Regulation of targets of mTOR (mammalian target of rapamycin) signalling by intracellular amino acid availability

Biochem J. 2003 Jun 1;372(Pt 2):555-66. doi: 10.1042/BJ20021266.

Abstract

In mammalian cells, amino acids affect the phosphorylation state and function of several proteins involved in mRNA translation that are regulated via the rapamycin-sensitive mTOR (mammalian target of rapamycin) pathway. These include ribosomal protein S6 kinase, S6K1, and eukaryotic initiation factor 4E-binding protein, 4E-BP1. Amino acids, especially branched-chain amino acids, such as leucine, promote phosphorylation of 4E-BP1 and S6K1, and permit insulin to further increase their phosphorylation. However, it is not clear whether these effects are exerted by extracellular or intracellular amino acids. Inhibition of protein synthesis is expected to increase the intracellular level of amino acids, whereas inhibiting proteolysis has the opposite effect. We show in the present study that inhibition of protein synthesis by any of several protein synthesis inhibitors tested allows insulin to regulate 4E-BP1 or S6K1 in amino-acid-deprived cells, as does the addition of amino acids to the medium. In particular, insulin activates S6K1 and promotes initiation factor complex assembly in amino-acid-deprived cells treated with protein synthesis inhibitors, but cannot do so in the absence of these compounds. Their effects occur at concentrations commensurate with their inhibition of protein synthesis and are not due to activation of stress-activated kinase cascades. Inhibition of protein breakdown (autophagy) impairs the ability of insulin to regulate 4E-BP1 or S6K1 under such conditions. These and other data presented in the current study are consistent with the idea that it is intracellular amino acid levels that regulate mTOR signalling.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenine / analogs & derivatives*
  • Adenine / pharmacology
  • Amino Acids / chemistry
  • Amino Acids / metabolism*
  • Animals
  • Anisomycin / pharmacology
  • Blotting, Western
  • CHO Cells / drug effects
  • CHO Cells / metabolism
  • Carrier Proteins / genetics
  • Carrier Proteins / metabolism*
  • Cricetinae
  • Cycloheximide / pharmacology
  • Enzyme Inhibitors / pharmacology
  • Gene Expression Regulation / drug effects
  • Immunosuppressive Agents / pharmacology
  • Insulin / pharmacology
  • JNK Mitogen-Activated Protein Kinases*
  • MAP Kinase Kinase 4
  • Mitogen-Activated Protein Kinase Kinases / antagonists & inhibitors
  • Mitogen-Activated Protein Kinase Kinases / metabolism
  • Mitogen-Activated Protein Kinases / antagonists & inhibitors
  • Mitogen-Activated Protein Kinases / metabolism
  • Peptide Initiation Factors / metabolism
  • Phosphoproteins / genetics
  • Phosphoproteins / metabolism*
  • Phosphorylation / drug effects
  • Protein Kinases / genetics
  • Protein Kinases / metabolism*
  • Protein Synthesis Inhibitors / pharmacology
  • Ribosomal Protein S6 Kinases / genetics
  • Ribosomal Protein S6 Kinases / metabolism*
  • Signal Transduction / drug effects
  • Sirolimus / pharmacology
  • TOR Serine-Threonine Kinases
  • p38 Mitogen-Activated Protein Kinases

Substances

  • Amino Acids
  • Carrier Proteins
  • Enzyme Inhibitors
  • Immunosuppressive Agents
  • Insulin
  • Peptide Initiation Factors
  • Phosphoproteins
  • Protein Synthesis Inhibitors
  • 3-methyladenine
  • Anisomycin
  • Cycloheximide
  • Protein Kinases
  • TOR Serine-Threonine Kinases
  • Ribosomal Protein S6 Kinases
  • JNK Mitogen-Activated Protein Kinases
  • Mitogen-Activated Protein Kinases
  • p38 Mitogen-Activated Protein Kinases
  • MAP Kinase Kinase 4
  • Mitogen-Activated Protein Kinase Kinases
  • Adenine
  • Sirolimus