Stimulation of hepatocytic AMP-activated protein kinase by okadaic acid and other autophagy-suppressive toxins

Abstract

Autophagic activity in isolated rat hepatocytes is strongly suppressed by OA (okadaic acid) and other PP (protein phosphatase)-inhibitory toxins as well as by AICAR (5-aminoimidazole-4-carboxamide riboside), a direct activator of AMPK (AMP-activated protein kinase). To investigate whether AMPK is a mediator of the effects of the toxin, a phosphospecific antibody directed against the activation of phosphorylation of the AMPK alpha (catalytic)-subunit at Thr172 was used to assess the activation status of this enzyme. AICAR as well as all the toxins tested (OA, microcystin-LR, calyculin A, cantharidin and tautomycin) induced strong, dose-dependent AMPKalpha phosphorylation, correlating with AMPK activity in situ (in intact hepatocytes) as measured by the AMPK-dependent phosphorylation of acetyl-CoA carboxylase at Ser79. All treatments induced the appearance of multiple, phosphatase-sensitive, low-mobility forms of the AMPK alpha-subunit, consistent with phosphorylation at several sites other than Thr172. The flavonoid naringin, an effective antagonist of OA-induced autophagy suppression, inhibited the AMPK phosphorylation and mobility shifting induced by AICAR, OA or microcystin, but not the changes induced by calyculin A or cantharidin. AMPK may thus be activated both by a naringin-sensitive and a naringin-resistant mechanism, probably involving the PPs PP2A and PP1 respectively. Neither the Thr172-phosphorylating protein kinase LKB1 nor the Thr172-dephosphorylating PP, PP2C, were mobility-shifted after treatment with toxins or AICAR, whereas a slight mobility shifting of the regulatory AMPK beta-subunit was indicated. Immunoblotting with a phosphospecific antibody against pSer108 at the beta-subunit revealed a naringin-sensitive phosphorylation induced by OA, microcystin and AICAR and a naringin-resistant phosphorylation induced by calyculin A and cantharidin, suggesting that beta-subunit phosphorylation could play a role in AMPK activation. Naringin antagonized the autophagy-suppressive effects of AICAR and OA, but not the autophagy suppression caused by cantharidin, consistent with AMPK-mediated inhibition of autophagy by toxins as well as by AICAR.

Proteomic Analysis of Membrane-Associated Proteins from Rat Liver Autophagosomes

“…100,106 Although this autophagy suppression correlates with, and is generally ascribed to, activation of the AMP-activated protein kinase (AMPK), 48,64,100 a contribution by phagophore-associated FBPase cannot be excluded. Considering that the functionally reciprocal (glycolytic) enzyme, phosphofructokinase (PFK1 a-subunit), has been found to be essential for micropexophagy in methylotropic yeast, 155 FBPase could equally well exert an autophagic function, independently of its role in gluconeogenesis.…”

“…Subsequent washing and processing was performed as previously described. 48 Two-dimensional gel electrophoresis. For two-dimensional gel electrophoretic analysis of the protein composition of autophagosomal or cellular membranes, the sedimented pellets from freeze-thawed PNS or autophagosomes were dissolved in a sample buffer containing 65 mM dithiothreitol (DTT), 2% (w/v) Pharmalyte (Pharmacia AB, Uppsala, Sweden), 6 M urea, 2 M thiourea and 2% CHAPS (all Sigma).…”

“…One-dimensional gel electrophoresis and immunoblotting was performed essentially as previously described, 48 except that, with LC3, 15% rather than 10% gels were used for electrophoresis. For optimal immunoblotting (Western blotting) with the LC3 antibody, 43 proteins were wet-blotted onto PVDF membranes (0.2 mm) for 60 min at 100 V and 0.35 A, using a CAPS-based blotting buffer (10 mM CAPS, 10% v/v methanol, pH 11).…”

AMP-Activated Protein Kinase and Autophagy

“…6 By contrast, activation of AMPK by addition of the cell-permeable nucleotide analogue AICAriboside (AICAR) in hepatocytes strongly inhibits autophagy. 7,8 Because autophagy is accelerated when cells have insufficient oxidizable substrate at their disposal, inhibition of autophagy by AMPK activation under these conditions was, however, considered to be counterproductive. 5 Using different mammalian cell types, we have therefore reexamined the possible role of AMPK in the control of autophagy, and the new data 9 indicate that AMPK, like in yeast, is required for autophagy.…”

T cell proliferation and adaptive immune responses are critically regulated by protein phosphatase 4

“…17 Recently, PP4 has also been implicated in DNA damage response via its ability to either permit cell cycle reentry, 18 dephosphorylate gH2AX, 19,20 regulate the activity of KAP1, 21 or control cell cycles in Drosophila 22 or yeast 23 ; however, its role in regulating mammalian cell proliferation has not been thoroughly investigated. Finally, it is worth noting that okadaic acid (OA), which is generally recognized as a specific inhibitor of PP2A, actually also suppresses PP4 activity with equal 24 or better 25 efficacy; these results then raise the possibility that many biological processes, such as IL-2 signaling modulation, 26,27 AMPK activation 28 and the regulation of T cell proliferation, 29 that have been linked to PP2A via OA treatments may actually be attributed to the functions of PP4.…”