Inhibition of hepatocytic autophagy by okadaic acid and other protein phosphatase inhibitors

Abstract

Autophagy, measured as the sequestration of electroinjected [3H]raffinose or endogenous lactate dehydrogenase, was inhibited in isolated rat hepatocytes by the protein phosphatase inhibitors okadaic acid, calyculin A and microcystin-LR. Okadaic acid, the most potent inhibitor, suppressed autophagy almost completely at 15 nM, suggesting inhibition of a protein phosphatase of type 2A. Okadaic acid had no effect on ATP levels, protein synthesis or cellular viability at this concentration, but caused a disruption of the hepatocytic cytoskeleton and a consequent reduction in organelle sedimentability, potentially interfering with the autophagy assay unless the necessary precautions are taken. Lysosomal (propylamine-sensitive) degradation of endogenous protein was inhibited by okadaic acid, whereas non-lysosomal (propylamine-resistant) degradation was unaffected. The autophagy-inhibitory effect of okadaic acid was not affected by inhibitors of CAMP-dependent protein kinase or protein kinase C (H-7, H-89, calphostin C) but eliminated by the non-specific inhibitor K-252a and its analogues (KT-5720, KT-5823, KT-5926) and by KN-62, a specific inhibitor of Ca2+/ calmodulin-dependent protein lunase 11. Protein phosphorylation by this kinase would thus seem to play a role in regulation of the autophagic-lysosomal degradation pathway.Autophagy, an intracellular mechanism for the bulk degradation of cytoplasm, plays an important role in protein metabolism and growth control [ 1 -31. The stepwise nature of the process has been suggested by ultrastructural and biochemical studies. In the first step, autophagic sequestration, a portion of the cytoplasm is wrapped up by a membraneous organelle which eventually forms a closed autophagic vacuole, an autophagosome [4]. This vacuole then matures into, or delivers the autophagocytosed cytoplasm to a second autophagic vacuole, an amphisome, so called because it can also receive material from endocytosis [ 5 ] . The amphisome in turn delivers its contents to a lysosome for degradation [61.The biochemistry of autophagy is largely uncharted. Regulation of the process occurs mainly at the initial sequestration step, which is sensitive to various hormones, growth factors and metabolites [3, 71. In rat hepatocytes the rate of autophagic sequestration and the formation of autophago- somes is influenced by several agents known to transmit their effects through protein kinases, such as glucagon [8, 91, insulin [lo, 111, adrenergic agonists [12, 131 and cyclic nucleotides [14]. An involvement of protein phosphorylation in the biological control of autophagy would, therefore, seem likeIn the present study we have examined this possibility by treating isolated rat hepatocytes with okadaic acid and other protein phosphatase inhibitors. Okadaic acid is a toxin associated with diarrhetic seafood poisoning, first isolated from the marine sponge Hulichondria okudui [15]. Its mechanism of action is a specific inhibition of protein phosphatases 1 (PP1) and 2A (PP2A), two of the major phosphatases responsi...

Naringin-sensitive Phosphorylation of Plectin, a Cytoskeletal Cross-linking Protein, in Isolated Rat Hepatocytes

“…for example the inhibition of the mitogen-activated protein kinase kinases MEK1 and MEK2 by PD-98059 (5Ј-methoxy-6Ј-aminoflavone) (69), and of cyclin-dependent protein kinases by flavopiridol (70)). The okadaic acid-antagonistic effect of naringin on hepatocytic autophagy can be mimicked by several inhibitors of CaMK-II (71,72), but as shown in the present study, naringin had no direct inhibitory effect on CaMK-II; nor did it affect PKA or PKC activity in in vitro assays. 2 However, by using a proteomic approach in combination with phosphospecific immunoblotting, we were able to identify a number of naringin-sensitive protein phosphorylations in intact hepatocytes.…”

“…The toxins examined in the present study differ in their absolute as well as their relative potencies toward PP1 and PP2A, yet all of them induced plectin phosphorylation as well as fragmentation of the plectin network. Okadaic acid is readily taken up by isolated hepatocytes (72), and its high absolute potency with regard to plectin phosphorylation and network disruption would be strongly indicative of a PP2A involvement, its affinity for PP1 being 2 orders of magnitude lower (1,2). Microcystin also behaves as a selective intracellular PP2A inhibitor by virtue of its specific binding to PP2A (42), although under cell-free conditions it is an equally potent inhibitor of PP1 (42,82).…”

Leucine Limitation Induces Autophagy and Activation of Lysosome-dependent Proteolysis in C2C12 Myotubes through a Mammalian Target of Rapamycin-independent Signaling Pathway

“…As is observed for protein synthesis, autophagy is also regulated by protein phosphorylation (22,48). When protein synthesis is inhibited, it was found that inhibition of autophagic proteolysis following amino acid supplementation, was correlated with the phosphorylation of ribosomal S6 protein in hepatocytes isolated from starved rats (22).…”

Autophagy and signaling: their role in cell survival and cell death

“…Shortly after a report indicating that pharmacological inhibition of protein phosphatases inhibited autophagy in hepatocytes, 25 an important step forward occurred in our understanding of the mechanism by which amino acids inhibit autophagy. This was the discovery that adding low concentrations of amino acids (leucine being particularly effective) to hepatocytes rapidly stimulated the phosphorylation of ribosomal protein S6 (S6), in synergy with either insulin or cell swelling induced by hypo-osmosis.…”