PRAS40 Regulates mTORC1 Kinase Activity by Functioning as a Direct Inhibitor of Substrate Binding

Lifu Wang,Thurl E Harris,Richard A Roth,John C Lawrence

doi:10.1074/jbc.m702376200

Abstract

Mammalian target of rapamycin (mTOR) functions in two distinct signaling complexes, mTORC1 and mTORC2. In response to insulin and nutrients, mTORC1, consisting of mTOR, raptor (regulatory-associated protein of mTOR), and mLST8, is activated and phosphorylates eukaryotic initiation factor 4E-binding protein (4EBP) and p70 S6 kinase to promote protein synthesis and cell size. Previously we found that activation of mTOR kinase in response to insulin was associated with increased 4EBP1 binding to raptor. Here we identify prolinerich Akt substrate 40 (PRAS40) as a binding partner for mTORC1. A putative TOR signaling motif, FVMDE, is identified in PRAS40 and shown to be required for interaction with raptor. Insulin stimulation markedly decreases the level of PRAS40 bound by mTORC1. Recombinant PRAS40 inhibits mTORC1 kinase activity in vivo and in vitro, and this inhibition depends on PRAS40 association with raptor. Furthermore, decreasing PRAS40 expression by short hairpin RNA enhances 4E-BP1 binding to raptor, and recombinant PRAS40 competes with 4E-BP1 binding to raptor. We, therefore, propose that PRAS40 regulates mTORC1 kinase activity by functioning as a direct inhibitor of substrate binding.

Highlights

Mycin-insensitive and contains Mammalian target of rapamycin (mTOR), rictor, mLST8, and hSIN [2]. mTORC1 catalyzes the phosphorylation of eIF4E binding protein-1 (4EBP1, known as PHAS-I) and p70 S6 kinase 1 (S6K1), whereas mTORC2 phosphorylates Ser-473 in the hydrophobic motif of Akt/PKB [3]
We have recently demonstrated that insulin produced a stable increase of mTORC1 kinase activity, and this response was associated with a marked increase in substrate binding to raptor [14]
To determine the component of mTORC1 associated with prolinerich Akt substrate 40 (PRAS40), extracts prepared with 0.2% of Tween 20, CHAPS, Nonidet P-40, and Triton X-100 were used for the immunoprecipitations of mTOR and raptor

Summary

EXPERIMENTAL PROCEDURES

Materials—Antibodies to mTOR [23], raptor [14], mLST8 [24], PRAS40 [22], eIF4E [25], and HA [14] have been described previously. Cells were cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum (Invitrogen) for 10 –12 days after adding the differentiation medium. HEK293T, HEK293E, NIH-3T3, and CHO-IR cells were cultured in Dulbecco’s modified Eagle’s medium containing 5% fetal bovine serum. The pGEX-4T-1 constructs encoding NH2-terminal GST-tagged PRAS40 and PRAS point mutations of Phe129 to Ala (F129A) and GST-FKBP12 were expressed in bacteria and purified as described previously [28]. 800 ␮l of cell extracts were incubated with 15 ␮l (Amersham Biosciences) of beads coupled with His-tagged 4EBP1 at 4 °C for 2 h with constant mixing and washed as described for immunoprecipitations. The GST-FKBP12coupled beads were incubated with 1 ␮M either rapamycin, FK506, or Me2SO for 30 min and washed with buffer A. Gel Filtration—Performed as previously described [14]

RESULTS

Insulin Promotes a Marked

DISCUSSION