Mammalian target of rapamycin-dependent phosphorylation of PHAS-I in four (S/T)P sites detected by phospho-specific antibodies

J Biol Chem. 2000 Oct 27;275(43):33836-43. doi: 10.1074/jbc.M006005200.

Abstract

The role and control of the four rapamycin-sensitive phosphorylation sites that govern the association of PHAS-I with the mRNA cap-binding protein, eukaryotic initiation factor 4E (eIF4E), were investigated by using newly developed phospho-specific antibodies. Thr(P)-36/45 antibodies reacted with all three forms of PHAS-I that were resolved when cell extracts were subjected to SDS-polyacrylamide gel electrophoresis. Thr(P)-69 antibodies bound the forms of intermediate and lowest mobility, and Ser(P)-64 antibodies reacted only with the lowest mobility form. A portion of PHAS-I that copurified with eIF4E reacted with Thr(P)-36/45 and Thr(P)-69 antibodies but not with Ser(P)-64 antibodies. Insulin and/or amino acids increased, and rapamycin decreased, the reactivity of all three antibodies with PHAS-I in both HEK293 cells and 3T3-L1 adipocytes. Immunoprecipitated epitope-tagged mammalian target of rapamycin (mTOR) phosphorylated Thr-36/45. mTOR also phosphorylated Thr-69 and Ser-64 but only when purified immune complexes were incubated with the activating antibody, mTAb1. Interestingly, the phosphorylation of Thr-69 and Ser-64 was much more sensitive to inhibition by rapamycin-FKBP12 than the phosphorylation of Thr-36/45, and the phosphorylation of Ser-64 by mTOR was facilitated by phosphorylation of Thr-36, Thr-45, and Thr-69. In these respects the phosphorylation of PHAS-I by mTOR in vitro resembles the ordered phosphorylation of PHAS-I in cells.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Adaptor Proteins, Signal Transducing
  • Amino Acid Sequence
  • Amino Acids / pharmacology
  • Antibodies / immunology*
  • Antibody Specificity
  • Carrier Proteins*
  • Cell Cycle Proteins
  • Cells, Cultured
  • Humans
  • Insulin / pharmacology
  • Molecular Sequence Data
  • Phosphoproteins / immunology
  • Phosphoproteins / metabolism*
  • Phosphorylation
  • Phosphotransferases (Alcohol Group Acceptor) / physiology*
  • Protein Kinases*
  • Sirolimus / pharmacology*
  • TOR Serine-Threonine Kinases
  • Tacrolimus Binding Protein 1A / pharmacology

Substances

  • Adaptor Proteins, Signal Transducing
  • Amino Acids
  • Antibodies
  • Carrier Proteins
  • Cell Cycle Proteins
  • EIF4EBP1 protein, human
  • Insulin
  • Phosphoproteins
  • Protein Kinases
  • Phosphotransferases (Alcohol Group Acceptor)
  • MTOR protein, human
  • TOR Serine-Threonine Kinases
  • Tacrolimus Binding Protein 1A
  • Sirolimus