Mammalian Target of Rapamycin-dependent Phosphorylation of PHAS-I in Four (S/T)P Sites Detected by Phospho-specific Antibodies

• Published in: The Journal of biological chemistry 2000-10, Vol.275 (43), p.33836-33843
• Main Authors: Mothe-Satney, Isabelle, Brunn, Gregory J., McMahon, Lloyd P., Capaldo, Christopher T., Abraham, Robert T., Lawrence, John C.
• Format: Article
• Language: English
• Subjects: 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; Tacrolimus Binding Protein 1A - pharmacology; TOR Serine-Threonine Kinases
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M006005200

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.