The results presented above strongly support the conclusion that PRAS40 is a physiological substrate of mTORC1 kinase, whose phosphorylation at Ser183
is enabled by its binding to raptor. The interaction of PRAS40 with endogenous raptor is heavily dependent on PRAS40 (Phe129
), inasmuch as mutation of this residue to Ala greatly reduces the binding of recombinant PRAS40 to raptor; whether, as with 4E-BP1, additional PRAS40 segments are necessary for optimal binding to raptor is not yet established. Vander Haar et al.
) observed that Myc-PRAS40-(1–150) and -(150–234) are each unable to co-immunoprecipitate raptor, whereas PRAS40-(103–258) and -(1–234) are able to do so; taken together with the present data, this suggests that a PRAS40 segment between 150 and 234 is needed together with the region surrounding Phe129
for optimal binding to raptor. As with the TOS motif, the four PRAS40 residues carboxyl-terminal to Phe129
) consist of two hydrophobic (ϕ
) and two acidic (Ac) amino acids; however, the arrangement of these residues in S6K1, 4E-BP1 (13
), and STAT3 is Phe-Ac-ϕ
. Given the compelling evidence that PRAS40 is an endogenous substrate for mTORC1, we propose that the definition of a TOS motif be extended to include the motif Phe-ϕ
The binding of PRAS40 to raptor was also greatly inhibited by mutation of PRAS40 Ser183
to Asp, whereas mutation to Ala had a modest inhibitory effect on the interaction. We propose that the phosphorylation of Ser183
, like its conversion from Ser to Asp, results in the dissociation of PRAS40 from raptor. A similar phenomenon was observed with 4E-BP1, i.e.
mutation of the five mTORC1-catalyzed Ser/Thr phosphorylation sites to Glu abolished 4E-BP1 binding to raptor (19
). We propose that the ability of amino acid withdrawal to inhibit PRAS40 (Ser183
) phosphorylation in vivo
may account for the ability of amino acid withdrawal to increase the binding of endogenous PRAS40 to raptor. By contrast, when PRAS40 was overexpressed, amino acid withdrawal caused little or no further increase in retrieval of endogenous raptor (, , and ), perhaps because the cells are then expressing a large excess of PRAS40, much of which may not be phosphorylated at Ser183
. This is also a likely explanation for why recombinant PRAS40 (S183A) does not exhibit increased binding to raptor as compared with recombinant wild-type PRAS40 ().
The mechanism responsible for the ability of rapamycin to interfere with the association of endogenous PRAS40 with endogenous raptor ( (bottom) and ) is less obvious. Based on the ability of rapamycin to inhibit mTORC1-catalyzed phosphorylation of PRAS40 (Ser183) in vivo, rapamycin might be expected to increase rather than diminish the raptor-PRAS40 association. In fact, when raptor was overexpressed and presumably was present far in excess to endogenous mTOR, rapamycin did cause a small increase in the retrieval of endogenous PRAS40 by the recombinant raptor (, top). Under those conditions, rapamycin, which (with FKBP12) binds only to mTOR, inhibited mTORC1 kinase and therefore diminished PRAS40 (Ser183) phosphorylation; we suggest that the excess raptor unassociated with mTOR is unaffected by the FKBP12-rapamycin complex, and the decreased phosphorylation of endogenous PRAS40 at Ser183 then results in increased binding of endogenous PRAS40 to the overexpressed recombinant raptor (, top). In contrast, when the endogenous interactions were examined ( (bottom) and ) or when only PRAS40 was overexpressed (e.g. , , and ), the inhibitory effect of rapamycin on the association of endogenous raptor with recombinant PRAS40 was highly pronounced. Under these conditions, most or all cellular raptor is in mTORC1 and thus susceptible to the inhibitory action of the FKBP12-rapamycin complex on the contiguous mTOR; inasmuch as the FKBP12-rapamycin complex binds only to mTOR, the effect of rapamycin on raptor must be exerted through mTOR. Although the concentrations of rapamycin employed in these experiments (200 nm) were sufficient to cause major dissociation of the mTOR-mLST8 heterodimer from raptor in vivo (), it is unlikely that the loss of mTOR-mLST8 is itself the explanation for the loss of PRAS40 binding to raptor, inasmuch as complete dissociation of mTOR-mLST8 from raptor in vitro using Nonidet P-40 did not diminish the amount of PRAS40 bound to raptor (). Thus, the ability of rapamycin (at 200 nm) to inhibit the binding of endogenous ( (bottom) and ) or recombinant PRAS40 (, , and ) to endogenous raptor is not due to the loss of mTOR-mLST8 per se or to the dephosphorylation of PRAS40 (Ser183); rather, we propose that it reflects a decrease in the affinity of endogenous, mTORC1-associated raptor for PRAS40, which cannot be overcome by PRAS40 overexpression; generalizing this hypothesis, we propose that rapamycin may diminish the affinity of mTORC1-associated raptor for mTORC1 substrates in general. The mechanism for such an effect is unknown; although it is not due to the rapamycin-induced dissociation of raptor from mTOR-mLST8 per se (), it may be that the rapamycin-induced dissociation of mTOR-mLST8 from raptor that occurs in vivo (, , and ) eliminates some input from mTOR to raptor (e.g. phosphorylation) that is important for substrate binding. Clearly, much further work will be required to explore the validity of this hypothesis and the biochemical mechanisms responsible.
The identification of PRAS40 as a likely physiological substrate of mTORC1 raises the question of the function of mTORC1-catalyzed PRAS40 (Ser183
) phosphorylation. Vander Haar et al.
) and Sancak et al.
) showed that PRAS40 overexpression reduces the size of a variety of cells, consistent with the ability of PRAS40 overexpression to suppress S6K1 and 4E-BP1 phosphorylation. This suggests (but does not establish) that endogenous PRAS40 is not a positive regulator of cell size. In our hands, neither transient nor stable overexpression or siRNA-mediated knockdown of PRAS40 affected the size of K562 cells (data not shown). Sancak et al.
) showed that PRAS40 inhibits mTORC1-catalyzed phosphorylation of 4E-BP1 and S6K1 in vitro
, and a PRAS40 phosphorylated by Akt was less inhibitory in vitro
, whereas a PRAS40 (T246A) mutant was somewhat more inhibitory than wild-type PRAS40 during transient expression in vivo
. Vander Haar et al.
) both observed that sustained reduction of endogenous PRAS40 by lentivirus-encoded short hairpin RNA is accompanied by diminished levels of endogenous IRS1 polypeptide and diminished insulin activation of both Akt and S6K1. Consequently, both Vander Haar et al.
) and Sancak et al.
) propose that a primary function of endogenous PRAS40 is to suppress mTOR and up-regulate insulin signaling to Akt. The responses observed with sustained suppression of PRAS40, however, are those that would be expected from removal of a significant physiological mTORC1 substrate other than S6K1, especially one whose primary function is unrelated to the up-regulation of cell growth. As shown in the present studies, removal of PRAS40 would initially up-regulate the phosphorylation/activity of S6K1 (and other mTORC1 substrates), which in turn has been shown to result in the phosphorylation and enhanced degradation of IRS1 (36
), reviewed in Ref. 39
; these actions at the level of IRS1 would result in reduced insulin activation of Akt and S6K1 itself. Thus, although the present results do not eliminate a regulatory function for PRAS40 on mTORC1 or Akt signaling, they indicate strongly that the negative effects of PRAS40 depletion on Akt activation are likely to be largely or entirely attributable to the removal of an mTORC1 substrate rather than to a primary regulatory function of PRAS40.
The physiological function(s) of PRAS40 remain uncertain, and therefore the significance of the Akt-catalyzed phosphorylation of PRAS40 (Thr246
), the subsequent binding of 14-3-3 (20
), and the mTORC1-catalyzed phosphorylation of Ser183
also remain obscure. It has been reported that amino acids are required for the binding of 14-3-3 to PRAS40 phosphorylated at Thr246
). It is unlikely that amino acid-stimulated phosphorylation at PRAS40 (Ser183
) is a priming signal for the phosphorylation at Thr246
inasmuch as the insulin-stimulated phosphorylation at Thr246
is largely unaltered in rapamycin-treated cells () (20
). Alternatively, stable 14-3-3 binding to PRAS40 may require binding of the 14-3-3 dimer to PRAS40 at two independent sites (41
), one of which might be (Ser(P)183
PRAS40 has been implicated in the regulation of cell survival and apoptosis (43
). The introduction of PRAS40 in mouse brain protects neuronal cells from apoptosis after ischemic injury, and the phosphorylation of PRAS40 at Thr246
by Akt in response to nerve growth factor is proposed to be important for the prevention of apoptosis (43
). PRAS40 phosphorylated at Thr246
has been detected in mouse cortical neurons after ischemia and reperfusion (43
), and the phosphorylation of PRAS40 (Thr246
) and the subsequent binding of 14-3-3 are reported to be important for the survival of neuronal cells from apoptosis after ischemic injury in vivo
). Lobe, a putative D. melanogaster
ortholog of PRAS40, is required for ventral growth of eye disc and cell survival during early eye development (46
). Therefore, PRAS40 seems to play an important role in cell survival conserved among different species. Phosphorylated PRAS40 (Thr246
) is detected predominantly in the nuclei of H9c2-E2 cardiomyocytes and A14 fibroblasts after insulin stimulation (48
). Thus, nuclear PRAS40, phosphorylated by mTORC1 and/or Akt may regulate the transcription levels of anti- and/or proapoptotic proteins. Moreover, the transcription of PRAS40 itself is enhanced in TSC1 knock-out mouse astrocytes in which the mTORC1 pathway is up-regulated (49
), indicating that the expression level as well as the phosphorylation of PRAS40 is up-regulated by mTORC1 signaling. Thus, PRAS40 is a transcriptional and post-transcriptional target of mTORC1 regulation and thus likely to play an important, if as yet undefined role in mTORC1 action.