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
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ABSTRACT |
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Key Words: rat; liver; hepatocyte; okadaic acid; microcystin; calyculin A; cantharidin; tautomycin; naringin; AICAR; S6 kinase; AMP-activated protein kinase.
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INTRODUCTION |
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Okadaic acid and other protein phosphatase-inhibitory toxins have been shown to induce hyperphosphorylation of cytoskeletal proteins like keratin (Blankson et al., 2000) and plectin (Ruud Larsen et al., 2002
) in isolated rat hepatocytes, and to cause disruption of the intracellular network of keratin intermediate filaments (Blankson et al., 1995
). The toxins also inhibit hepatocellular processes like autophagy, endocytosis, and protein synthesis (Gordon et al., 1995
; Holen et al., 1993
) and elicit apoptotic cell death when administered to hepatocytes in culture (Blankson et al., 2000
; Bøe et al., 1991
). 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., 1995
; Holen et al., 1993
; Ruud Larsen et al., 2002
). 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., 2002
). 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., 2002
; Samari and Seglen, 1998
). 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., 1998
). 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., 2002). 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, 1999
). S6 and S6K have also been implicated in the regulation of autophagy, which is suppressed by S6K-activating amino acids (Blommaart et al., 1995
). 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.
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MATERIALS AND METHODS |
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Animals and cells. Isolated hepatocytes were prepared from 18-h starved male Wistar rats (250300 g; Harlan UK Ltd., Shaws Farm, Oxon, UK) by two-step collagenase perfusion (Seglen, 1976), purified by differential centrifugation, and resuspended in suspension buffer fortified with 2 mM Mg2+ and 15 mM pyruvate (Seglen, 1976
). 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 TrisHCl, 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, 1976), 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, 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).
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RESULTS |
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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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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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DISCUSSION |
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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., 1985), 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., 2002
). (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., 2002
). 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., 2002
) (Fig. 13).
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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., 1995; Moore et al., 1991
), but there is some evidence that the latter may regulate dephosphorylation of the PP2C
subunit (Kobayashi et al., 1998
) 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., 2003b
), or that they somehow promote T172 phosphorylation of AMPK by its upstream protein kinase, LKB1 (Hawley et al., 2003
; Hong et al., 2003
; Woods et al., 2003a
).
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, 2002; Kimura et al., 2003
; Krause et al., 2002
). This effect of AMPK could be secondary to an inhibitory phosphorylation of mTOR, an S6K kinase (Cheng et al., 2004
). 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., 1995). 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., 2003
).
Since both algal toxins and AICAR have been shown to elicit hepatocellular apoptosis (Blankson et al., 2000; Meisse et al., 2002
), 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., 2000
; Harada et al., 2001
; Tee and Proud, 2001
); 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., 1998; Shima et al., 1998
), 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, 1999
). Thirdly, although protein kinases in the related RSK family (p90RSK 13) generally serve other cellular functions (Frödin and Gammeltoft, 1999
), 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., 2000
), in okadaic acid-treated maturing oocytes (Gavin and Schorderet-Slatkine, 1997
) or in mutant mice that lack both S6K and S6K2 (Pende et al., 2004
).
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.
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ACKNOWLEDGMENTS |
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NOTES |
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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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