Autophagy is a process used by eukaryotic cells to degrade their own cytoplasm under conditions of nitrogen starvation or stress, in order to obtain amino acids and other small molecules needed for the maintenance of essential cell functions. A variety of biological agents have been described to take part in the regulation of mammalian autophagy.
Naringin, a flavanone isolated from grapefruit peel as the bitter principle, is known to exhibit autophagy-preserving effects in isolated hepatocytes. We employed various autophagy inhibitors (e.g., protein phosphatase-inhibitory toxins and AICAR) to study naringin-sensitive protein phosphorylation pathways that could be implicated in the regulation of hepatocytic autophagy.
By using a proteomic approach in combination with phosphospecific immunoblotting and radioactive labeling we were able to identify a number of naringin-sensitive protein phosphorylations.
We found that okadaic acid induced a naringin-sensitive phosphorylation of glycine N-methyltransferase (GNMT), a key enzyme in the regulation of methionine metabolism and cellular methylation processes. Homocysteine, a metabolite of methionine, was a strong autophagy suppressant.
Similarly, we identified plectin as a high molecular weight naringin-sensitive phosphoprotein known to be of prime importance for the maintenance of intermediate filament networks. We also found that several protein kinases were subject to dynamic, naringin-sensitive phosphorylation, including S6 kinase, AMP-activated kinase (AMPK), JNK and SEK1.
Our results indicate a complex involvement of AMPK in S6 kinase regulation following toxin treatment, with evidence for a naringin-sensitive as well as a naringin-resistant mechanism, involving protein phosphatase PP2A and PP1, respectively. Furthermore, our results suggest that the toxins induce an AMPK-dependent phosphorylation of the S6K tail region in the absence of any concomitant enzyme activation. As a hypothetical model it is considered that S6K tail phosphorylation may convey a toxic signal independently of S6K enzymatic activity, causing, e.g. autophagy suppression through a putative LKB1/AMPK/SEK1/JNK/S6K signaling pathway.
List of papers. Papers I and III are removed from the thesis due to publisher restrictions.
Paper I: Møller, M.T.N., Samari, H.R., Fengsrud, M, Strømhaug, P.E., Østvold, A.C. and Seglen, P.O. Okadaic acid-induced, naringin-sensitive phosphorylation of glycine Nmethyltransferase in isolated rat hepatocytes. Biochem. J. (2003) 373: 505-513. doi:10.1042/BJ20030502
Paper II: Møller, M.T.N., Samari, H.R. and Seglen, P.O. (2004). Suppression of hepatocellular autophagy and ATP levels by homocysteine. Unpublished. Some of the findings are published in: Michael T.N. Møller, Hamid R. Samari, Lise Holden and Per O. Seglen. (2003). Regulation of Mammalian Autophagy by Protein Phosphorylation. In Daniel Klionsky (ed), Autophagy (pages.48-59). Landes Bioscience.
Paper III: Ruud Larsen, A.-K., Møller, M.T.N., Blankson, H., Samari, H.S., Holden, L. and Seglen, P.O. Naringin-sensitive phosphorylation of plectin, a cytoskeletal cross-linking protein, in isolated rat hepatocytes. J. Biol. Chem. (2002) 277:34826-34835. doi:10.1074/jbc.M205028200
Paper IV: Møller, M.T.N., Samari, H.R., Hildonen, S. and Seglen P.O. (2004). Toxin-induced Tail Phosphorylation of Hepatocellular S6 Kinase: Evidence for a dual Involvement of the AMP-activated protein Kinase in S6 Kinase Regulation. This is a pre-copyedited, author-produced PDF of an article accepted for publication in Toxicological Sciences following peer review. The version of record Toxicol Sci. 2004 Dec;82(2):628-37. is available online at: doi:10.1093/toxsci/kfh273
Paper V: Samari, H.R., Møller, M.T.N., Holden, L., Tonje Asmyhr and Seglen P.O. (2004). Stimulation of Hepatocytic AMP-Activated Protein Kinase by Okadaic Acid and other Autophagy-suppressive Toxins. Biochem. J. 2005 Mar 1;386(Pt 2):237-44. The final version of record is available at doi:10.1042/BJ20040609