The proline-rich Akt substrate of 40 kilodaltons (PRAS40) was identified as a raptor binding protein that is phosphorylated directly by mTOR complex (mTORC) 1 but not mTORC 2 in vitro, predominantly at PRAS40 (Ser183).
The binding of S6K1 and 4E-BP1 to raptor requires a “TOR signaling” (TOS) motif which contains an essential Phe followed by four alternating acidic and small hydrophobic amino acids; PRAS40 binding to raptor was severely inhibited by mutation of PRAS40 (Phe129) to Ala; immediately carboxy-terminal to Phe129 are two small hydrophobic amino acid followed by two acidic residues, an arrangement different from that of the canonical TOS motif.
PRAS40 binding to raptor was also abolished by mutation of the major mTORC1 phosphorylation site Ser183, to Asp.
PRAS40 (Ser183) was phosphorylated in intact cells; as with S6K1 and 4E-BP1, PRAS40 (Ser183) phosphorylation was inhibited by rapamycin, by 2-deoxyglucose and by overexpression of the tuberous sclerosis complex heterodimer.
PRAS40 (Ser183) phosphorylation was also inhibited reversibly by withdrawal of all or of only the branched-chain amino acids; this inhibition was reversed by overexpression of the Rheb GTPase.
Overexpressed PRAS40 suppressed the phosphorylation of S6K1 and 4E-BP1 at their rapamycin-sensitive phosphorylation sites, and reciprocally, overexpression of S6K1 or 4E-BP1 suppressed phosphorylation of PRAS40 (Ser183) and its binding to raptor.
More importantly, RNAi-induced depletion of PRAS40 enhanced the amino-acid stimulated phosphorylation of both S6K1 and 4E-BP1.
These results establish PRAS40 as a physiological mTORC1 substrate that contains a variant TOS motif.
Moreover, they indicate that the capacity or ability of raptor to bind endogenous substrates is limiting for the activity of mTORC1 in vivo, and is therefore a potential locus of regulation, as suggested by others.
Applications:Suitable for use in Western Blot.
Other applications not tested.
Recommended Dilution:Western Blot: 1:1000 detected phosphorylated PRAS40 (Thr246) in RIPA lysates of unstimulated and PDGF stimulated NIH3T3 cells
Optimal dilutions to be determined by the researcher.
Positive Control:PDGF stimulated NIH3T3 cells.
Storage and Stability:May be stored at 4°C for short-term only.
Aliquot to avoid repeated freezing and thawing.
Store at -20°C.
Aliquots are stable for 12 months after receipt.
For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.
仕様
Size：100ul
Host：rabbit
Source Antibody：human
Grade：Affinity Purified
Purity：Purified by affinity chromatography.
Form：Supplied as a liquid in PBS (without Mg2+ and Ca2+), pH 7.3, 1mg/ml BSA (IgG, protease free), 0.05% sodium azide, 50% glycerol.
Specificity：Recognizes phosphorylated human PRAS40 (Thr246). Species Crossreactivity: mouse
Isotype：IgG
Calc Applications Abbrev：WB
Calc Crossreactivity：Hu Mo
Immunogen：Peptide corresponding to amino acid region encompassing the human phospho-PRAS40 (Thr246).
