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

Isabelle Mothe-Satney,Gregory J Brunn,Lloyd P Mcmahon,Christopher T Capaldo,Robert T Abraham,John C Lawrence

doi:10.1074/jbc.m006005200

Isabelle Mothe-Satney, Gregory J Brunn + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m006005200

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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.

Highlights

PHAS-I is a key element in a regulatory system that governs translation initiation by controlling the availability of eIF4E1 [1, 2]
The kinases participating in the ordered phosphorylation of PHAS-I are not known, it is clear that the phosphorylation of PHAS-I in cells is controlled in part by the mammalian target of rapamycin (mTOR) signaling pathway [1]. mTOR is a member of the family of protein kinases that have catalytic domains more homologous to phosphatidylinositol 3-OH-kinase than to members of the much larger protein kinase family, of which the catalytic subunit of cAMP-dependent protein kinase is the prototypic member [13]
Phosphorylation of PHAS-I becomes insensitive to rapamycin following expression of mTOR having a point mutation that disrupts high affinity binding to rapamycin-FKBP12. mTOR rendered kinase-dead by a point mutation in the kinase domain is ineffective in stimulating PHAS-I phosphorylation [16]

Summary

Introduction

PHAS-I ( known as 4E-BP1) is a key element in a regulatory system that governs translation initiation by controlling the availability of eIF4E1 [1, 2]. MTOR phosphorylated Thr-69 and Ser-64 but only when purified immune complexes were incubated with the activating antibody, mTAb1.

Results

Conclusion