Toxin-Induced Tail Phosphorylation of Hepatocellular S6 Kinase: Evidence for a Dual Involvement of the AMP-Activated Protein Kinase in S6 Kinase Regulation

Michael T.N. Møller, Hamid R. Samari and Per O. Seglen1

Proteomics & Mammalian Cell Biology Section, Department of Cell Biology, Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, N-0310 Oslo, Norway

Received July 8, 2004; accepted August 25, 2004


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Several protein phosphatase-inhibitory toxins (okadaic acid, microcystin, calyculin A, cantharidin, tautomycin) administered to isolated rat hepatocytes were found to induce phosphorylation in the tail region of S6 kinase (S6K; p70S6K1) as detected with a phosphospecific antibody against doubly phosphorylated Thr-421/Ser424. 5-Aminoimidazole-4-carboxamide riboside (AICAR), an adenosine analogue that elicits activation of the hepatocellular AMP-activated protein kinase (AMPK), similarly stimulated S6K tail phosphorylation. The flavonoid naringin prevented the effects of AICAR, okadaic acid, and microcystin on AMPK activation as well as on S6K tail phosphorylation, suggesting AMPK as a mediator of the latter. The effects of AICAR and the toxins were rapamycin resistant; in contrast, amino acids induced an S6K tail phosphorylation that was rapamycin sensitive, suggesting mediation by the protein kinase mammalian target of rapamycin (mTOR). Amino acids activated S6K by phosphorylation at Thr-389, but the toxins did not, and AICAR in fact suppressed the activating phosphorylation induced by the amino acids. The possibility thus must be considered that the phosphorylated S6K tail may transmit a toxin-induced signal independently of S6K enzymatic activity. Despite their inability to activate S6K, the toxins (but not AICAR) stimulated phosphorylation of the ribosomal protein S6, presumably by activating some other S6-phosphorylating protein kinase.

Key Words: rat; liver; hepatocyte; okadaic acid; microcystin; calyculin A; cantharidin; tautomycin; naringin; AICAR; S6 kinase; AMP-activated protein kinase.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A variety of protein phosphatase-inhibitory toxins of biological origin have been shown to exert toxic effects on mammalian organisms and cells. Microcystins from blue-green algae are acutely hepatotoxic when administered to experimental animals or inadvertently ingested by livestock (Runnegar et al., 1986Go), and their presence in drinking water is suspected to contribute to liver cancer in certain parts of China (Carmichael, 1994Go; Ueno et al., 1996Go). Okadaic acid from marine dinoflagellates is well documented to be a major contributant to diarrhetic shellfish poisoning (Tester, 1994Go). However, when administered intravenously, okadaic acid evokes hepatotoxic rather than enterotoxic symptoms (Berven et al., 2001Go). Isolated rat hepatocytes (Seglen, 1976Go) would, therefore, seem to be a suitable experimental system in which to study the mechanisms of action of protein phosphatase-inhibitory environmental toxins in general (Fladmark et al., 1998Go).

Okadaic acid and other protein phosphatase-inhibitory toxins have been shown to induce hyperphosphorylation of cytoskeletal proteins like keratin (Blankson et al., 2000Go) and plectin (Ruud Larsen et al., 2002Go) in isolated rat hepatocytes, and to cause disruption of the intracellular network of keratin intermediate filaments (Blankson et al., 1995Go). The toxins also inhibit hepatocellular processes like autophagy, endocytosis, and protein synthesis (Gordon et al., 1995Go; Holen et al., 1993Go) and elicit apoptotic cell death when administered to hepatocytes in culture (Blankson et al., 2000Go; Bøe et al., 1991Go). Two toxic mechanisms can be distinguished on the basis of toxin dose-response characteristics and antagonistic effects of the grapefruit flavonoid, naringin: (1) a naringin-sensitive mechanism that seems to involve protein phosphatase PP2A, used by microcystin and low concentrations of okadaic acid, and (2) a naringin-resistant mechanism used by calyculin A, cantharidin, and tautomycin that probably involves PP1 (Gordon et al., 1995Go; Holen et al., 1993Go; Ruud Larsen et al., 2002Go). Such differential naringin sensitivity is also seen in the activating effects of toxins on the AMP-activated protein kinase (AMPK), suggesting AMPK as a mediator of the toxicity (Ruud Larsen et al., 2002Go). This hypothesis is strengthened by the ability of 5-aminoimidazole-4-carboxamide riboside (AICAR), an adenosine analogue that activates AMPK after being converted intracellularly to an AMP analogue, to mimic many of the hepatocellular effects of the toxins (Ruud Larsen et al., 2002Go; Samari and Seglen, 1998Go). The major function of AMPK is to save energy when cellular ATP levels are low (and AMP levels correspondingly high), a goal achieved by phosphorylation of key metabolic enzymes (Hardie et al., 1998Go). AMPK may conceivably exert a similar function in the preservation of cellular resources under conditions of toxic stress.

In a search for mediators of toxin-induced signaling downstream of AMPK, we have used proteomic methods and phosphospecific antibodies to identify toxin- and naringin-sensitive hepatocellular phosphoproteins (Ruud Larsen et al., 2002Go). One protein thus identified is S6 kinase (S6K), which plays an essential role in cellular mass growth by phosphorylating the ribosomal protein S6, thereby directing the ribosomes to mRNAs specifically associated with protein synthetic capacity (ribosomal proteins and translation factors) (Dufner and Thomas, 1999Go). S6 and S6K have also been implicated in the regulation of autophagy, which is suppressed by S6K-activating amino acids (Blommaart et al., 1995Go). An AMPK-dependent shutdown of these energy-requiring processes may well be a functional adaptive response to toxic stress.

In the present study we have investigated the effects of toxins on S6K phosphorylation, using a phosphospecific antibody directed against the tail region of the enzyme (where an activation-priming phosphorylation at T421/S424 takes place) as well as an antibody detecting the enzyme-activating phosphorylation at T389. The results suggest that the protein phosphatase-inhibitory toxins induce an AMPK-dependent phosphorylation of the S6K tail in the absence of any concomitant enzyme activation. The possibility thus must be considered that S6K tail phosphorylation may convey a toxin-induced signal independently of S6K enzymatic activity, causing, for example, autophagy suppression.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Reagents. Phosphospecific polyclonal rabbit antibodies against Thr389-phosphorylated or Thr421/Ser424-phosphorylated S6K, and horseradish peroxidase-linked anti-rabbit IgG antibody, were purchased from Cell Signaling Technology Inc. (Beverly, MA). Okadaic acid and microcystin-LR were from Alexis Biochemicals (Läufelfingen, Switzerland); calyculin A, cantharidin, and tautomycin from Calbiochem (San Diego, CA), and 5-aminoimidazole-4-carboxamide riboside (AICAR) from Toronto Research Chemicals (North York, ON). Rainbow molecular weight markers (RPN 756) and the ECL Western blotting detection kit were from Amersham Biosciences (Little Chalfont, Buckinghamshire, UK). Sodium dodecyl sulfate (SDS), acrylamide, and bisacrylamide were obtained from BioRad (Hercules, CA). Dry milk powder was from Nestlé (Vevey, Switzerland), and nitrocellulose membranes from Osmonics (Westborough, MA). Methanol and acetic acid were from Merck (Whitehouse Station, NJ), and alkaline phosphatase (MB grade) from Roche Appl. Sci., Penzberg, Germany. Other biochemicals were purchased from Sigma Chem. Co. (St. Louis, MO).

Animals and cells. Isolated hepatocytes were prepared from 18-h starved male Wistar rats (250–300 g; Harlan UK Ltd., Shaws Farm, Oxon, UK) by two-step collagenase perfusion (Seglen, 1976Go), purified by differential centrifugation, and resuspended in suspension buffer fortified with 2 mM Mg2+ and 15 mM pyruvate (Seglen, 1976Go). Aliquots of cell suspension, each containing about 30 mg cells (wet mass) in a volume of 0.4 ml, were incubated for 60 min at 37°C in shaking centrifuge tubes.

Gel electrophoresis and immunoblotting. Cell incubations were terminated by adding 2 ml of ice-cold Tris-buffered saline (TBS; 20 mM Tris-base, 0.1% Tween-20, pH 7.6) to each tube, followed by centrifugation of the cells at 1600 rev/min for 4 min in the cold (4°C); this washing was repeated once. The cells were lysed for 30 min on ice in one ml of lysis buffer containing 0.4% SDS, 5 mM EDTA, 5 mM EGTA, 10 mM sodium pyrophosphate, and 20 mM Tris-base, pH 7.2. The resulting whole-cell extracts were diluted 1:2 in double-strength SDS gel-loading buffer (single strength, 2% SDS, 1 M mercaptoethanol, 0.1% bromophenol blue, 10% glycerol, 50 mM Tris–HCl, pH 6.8) and heated boiled for 5 min at 95°C. After measuring the protein contents of the extracts by the method of Bradford (Bradford, 1976Go), using the BCA protein assay kit from Pierce (Rockford, IL), samples containing 20 µg were separated by SDS gel electrophoresis for approximately 40 min at 200 V in 10% polyacrylamide gels containing 0.1% SDS. Molecular weight markers were included in all gels.

Gel-separated proteins were transferred to nitrocellulose blotting membranes using a semi-dry transfer unit (Bio-Rad Laboratories, Hercules, CA) with Towbin's blotting buffer (192 mM glycine, 20% methanol, 25 mM Tris-base, pH 8.3). The membranes were blocked by overnight incubation at 4°C with 5% dry milk in TBS containing 0.2% Tween-20 (TBS-T), and washed three times for 10 min in TBS-T. For detection of total or phosphorylated AMPK{alpha}, the membranes were first incubated overnight at 4°C with the respective antibodies (diluted 1:1000 in TBS-T). After washing three times with TBS-T the membranes were incubated for 1 h at room temperature with anti-rabbit-horseradish peroxidase (diluted 1:2000 in TBS-T), washed three times, and visualized by chemiluminescence using the ECL Western Detection Kit (Amersham Biosciences).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Toxin-Induced Phosphorylation of the S6K Tail Region
The availability of a phosphospecific antibody that recognizes simultaneous phosphorylation of T421 and S424 in the C-terminal tail region of S6K, where multiple phosphorylations are involved in conformational changes required for activation of this enzyme (Dufner and Thomas, 1999Go), has allowed us to investigate the effects of protein phosphatase-inhibitory toxins on S6K tail phosphorylation. Okadaic acid, microcystin-LR, calyculin A, cantharidin, or tautomycin all induced a dose-dependent immunoreactivity in the upper of two adjacent bands in the 70-kDa region of the gel when administered to isolated rat hepatocytes incubated at 37°C (Fig. 1), okadaic acid being the most potent of the toxins. The present study has concentrated on this 70-kDa band, taken to represent the cytosolic p70S6K (henceforth referred to as S6K), although some toxin-induced immunostaining could be observed at higher molecular mass values as well.



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FIG. 1. Toxin-induced S6K tail phosphorylation. Freshly isolated rat hepatocytes were incubated for 1 h at 37°C with okadaic acid, microcystin-LR, calyculin A, cantharidin, or tautomycin at the concentrations indicated. Gel-separated cell extracts were immunoblotted with a phosphospecific antibody against Thr421/Ser424-phosphorylated S6K.

 
Immunoblotting with a general S6K antibody (Fig. 2A) showed that only the upper of the two 70 kDa bands was immunoreactive. A reduction in the mobility of this band, sometimes evident as an upward-directed band broadening, was observed at increasing concentrations of the toxins tested (okadaic acid, microcystin, calyculin A, cantharidin, and tautomycin). To check if the mobility shift might reflect toxin-induced S6K phosphorylation, cell extracts were treated with alkaline phosphatase prior to gel electrophoresis and immunoblotting. As shown in Figure 2B (upper lane), the phosphatase treatment abolished the mobility shifts induced by selected toxins (okadaic acid, microcystin, and tautomycin), and strongly reduced the signal obtained with the phosphospecific pThr421/pSer424 S6K antibody (lower lanes), thus demonstrating that the mobility shift was indeed due to S6K phosphorylation.



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FIG. 2. Phosphorylation-induced S6K mobility shift. (A) Hepatocytes were incubated for 1 h at 37°C with okadaic acid, microcystin, calyculin A, cantharidin, or tautomycin at the concentrations indicated. Cell extracts were immunoblotted with a general antibody against S6K. (B) Extracts of cells incubated with okadaic acid (300 nM), microcystin (3 µM,) or tautomycin (1 µM) were treated with alkaline phosphatase (150 units/ml) for 3 h at 37°C as indicated. The treated extracts were gel-separated and immunoblotted with a general S6K antibody (upper lane) or a T421/S424-phosphospecific antibody (lower lanes).

 
Differential Naringin Sensitivity of Toxin-Induced S6K Tail Phosphorylation
The toxins used have previously been shown to induce activating phosphorylations of the AMP-activated protein kinase (AMPK) by two different mechanisms: a naringin-sensitive mechanism associated with PP2A inhibition (okadaic acid, microcystin) and a naringin-resistant mechanism associated with PP1 inhibition (calyculin A, cantharidin, tautomycin) (Ruud Larsen et al., 2002Go). To see if S6K tail phosphorylation might similarly exhibit differential naringin sensitivity, the effect of this flavonoid was examined. As shown in Figure 3, naringin, while having no effect on its own, antagonized the phosphorylation-stimulatory effects of okadaic acid and microcystin, but not the effects of calyculin A, cantharidin, or tautomycin. The two toxin mechanisms operating on AMPK can thus also be observed at the level of S6K tail phosphorylation, consistent with the possibility of the latter phosphorylation being mediated by AMPK.



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FIG. 3. Effect of naringin on toxin-induced S6K tail phosphorylation. Hepatocytes were incubated for 1 h at 37°C with toxins and/or naringin at the concentrations indicated. Cell extracts were gel-separated and immunoblotted with an antibody against Thr421/Ser424-phosphorylated S6K.

 
Effects of AICAR and Amino Acids on S6K Tail Phosphorylation
To further examine the possibility of an AMPK involvement, we tested the effect of AICAR, an adenosine analogue converted by hepatocytes to an AMP analogue that activates AMPK (Samari and Seglen, 1998Go) in a naringin-sensitive manner (Ruud Larsen et al., 2002Go). AICAR had no effect on the absolute S6K levels as indicated by immunoblotting with the general S6K antibody (results not shown), but induced a dose-dependent phosphorylation of S6K at T421/S424 that was antagonized by increasing concentrations of naringin (Fig. 4A). This observation would be consistent with an involvement of AMPK in S6K tail phosphorylation. In contrast, a mixture of amino acids, which activate S6K through a mammalian target of rapamycin (mTOR)-dependent pathway (Blommaart et al., 1995Go; Shigemitsu et al., 1999Go), elicited a dose-dependent S6K tail phosphorylation that was resistant to naringin (Fig. 4B). Amino acids induced a more pronounced mobility shift than did the other phosphorylation stimulants, effectively causing a split of the upper 70-kDa band, probably indicating phosphorylation at more sites than with AICAR or toxins.



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FIG. 4. AICAR- and amino acid-induced S6K tail phosphorylation: effect of naringin. Hepatocytes were incubated for 1 h at 37°C with a physiological amino acid mixture, AICAR, and/or naringin at the concentrations indicated. Cell extracts were gel-separated and immunoblotted with an antibody against Thr421/Ser424-phosphorylated S6K.

 
The ability of AICAR to induce phosphorylation of S6K in a position generally associated with activation of the enzyme is surprising, since several previous studies have shown that AICAR inhibits S6K activity, preventing an activating phosphorylation at T389 (Dubbelhuis and Meijer, 2002Go; Kimura et al., 2003Go; Krause et al., 2002Go). We examined, therefore, if AICAR might antagonize the effects of amino acids and toxins on S6K tail phosphorylation. However, as shown in Figure 5, AICAR did not have any antagonistic effects on tail phosphorylation induced by the other effectors.



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FIG. 5. Lack of effect of AICAR on S6K tail phosphorylation induced by amino acids or toxins. Hepatocytes were incubated for 1 h at 37°C with amino acids or toxins in the absence or presence of AICAR at the concentrations indicated. Cell extracts were gel-separated and immunoblotted with an antibody against Thr421/Ser424-phosphorylated S6K.

 
The Toxin-Induced S6K Tail Phosphorylations Are Rapamycin- and Wortmannin-Resistant
The above results suggest that the naringin-sensitive S6K tail phosphorylations induced by okadaic acid, microcystin, and AICAR proceed by a mechanism different from the naringin-resistant phosphorylation induced by amino acids. However, since amino acids could conceivably share the mechanism used by the naringin-resistant toxins, the effect of rapamycin, an inhibitor of mTOR-mediated amino acid effects on S6K (Blommaart et al., 1995Go; Shigemitsu et al., 1999Go), was examined. As shown in Figure 6, rapamycin effectively antagonized the amino acid-induced S6K tail phosphorylation, but had no detectable effect on the phosphorylations induced by AICAR or any of the toxins. Thus, whereas amino acids seem to induce S6K tail phosphorylation through a rapamycin-sensitive, mTOR-mediated mechanism, AICAR and the toxins would appear to use a different, probably AMPK-mediated mechanism.



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FIG. 6. Effects of rapamycin on S6K tail phosphorylation induced by amino acids, AICAR or toxins. Hepatocytes were incubated for 1 h at 37°C with amino acids or toxins in the absence or presence of AICAR at the concentrations indicated. Cell extracts were gel-separated and immunoblotted with an antibody against Thr421/Ser424-phosphorylated S6K.

 
In addition to the two mechanisms discussed above, S6K is known to be regulated by hormones and growth factors (e.g., by insulin) through a wortmannin-sensitive pathway that involves PI 3-kinase (Shigemitsu et al., 1999Go). However, wortmannin did not antagonize any of the hepatocellular S6K tail phosphorylations, be they induced by amino acids, AICAR, okadaic acid, microcystin, or cantharidin, nor did wortmannin have any effect on its own (Fig. 7). Furthermore, we have not been able to observe any effects of toxins on the phosphorylative activation of protein kinase B, a common effector in the PI 3-kinase pathway (results not shown). The PI 3-kinase pathway would thus seem unlikely to be involved in toxin-induced S6K phosphorylation.



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FIG. 7. Lack of effect of wortmannin on S6K tail phosphorylation induced by amino acids, AICAR, or toxins. Hepatocytes were incubated for 1 h at 37°C with amino acids, AICAR, or toxins in the absence or presence of wortmannin at the concentrations indicated. Cell extracts were gel-separated and immunoblotted with an antibody against Thr421/Ser424-phosphorylated S6K.

 
Effects of Toxins, Amino Acids, and AICAR on S6 Phosphorylation
To see if the toxin-induced S6K tail phosphorylation was accompanied by S6K activation, S6K activity in the hepatocytes was examined as in situ phosphorylation of the enzyme's substrate, ribosomal protein S6. Using a phosphospecific antibody directed against double phosphorylation of the adjacent S6 positions S235 and S236, it could be shown that all of the toxins tested (okadaic acid, microcystin, calyculin A, cantharidin, and tautomycin), as well as amino acids, induced a dose-dependent phosphorylation of a double band around 32 kDa, corresponding to the position of S6 (Fig. 8), consistent with activation of S6K (or some other S6-phosphorylating enzyme). In contrast, AICAR failed to induce S6 phosphorylation (some inhibition of the basal S6 phosphorylation was in fact indicated, but the absolute levels were too low for reliable interpretation), indicating that the drug did not stimulate hepatocellular S6K activity.



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FIG. 8. Effects of toxins, amino acids, and AICAR on S6 phosphorylation. Hepatocytes were incubated for 1 h at 37°C with toxins, amino acids, or AICAR at the concentrations indicated. Cell extracts were gel-separated and immunoblotted with an antibody against Ser235/236-phosphorylated ribosomal protein S6.

 
These and the following experiments also included immunoblotting with a phosphospecific antibody directed against S6 doubly phosphorylated at Ser240 and Ser244, with results more or less identical to those obtained with the pSer235/236 antibody.

Differential Naringin Sensitivity of S6 Phosphorylation
S6 phosphorylation induced by okadaic acid or microcystin was antagonized by naringin, whereas phosphorylation induced by calyculin A, cantharidin, or tautomycin was not (Fig. 9), paralleling the differential naringin sensitivity of the AMPK activation and S6K tail phosphorylation elicited by these two groups of toxins. The amino acid-induced S6 phosphorylation was, as expected, unaffected by naringin (Fig. 9). No antagonism between AICAR and naringin could be reliably detected at the low, basal levels of S6 phosphorylation.



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FIG. 9. Effect of naringin on S6 phosphorylation induced by toxins, or amino acids. Hepatocytes were incubated for 1 h at 37°C with toxins, amino acids, or AICAR in the absence or presence of naringin at the concentrations indicated. Cell extracts were gel-separated and immunoblotted with an antibody against Ser235/236-phosphorylated ribosomal protein S6.

 
Effects of AICAR, Rapamycin, and Wortmannin on S6 Phosphorylation
In contrast to the inability of AICAR to antagonize the S6K tail phosphorylation elicited by amino acids (Fig. 5), this AMPK activator completely suppressed the amino acid-induced S6 phosphorylation at S235/236 (Fig. 10A). The inhibitory effect of AICAR was markedly antagonized by naringin, consistent with mediation by AMPK. These results thus corroborate previous studies, which have indicated that AICAR induces an AMPK-dependent inhibition of hepatocellular S6K activity (Dubbelhuis and Meijer, 2002Go; Kimura et al., 2003Go; Krause et al., 2002Go).



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FIG. 10. Effect of AICAR on S6 phosphorylation induced by toxins or amino acids. Hepatocytes were incubated for 1 h at 37°C with (A) amino acids (with or without naringin) or (B) toxins in the absence or presence of AICAR at the concentrations indicated. Cell extracts were gel-separated and immunoblotted with an antibody against Ser235/236-phosphorylated ribosomal protein S6.

 
The apparent ability of AMPK both to inhibit S6 phosphorylation (elicited by amino acids) and to stimulate S6 phosphorylation (elicited by toxins) raises a paradox. As shown in Figure 10B, toxin-induced S6 phosphorylation was not much affected by S6K-inhibitory concentrations of AICAR, possibly indicating that the toxins stimulated S6 phosphorylation by an S6K-independent mechanism.

Rapamycin, at 300 nM, completely abolished the amino acid-induced S6 phosphorylation, while having no effect on toxin-induced phosphorylation (Fig. 11). These observations are consistent with an mTOR-mediated amino acid effect, whereas the toxins apparently cause S6 phosphorylation by a different mechanism. Wortmannin had no effect on S6 phosphorylation elicited by either amino acids or toxins (results not shown).



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FIG. 11. Effect of rapamycin on S6 phosphorylation induced by amino acids or toxins. Hepatocytes were incubated for 1 h at 37°C with amino acids or toxins in the absence or presence of rapamycin at the concentrations indicated. Cell extracts were gel-separated and immunoblotted with an antibody against Ser235/236-phosphorylated ribosomal protein S6.

 
Effects of Toxins, Amino Acids, and AICAR on S6K-Activating Phosphorylation at Thr-389
Phosphorylation at T389 is absolutely required for S6K activity and can be used as a convenient indicator of S6K activation. As shown in Figure 12A, an amino acid mixture significantly stimulated T389 phosphorylation in isolated hepatocytes, as detected with a phosphospecific antibody directed against this site. The phosphorylation was rapamycin sensitive, indicating mediation through mTOR, and eliminated by AICAR as previously observed (Dubbelhuis and Meijer, 2002Go; Kimura et al., 2003Go; Krause et al., 2002Go), whereas naringin had no effect. The amino acid-antagonistic effect of AICAR was overcome by naringin (Fig. 12B), supporting the contention that AICAR inhibits S6K activity by signaling through the AMPK pathway.



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FIG. 12. Effects of amino acids, AICAR, toxins, and naringin on the phosphorylative activation of S6K. Hepatocytes were incubated for 1 h at 37°C with (A, B) amino acids, rapamycin, AICAR, naringin, or (C) toxins at the various combinations and concentrations indicated. Cell extracts were gel-separated and immunoblotted with an antibody against Thr389-phosphorylated S6K.

 
Okadaic acid and other toxins did not stimulate T389 phosphorylation (Fig. 12C), despite their ability to promote S6 phosphorylation as demonstrated above. This surprising observation suggests that the toxins do not activate S6K, indicating that some other kinase must be responsible for the toxin-induced S6 phosphorylation.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The present study shows that protein phosphatase-inhibitory toxins stimulate S6K tail phosphorylation at T421/S424 in isolated rat hepatocytes. Tail phosphorylation is also elicited by an amino acid mixture, but by a different mechanism: whereas the amino acid-induced tail phosphorylation is blocked by rapamycin, suggesting mediation by the protein kinase mTOR, the toxin-induced phosphorylation is not.

Two types of evidence indicate that the toxin-induced S6K tail phosphorylation may be mediated by the AMP-activated protein kinase, AMPK: (1) AICAR, an adenosine analogue that activates AMPK after being converted intracellularly to the AMP analogue, 5-amino-4-imidazolecarboxamide riboside 5'-monophosphate (ZMP) (Sabina et al., 1985Go), causes S6K tail phosphorylation. The phosphorylation is prevented by naringin, a flavonoid previously shown in our laboratory to antagonize AICAR-induced AMPK activation (Ruud Larsen et al., 2002Go). (2) S6K tail phosphorylation induced by the toxins okadaic acid or microcystin is similarly prevented by naringin, which also inhibits okadaic acid-induced AMPK activation (Ruud Larsen et al., 2002Go). Although other toxins (calyculin A, cantharidin, and tautomycin) stimulate S6K tail phosphorylation through a naringin-insensitive mechanism, this naringin-insensitivity is paralleled by the effects of these toxins on AMPK activation (our unpublished results). Thus both groups of toxins may stimulate S6K tail phosphorylation through the activation of AMPK. The naringin-sensitive and naringin-resistant mechanisms are thought to reflect the differential effects of the toxins on the inhibition of protein phosphatases PP2A and PP1, respectively (Ruud Larsen et al., 2002Go) (Fig. 13).



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FIG. 13. Toxin-induced signaling through AMPK and S6K: A hypothetical scheme. Two mechanisms for AMPK regulation are postulated, corresponding to two different regulatory AMPK phosphorylations (performed by unknown protein kinases) that are permissive for the activating phosphorylation at Thr172 (performed by the protein kinase LKB1). The naringin-sensitive mechanism, shared by okadaic acid and microcystin, is thought to involve inhibition of protein phosphatase 2A (PP2A), which may directly dephosphorylate the first permissive site on AMPK (as indicated here) or somehow indirectly stimulate phosphorylation of this site. AICAR acts through its phosphorylated derivative, ZMP, an AMP analogue that binds to AMPK and probably sensitizes it to phosphorylation at the permissive site. Naringin may prevent phosphorylation at this site (e.g., by competing with AMP/ZMP, or by inhibiting the responsible protein kinase). The naringin-resistant mechanism, shared by calyculin A, cantharidin, and tautomycin, is thought to involve PP1, which may directly dephosphorylate the second permissive site, or indirectly stimulate its phosphorylation. The activated AMPK sends a downstream signal, probably through several intermediary protein kinases, that results in (1) inhibition of the amino acid-inducible, rapamycin-sensitive, activating phosphorylation of S6K at Thr389, mediated by the protein kinase mTOR; (2) phosphorylation of S6K in the tail region (Thr421 and Ser424); and (3) phosphorylation of an unknown protein kinase (e.g., S6K2 or RSK) capable of phosphorylating the ribosomal protein S6. The S6K tail phosphorylation could possibly act as a mediating signal for toxin-induced apoptosis, inhibition of autophagy and disruption of the intracellular plectin/keratin cytoskeletal networks.

 
In vascular smooth muscle cells, the MAP kinase pathway and the PI 3-kinase-mTOR pathway have been shown to cooperate in phosphorylating S6K at S411 (Eguchi et al., 1999Go), but MAP kinase does not seem to be capable of phosphorylating rat liver S6K at T421/S424 (Mukhopadhyay et al., 1992Go), and as shown in the present study, inhibitors of PI 3-kinase (wortmannin) or mTOR (rapamycin) do not affect toxin-induced S6K tail phosphorylation. The protein kinase Cdc2 may also play a role in S6K tail phosphorylation at S411 (Mukhopadhyay et al., 1992Go; Papst et al., 1998Go), but this site seems to be phosphorylated independently of T421/S424 (Le et al., 2003Go). A more likely candidate would be the stress-activated protein kinase, c-Jun N-terminal protein kinase (JNK), which has been strongly implicated in T421/S424 phosphorylation in several epithelial cancer cell lines (Le et al., 2003Go), and which is activated in rat hepatocytes by AICAR and okadaic acid (Ruud Larsen et al., 2002Go) as well as by the other toxins studied here (our unpublished results).

The mechanism by which toxin-induced protein phosphatase inhibition causes activation of AMPK, a protein kinase, is not clear. AMPK is reportedly dephosphorylated at pT172 in vivo by PP2C rather than by the toxin-sensitive phosphatases, PP1 and PP2A (Davies et al., 1995Go; Moore et al., 1991Go), but there is some evidence that the latter may regulate dephosphorylation of the PP2C {alpha} subunit (Kobayashi et al., 1998Go) and, hence, control AMPK dephosphorylation indirectly. An alternative possibility is that the toxins prevent dephosphorylation of other, as yet uncharacterized sites involved in the regulation of AMPK activity (Woods et al., 2003bGo), or that they somehow promote T172 phosphorylation of AMPK by its upstream protein kinase, LKB1 (Hawley et al., 2003Go; Hong et al., 2003Go; Woods et al., 2003aGo).

The ability of AICAR to induce S6K tail phosphorylation is somewhat surprising, since this AMPK activator has previously been shown to inhibit S6K activity by preventing the activating phosphorylation of the enzyme at T389, elicited, for example, by amino acids (Dubbelhuis and Meijer, 2002Go; Kimura et al., 2003Go; Krause et al., 2002Go). This effect of AMPK could be secondary to an inhibitory phosphorylation of mTOR, an S6K kinase (Cheng et al., 2004Go). The present study confirms that AICAR does, in fact, inhibit both the amino acid-induced T389 phosphorylation and the activity of S6K as indicated by reduced S6 phosphorylation in intact cells. Both of these inhibitory effects of AICAR are antagonized by naringin, suggesting that they are mediated by AMPK. AMPK would thus appear to have a dual effect on S6K: it promotes tail phosphorylation, but suppresses T389 phosphorylation and S6K activity (Fig. 13).

Could the S6K tail phosphorylation serve an independent physiological function, unrelated to the S6-phosphorylating role of S6K? It is noteworthy that agents that either stimulate (amino acids), inhibit (AICAR), or have no effect (toxins) on S6K activity can all induce S6K tail phosphorylation. These diverse effectors have one other property in common: they all inhibit hepatocytic autophagy, in the same dose ranges that induce S6K tail phosphorylation. Previous investigators have pointed to a correlation between the abilities of amino acids to induce S6 phosphorylation and to suppress autophagy, suggesting that phosphorylated S6 might somehow act as an autophagy inhibitor (Blommaart et al., 1995Go). Although the ability of AICAR to inhibit autophagy while suppressing S6 phosphorylation does not support an autophagy-regulatory role for S6, the effect of AICAR on S6K tail phosphorylation would be compatible with a nonenzymatic, autophagy-regulatory role for S6K. The possibility is not entirely unprecedented: in yeast, the protein kinase Atg1 has been found to regulate autophagy (albeit in a stimulatory capacity) independently of its kinase activity (Abeliovich et al., 2003Go).

Since both algal toxins and AICAR have been shown to elicit hepatocellular apoptosis (Blankson et al., 2000Go; Meisse et al., 2002Go), the possibility should also be considered that AMPK-induced S6K modifications might play a role in apoptotic signaling. Inactivation of S6K has been shown to favor apoptosis under several conditions (Bonatti et al., 2000Go; Harada et al., 2001Go; Tee and Proud, 2001Go); in addition, phosphorylation of the S6K tail could conceivably act as a separate pro-apoptotic signal. Whether tail-phosphorylated S6K can promote apoptosis or suppress autophagy independently of the enzyme's enzymatic (S6-phosphorylating) activity will need to be investigated by means of S6K mutations. Unfortunately, such mutations cannot presently be generated in hepatocytes, due to the extremely limited proliferation capacity of these cells in culture.

Future studies are also required to resolve another paradox arising from the present results: how can the toxins stimulate S6 phosphorylation without activating S6K? The most likely answer is that another kinase, activated by the toxins, is responsible for the S6 phosphorylation. One candidate would be the closely related enzyme, S6K2 (p70ßS6K), which differs sufficiently from S6K (S6K1) to preclude immunological cross-reactivity (Gout et al., 1998Go; Shima et al., 1998Go), and which might, therefore, have escaped detection by the pT389 antibody. Furthermore, both the S6K and the S6K2 transcripts generate larger (p85) translation products with a nuclear localization, probably engaged in the phosphorylation of S6 at nucleolar sites (Dufner and Thomas, 1999Go). Thirdly, although protein kinases in the related RSK family (p90RSK 1–3) generally serve other cellular functions (Frödin and Gammeltoft, 1999Go), they may be capable of phosphorylating S6, as observed, for example, in human peripheral blood mononuclear cells after leptin treatment (van den Brink et al., 2000Go), in okadaic acid-treated maturing oocytes (Gavin and Schorderet-Slatkine, 1997Go) or in mutant mice that lack both S6K and S6K2 (Pende et al., 2004Go).

Given the numerous signaling pathways and cellular processes regulated by toxin-sensitive protein phosphatases, it is perhaps not surprising that the toxins may have complex effects even on a single biochemical event such as S6 phosphorylation. Whether the toxin effects on S6 and S6K are a cause or a manifestation of cellular toxicity remains to be shown.


    ACKNOWLEDGMENTS
 
This study has been generously supported by The Norwegian Cancer Society. The excellent technical assistance of Frank Sætre and Suphawadee Finsnes is gratefully acknowledged.


    NOTES
 
M. T. N. Møller and H. R. Samari contributed equally to the present study.

1 To whom correspondence should be addressed. Fax: +47 22 93 45 80. E-mail: p.o.seglen{at}4labmed.uio.no.


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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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