In isolated rat hepatocytes, okadaic acid and cantharidin activate AMP-activated protein kinase (AMPK) by a naringin-sensitive and a naringin-resistant mechanism respectively, probably through inhibition of protein phosphatases PP2A and PP1, respectively.
The activating phosphorylation of AMPKá at Thr172 (detected by immunoblotting with a pThr172-specific antibody) induced by a 1-h treatment with okadaic acid was subsequently very stable at all toxin concentrations tested (30-100 nM), persisting even 2 hafter drug removal. AMPK phosphorylation induced by cantharidin (30 µM) was also completely stable for 2 h after removal of the toxin. Similarly, in correlation with the stable (Thr172) AMPKá phosphorylation, autophagy was inhibited in an irreversible manner upon incubation with 30 or 50 nM okadaic acid.
The stress-activated protein kinases, SAPK/Erk kinase (SEK1) and C-Jun NH2 terminal kinase (JNK), as well as S6 kinase and its substrate, S6, have been suggested as downstream components in a LKB1/AMPK-initiated signalling pathway and are phosphorylated following toxin treatment. However, unlike AMPK, all these phosphoproteins showed a reversal of phosphorylation upon toxin removal, questioning their roles as downstream mediators in an autophagy-regulating pathway.
At 30 nM and higher okadaic concentrations, AMPK phosphorylation was evident in immunoblots as at least three distinct and adjacent bands, the uppermost low-mobility form being predominant. This multi-phosphorylated AMPK was clearly enzymatically active in situ, as shown by the effective phosphorylation of its direct substrate, acetyl-CoA carboxylase (ACC) at Ser79. With a shorter incubation time (20 min) as well as at lower okadaic acid concentrations (10-15 nM), the monophosphorylated high-mobility form was the only bandpresent. This monophosphorylated AMPK appeared to be enzymatically inactive in relation to ACC phosphorylation, but still seemed to be able to suppress autophagic sequestration significantly. In addition, monophosphorylated AMPK allowed the phosphorylation of SEK1(pThr261), JNK (pThr183/Tyr185) and S6K (pThr421/Ser424), but not of S6 (pSer235/236). Thus, mono- and multi- (full) phosphosphorylated AMPK seem to have slightly different substrate preferences.
For further examination of the Thr172 AMPK immuno-stained bands, isoelectric focusing (IEF) and 2-D gel electrophoresis were utilized as protein separation methods. Identification by these techniques has been a difficult task, since phosphorylated AMPK has not been detectable. By using a general antibody against the AMPKá subunit, three distinct spots were detected after IEF. Two of these were identified as the á2 isoform.