Stimulation of hepatocytic AMP-activated protein kinase by okadaic acid and other autophagy-suppressive toxins.
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.
Proteomics and Mammalian Cell Biology Section, Department of Cell Biology, Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, 0310 Oslo, Norway.
Hamid R
HR
Samari
Michael T N
MT
Møller
Lise
L
Holden
Tonje
T
Asmyhr
Per O
PO
Seglen
D055372:AMP-Activated Protein Kinases; D000596:Amino Acids / Q000494:pharmacology; D000620:Aminoimidazole Carboxamide / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D001343:Autophagy / Q000187:drug effects*; D024202:Electrophoretic Mobility Shift Assay / Q000379:methods; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D044950:Flavanones / Q000494:pharmacology; D008297:Male; D052998:Microcystins; D009097:Multienzyme Complexes / Q000378:metabolism*; D019319:Okadaic Acid / Q000494:pharmacology*; D010456:Peptides, Cyclic / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D021122:Protein Subunits / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D012265:Ribonucleotides / Q000494:pharmacology; D014118:Toxins, Biological / Q000494:pharmacology*
D000596:Amino Acids; D004791:Enzyme Inhibitors; D044950:Flavanones; D052998:Microcystins; D009097:Multienzyme Complexes; D010456:Peptides, Cyclic; D021122:Protein Subunits; D012265:Ribonucleotides; D014118:Toxins, Biological; D019319:Okadaic Acid; D000620:Aminoimidazole Carboxamide; C078588:microcystin; D017346:Protein Serine-Threonine Kinases; D055372:AMP-Activated Protein Kinases; C031143:AICA ribonucleotide; C005274:naringin