Phosphorylation of Ribosomal Protein S6 Is Inhibitory for Autophagy in Isolated Rat Hepatocytes

Abstract

In rat hepatocytes, autophagy is known to be inhibited by amino acids. Insulin and cell swelling promote inhibition by amino acids. Each of the conditions leading to inhibition of autophagic proteolysis was found to be associated with phosphorylation of a 31-kDa protein that we identified as ribosomal protein S6. A combination of leucine, tyrosine, and phenylalanine, which efficiently inhibits autophagic proteolysis, was particularly effective in stimulating S6 phosphorylation. The relationship between the percentage inhibition of proteolysis and the degree of S6 phosphorylation was linear. Thus, inhibition of autophagy and phosphorylation of S6 are under the control of the same signal transduction pathway. Stimulation of S6 phosphorylation by the presence of amino acids was due to activation of S6 kinase and not to inhibition of S6 phosphatase. The inhibition by amino acids of both autophagic proteolysis and autophagic sequestration of electro-injected cytosolic [14C]sucrose was partially prevented by rapamycin, a compound known to inhibit activation of p70 S6 kinase. In addition, rapamycin partially inhibited the rate of protein synthesis. We conclude that the fluxes through the autophagic and protein synthetic pathways are regulated in an opposite manner by the degree to which S6 is phosphorylated. Possible mechanisms by which S6 phosphorylation can cause inhibition of autophagy are discussed.

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

“…32 The ability of amino acids to stimulate S6 phosphorylation (which reflects 70 kDa S6 kinase (S6K) activity in situ) in hepatocytes in a rapamycin-and wortmannin-sensitive manner, and their synergy with insulin, not only provided a possible clue to a mechanism by which amino acids control autophagy but also was, in fact, the first demonstration that amino acids are able to stimulate insulin signaling. 10 A few years later, this property of amino acids was confirmed for other insulin-sensitive cell types and, once again, leucine (but not the other branched-chain amino acids) was found to be the most effective amino acid. 33 In addition to S6, downstream targets of mTOR, such as S6K, eukaryotic translational initiation factor 4E-binding protein-1 (4E-BP1), eukaryotic initiation factor-2 alpha (eIF2a) kinase (the equivalent of general control nondepressible 2 (Gcn2) in yeast), and eEF2kinase, were found to be phosphorylated in response to amino-acid addition in a wortmannin-sensitive or LY294002-sensitive manner.…”

“…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. 10 Insulin alone did not stimulate S6 phosphorylation, and S6 phosphorylation induced by high concentrations of amino acids did not require the presence of insulin. Unlike insulin, glucagon inhibited S6 phosphorylation at intermediate concentrations of amino acids and stimulated proteolysis.…”

“…Since rapamycin also produced a partial inhibition of protein synthesis in the absence of cycloheximide, it was proposed that the opposing processes of (global) protein synthesis and (autophagic) protein degradation may both be controlled by the same signaling pathway, which would be an efficient solution to metabolic regulation. 10 The ability of rapamycin to stimulate autophagy has also been observed in other cell types, including yeast. 13 Amino-acid stimulation of S6 phosphorylation was inhibited by the phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin or LY294002.…”

“…Under various conditions, a linear relationship was found between the percentage inhibition of autophagic proteolysis (measured in the presence of cycloheximide to inhibit simultaneous protein synthesis) and the degree of phosphorylation of S6. 10 Amino-acidinduced S6 phosphorylation was completely prevented by rapamycin, indicating that mTOR and p70S6 kinase were components of the signaling pathway. Rapamycin partly, but not completely, abolished the inhibition of autophagy by amino acids.…”

“…8 In yeast, the kinase complex, Atg1, plays a central role in integrating signals downstream of the nutrient sensor TOR. 9 Despite the fact that (mammalian) target of rapamycin (mTOR) is involved in the control of autophagy in mammalian cells, 10 its connection with the molecular machinery remains to be clarified.…”