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.
Samari
Hamid R
HR
Proteomics and Mammalian Cell Biology Section, Department of Cell Biology, Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, 0310 Oslo, Norway.
Møller
Michael T N
MT
Holden
Lise
L
Asmyhr
Tonje
T
Seglen
Per O
PO
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