMP-dependent uPA mRNA accumulation was strongly enhanced by cycloheximide, while TPA-dependent accumulation was delayed but not suppressed at all. These results show that protein synthesis is required for the inductive effect of okadaic acid. This provides further support for the possibility that okadaic acid activates a different signalling pathway than either Br-cAMP or TPA. Effect of okadaic acid on cAMP-dependent protein kinase The results described above argue against the likelihood that the factor responsible for okadaic acid-dependent uPA mRNA induction is identical to the one that mediates the action of cAMP-PK or PK-C in uPA gene regulation. However, these results do not rule out the possibility that okadaic acid treatment leads to the activation of cAMP-PK or PK-C, which in turn increases expression of the uPA gene. To address this possibility we examined the effect of okadaic acid on cAMP-PK activity. Cells were treated with okadaic acid in the presence or absence of cycloheximide, cell-extracts were prepared, and cAMP-PK activity in the extracts was measured in the presence or absence of cAMP using the synthetic substrate kemptide. The activity ratio, defined as the activity in the absence of cAMP divided by that in the presence of cAMP, ai D at

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provides an estimate of the amount of active enzyme in the extracts and presumably the amount of active enzyme in cells at the time the extracts were prepared. As shown in Figure 4, treatment with okadaic acid resulted in a transient activation of cAMP-PK. However, pretreatment with cycloheximide, which strongly prevented okadaic acid-induced uPA mRNA accumulation (see Figure 3), did not inhibit but rather enhanced the okadaic acid-dependent activation of cAMP-PK. Therefore, it is unlikely that the activation of cAMP-PK is the primary cause of okadaic acid-dependent uPA niRNA induction. Furthermore, 0.3 mM Br-cAMP induced uPA mRNA accumulation much less than 125 nM okadaic acid (Figure 2), but still induced the activation of cAMP-PK to an activity ratio of 0.16 within 1 h (data not shown), implying that this amount of activation is not enough to account for the okadaic

acid-dependent induction. Effect of protein kinase C down-regulation on okadaic acid-dependent uPA mRNA induction To determine whether the okadaic acid-dependent induction was mediated by PK-C, cells were pretreated for 48 h with 100 ng/ml of TPA to down-regulate the PK-C (Nishizuka, 1986) and then treated with okadaic acid. Figure 5 shows that the cells treated with the second challenge of TPA after

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uPA mRNA accumulation. Cells were treated with or without TPA (100 ng/ml) for 48 h, and then treated with okadaic acid (125 nM) or additional TPA (100 ng/ml) for the time indicated. Total RNA was prepared and analysed as described in the legend to Figure 1.

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long-term treatment with TPA did not accumulate uPA mRNA (compare lanes 13 and 14). After down-regulation, no PK-C antigen could be detected using anti-alpha-type PK-C antibody (D.Fabbro, personal communication). Under these conditions, okadaic acid still induced uPA mRNA accumulation. At the maximum point of induction, PK-C down-regulation reduced the okadaic acid-dependent uPA mRNA induction by 35 % (lane 11, 368 c.p.m. versus lane 5, 561 c.p.m.). These results indicate that okadaic aciddependent uPA mRNA induction does not require PK-C. -

Discussion Okadaic acid is a potent inhibitor of both protein phosphatases 1 and 2A. We have provided evidence that okadaic acid induces uPA mRNA accumulation in LLCPK, cells. Induction was observed at 125 nM of okadaic acid but not at lower concentrations. Furthermore, this concentration of okadaic acid potentiated the effect of both Br-cAMP- and TPA-dependent increase of uPA mRNA. In cell-free extracts, okadaic acid inhibits protein phosphatase 2A at lower concentrations (-- 0.1 nM) and protein phosphatase 1 at higher concentrations (- 10 nM) (MacKintosh and Cohen, 1989). However, we do not know which protein phosphatase is involved in okadaic acid-dependent uPA induction, because the intracellular concentration of these phosphatases is usually in the range of 0.1-1 M (Cohen et al., 1990), and the efficiency of penetration of okadaic acid through the cytoplasmic membrane is not known. To answer this question we must wait until methods are developed to prepare cell extracts preserving the in vivo activity-state of phosphatase and differentially quantitate each protein phosphatase. It is also possible that an unidentified protein phosphatase is sensitive to okadaic acid and responsible for uPA mRNA induction. uPA gene expression in LLC-PKI cells is induced independently by cAMP-elevating agents (Nagamine et al., 1983; Andrus et al., 1988) and tumor promoter phorbol esters (Degen et al., 1985; D.Pearson and Y.Nagamine, unpublished). As these inducers activate cAMP-PK and PK-C, which phosphorylated serine or threonine residues of various proteins, we reasoned that any influence of okadaic acid on uPA gene expression would be through a signalling pathway involving one of these kinases. However, several lines of evidence indicate that okadaic acid-dependent uPA mRNA induction in LLC-PKI cells is mediated by a factor not involved in either cAMP-PK- or PK-C-dependent signal transduction. First, okadaic acid-dependent induction was extremely sensitive to protein synthesis inhibition, which was the opposite to either Br-cAMP-dependent or TPAdependent induction; Br-cAMP-dependent induction was enhanced and TPA-dependent induction was only delayed but not suppressed. Second, although okadaic acid treatment caused the activation of cAMP-PK, the extent of activation was much less than that caused by 0.3 mM Br-cAMP, which induced comparable amounts of uPA mRNA. Moreover, the activation of cAMP-PK by okadaic acid was not suppressed but enhanced by protein synthesis inhibition. Third, PK-C down-regulation did not abrogate okadaic acid-dependent induction of uPA mRNA. Therefore, it appears that okadaic acid-dependent uPA mRNA induction reflects a novel signal transduction pathway involving protein phosphorylation. As mentioned, okadaic acid induced a small but significant 120

activation of cAMP-PK. In this context, it is noteworthy that the regulatory subunit (Carmichael et al., 1982; Hemmings et al., 1982) as well as the catalytic subunit (Shoji et al., 1979) of the cAMP-PK can be phosphorylated in the cell, although neither the role nor consequence of this is known. It would be interesting to know whether the okadaic acidinduced activation of cAMP-PK is related to the phosphorylation of the subunits. However, in this work we did not pursue this aspect of the effects of okadaic acid. It is also possible that okadaic acid activates other kinases which can use kemptide as a substrate in the in vitro assay. Phosphorylation that may not be medited by cAMP-PK or PK-C has also been implicated in gene regulation. Both the DNA-binding and trans-activating activity of the serum response factor have been shown to be regulated by phosphorylation (Prywes et al., 1988). In yeast the activity of heat-shock factor mediating heat-induced gene transcription is modulated by temperature-dependent phosphorylation (Sorger and Pelham, 1988). However, it is unlikely that one of these particular processes is involved in okadaic acid-dependent uPA mRNA induction, as the uPA gene is not induced by serum (D.Pearson and Y.Nagamine, unpublished), or by heat (Andrus et al., 1988). A number of nuclear oncoproteins and transcription factors including Myc, Myb, Fos and CREB, contain casein kinase II phosphorylation sites near or within domains crucial for transforming and DNA-binding activity (Gonzalez et al., 1989; Luscher et al., 1989; and reviewed by Witters, 1990), although the relevance of phosphorylation in gene regulation in these cases is unknown. Because there are no specific inhibitors or stimulators of casein kinase II in LLC-PKI cells, a link between casein kinase II and okadaic acidsensitive phosphatase, although an interesting possibility, will not be clarified until an okadaic acid-mediating transcription factor is isolated. Okadaic acid-dependent uPA mRNA induction is dependent on ongoing protein synthesis. This raises two possibilities. First, okadaic acid induces the de novo synthesis of a factor that in turn activates the uPA gene; this provides a plausible explanation of why uPA mRNA induction by okadaic acid was slower than that by Br-cAMP and TPA, which does not require new protein synthesis. Second, the uPA gene is directly induced by the okadaic acid-dependent signal transduction pathway but may require an additional labile protein for activation. At the moment we have no evidence that allows us to discriminate between these two possibilities. In addition to the pathways involving cAMP-PK and PK-C, reorganization of the cytoskeleton of LLC-PKI cells by colchicine, nocodazole or cytochalasin B also induces uPA gene activation (Botteri et al., 1990). The cytoskeletondependent uPA gene induction is sensitive to protein synthesis inhibition and partly suppressed by PK-C downregulation, which is similar to the okadaic acid-dependent induction. It is tempting to speculate that these two pathways are related. We are currently examining this possibility. Okadaic acid induces uPA mRNA accumulation independently of cAMP-PK and PK-C; however, it appears that pathways involving these kinases are also affected by okadaic acid. The effect of okadaic acid on Br-cAMP-dependent and TPA-dependent uPA mRNA accumulation was more than additive, suggesting the functional interaction between an okadaic acid-sensitive protein phosphatase and cAMP-

Okadaic acid-dependent uPA gene regulation

dependent and PK-C-dependent signalling pathways. These results may be interpreted as the direct influence of an okadaic acid-sensitive protein phosphatase on factors mediating the action of cAMP-PK and PK-C. Nevertheless, the apparent synergism might also result from multiple but independent interactions of trans-acting factors mediating the action of okadaic acid, Br-cAMP and TPA with their regulatory sequences in the uPA gene promoter. We have no evidence to distinguish between these two possibilities. Originally, we postulated that the phosphorylation state of factors mediating the cAMP-PK and PK-C action on uPA gene regulation were in an equilibrium between phosphorylation and dephosphorylation at the basal state of cells (i.e. before treating with cAMP or TPA). The fact that the majority of uPA mRNA accumulation induced by the okadaic acid-dependent pathway was abrogated by cycloheximide does not favour this idea, because the pathway involving cAMP-PK and TPA is not down-regulated by protein synthesis inhibition. Alternatively, the factors that are phosphorylated by cAMP-PK and PK-C, are dephosphorylated by a phosphatase other than protein phosphatase 1 or 2A. Our study clearly supports the view that gene regulation by protein phosphorylation is achieved not only by modulating phosphorylating activity but also by modulating phosphatase activity. In view of the variety and complexity of protein phosphatases (reviewed by Cohen, 1989; Cohen and Cohen, 1989), there may be a signal transductional pathway that is primarily linked to a protein phosphatase. Identification of the protein phosphatase, kinase, and transcription factor involved in okadaic acid-dependent uPA gene regulation is awaited to unravel this new aspect of signal transduction.

Materials and methods Materials A stock solution of TPA (Pharmacia LKB Biotechnology Inc., 2 mg/ml) was made in ethyl acetate and stored at -20°C. An aliquot was air-dried and redissolved in DMSO at a concentration of 0.1 mg/mi. Okadaic acid was purchased from Moana BioProducts (Hawaii) and provided in dimethyl formamide at a concentration of 25 jg/ml (125 AM). 8-bromo-cAMP (BrcAMP) was purchased from Boehringer Mannheim, and cycloheximide was from Sigma. Escherichia coli DNA polymerase I Klenow fragment was obtained from Biofinex, and 'Gene screenplus' nylon filter from Du Pont-New England Nuclear. [a-32P]dATP was obtained from Amersham Corp.

Cell culture

LLC-PKI cells (Hull et al., 1976) were cultured in Dulbecco's modified

Eagle's medium supplemented with 10% fetal calf serum (Gibco, BRL) at 37°C in a humidified CO2 (6%) incubator. Cells were plated at 106 cells per 35 mm (diameter) plastic dish with 2 ml medium (with 10% fetal calf serum) and after 24 h treated with the reagents indicated. Probes cDNA clones for pig mPA, pYN15, and for pig ,B-actin have been described (Nagamine et al., 1984; Andrus et al., 1988). Radioactive probes were prepared by the random priming method (Feinberg and Vogelstein, 1983) using purified cDNA inserts. RNA analysis Total RNA was prepared according to Chomczynski and Sacchi (1987). Total RNA (5 jig) was resolved by electrophoresis under denaturing conditions and transferred to nylon filters as described (Nagamine et al., 1983). To confirm the loading and transfer of similar amounts of RNA, ribosomal RNA was visualized on nylon filters by staining the membrane with methylene blue (Herrin and Schmidt, 1988). Prehybridization and

hybridization were done as described (Andrus et al., 1988), and filters were exposed to Kodak XAR films with intensifying screens at -70°C.

cAMP-PK activity After treating cells as indicated in the figures, cell extracts were prepared and protein kinase activities were measured in the absence or presence of 10 AM cAMP using the synthetic substrate kemptide (Hemmings, 1986). The activity ratio ([-cAMP]/[+cAMP]) is a measure of the amount of in vivo-activated cAMP-PK.

Acknowledgements We thank Drs Brian Hemmings, Janet Lee, Robert Medcalf, Frederick Meins, Jr and George Thomas for critical reading of the manuscript, and Dr Brian Hemmings for many stimulating discussions. We thank Birgitta Kiefer for her excellent technical assistance.

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