In this study, we show that Rictor is regulated by multisite phosphorylation in a C-terminal region, with a sequence conserved only in vertebrate orthologs of Rictor. If we combine previous phospho-proteomic data with the 21 unique high-confidence sites identified in this study, Rictor appears to be phosphorylated on as many as 37 different sites, primarily Ser or Thr residues within a putative regulatory region (summarized in Table S2 in the supplemental material). While we focus here on the regulation of sites likely to be phosphorylated by AGC family kinases, the identities of the remaining sites will greatly facilitate future studies of the regulation and functions of Rictor and mTORC2. We identify T1135 as a major growth factor-regulated site on Rictor that is sensitive to rapamycin. It has been previously reported that rapamycin treatment causes an electrophoretic mobility shift in Rictor to a faster-migrating form, suggestive of dephosphorylation (1
). However, these experiments were done using prolonged treatment with rapamycin, which results in diminished assembly of mTORC2 (51
), thereby eliminating any Rictor phosphorylation site that is dependent on complex formation. Acute rapamycin treatment (e.g., 5 to 15 min), which eliminates T1135 phosphorylation, does not affect mTORC2 stability, nor does it cause a mobility shift on Rictor immunoblots. We identified several proline-directed phosphorylation sites in our LC/MS/MS analyses that could be sites of mTOR-mediated phosphorylation of Rictor within mTORC2 (i.e., autophosphorylation of the complex), and these sites might be primarily responsible for the previously reported shifts in Rictor mobility in response to prolonged rapamycin.
Our data demonstrate that the T1135 residue on Rictor is an S6K1-specific substrate. It remains possible that additional AGC family kinases can phosphorylate this site under specific conditions. However, the facts that Rictor-T1135 phosphorylation is acutely sensitive to rapamycin in numerous cell lines and that S6K1 knockdown blocks phosphorylation in response to both insulin and full serum suggest that S6K1 is the primary T1135 kinase in vivo. This site is phosphorylated on Rictor within mTORC2 and stimulates binding of 14-3-3 to Rictor. However, the precise molecular effects of this regulation are unknown. This site does not affect complex integrity or the intrinsic kinase activity of mTORC2, but a nonphosphorylatable mutant of Rictor (T1135A) reproducibly increases the ability of mTORC2 to phosphorylate Akt on S473 in cells. While this phosphorylation event is certainly not an on/off switch for mTORC2, it appears to play a role in mTORC2 regulation within cells, at least for its phosphorylation of Akt. It was somewhat surprising to find that this site does not affect other known mTORC2 targets, such as SGK1, PKCα, or the turn motif on Akt (T450). The sparse nature of our current understanding of growth factor-mediated activation of mTORC2 and its spatial regulation of Akt and its other substrates limits our ability to define the molecular mechanism of this regulatory effect at this time. However, our findings suggest that Rictor-T1135 phosphorylation and subsequent 14-3-3 binding specifically dampen the ability of mTORC2 to phosphorylate Akt on S473 in response to growth factors.
Our results are consistent with S6K1-mediated phosphorylation of Rictor on T1135 being a mechanism of feedback regulation or cross talk between mTORC1 and mTORC2. Like S6K-mediated regulation of IRS-1 upstream of PI3K, Rictor-T1135 phosphorylation represents another mechanism by which the status of mTORC1 activation leads to feedback regulation of Akt signaling (see the model in Fig. ). Our direct comparison of the acute effects of rapamycin to the effects of the Rictor-T1135A mutant on Akt phosphorylation suggests that the mTORC1-dependent feedback regulation of mTORC2 acts in concert with the well-documented effects on IRS-1. While the effects of the T1135A mutant on Akt-S473 phosphorylation are modest, they are comparable in scale to the effects of rapamycin, which should block all mTORC1-mediated feedback mechanisms. The parallel and cooperative nature of these two feedback mechanisms is illustrated by the fact that rapamycin treatment of cells expressing the Rictor-T1135A mutant inhibits IRS-1 feedback and results in a further increase in insulin-stimulated Akt phosphorylation.
FIG. 8. Model of mTORC1-dependent feedback mechanisms affecting insulin-stimulated activation of Akt. Insulin and IGF1 stimulate IRS-1 binding to PI3K, thereby activating PI3K and increasing its production of phosphotidylinositol-3,4,5-trisphosphate (PIP3). PIP3 (more ...)
While feedback mechanisms affecting IRS-1 have been intensely studied for years, the effects of individual phosphorylation sites on insulin-stimulated Akt activation have not been thoroughly examined. A few studies have overexpressed phosphorylation site mutants of IRS-1 affecting specific mTORC1- or S6K1-regulated sites, in otherwise wild-type backgrounds, but only rarely have the effects of these mutants on Akt phosphorylation been analyzed. One study, which found IRS-1-S1101 to be phosphorylated by S6K1, detected a modest increase in insulin-stimulated Akt-S473 phosphorylation upon overexpression of an S1101A mutant in CHO cells (59
). We are aware of only one study that has reconstituted an Irs
null cell line with a mutant affecting a residue known to be phosphorylated by S6K1 (13
). Surprisingly, in this particular study, expression of this mutant (IRS-1-S302A) actually decreased insulin-stimulated Akt-S473 phosphorylation relative to that in wild-type-reconstituted cells. Given our current knowledge of IRS-1 feedback mechanisms, it seems feasible that Rictor-T1135 phosphorylation contributes to the acute effects of mTORC1 signaling on Akt activation to an extent similar to that of IRS-1 serine phosphorylation. Whether additional mTORC1-mediated feedback mechanisms affecting PI3K-Akt signaling exist is unknown. It is interesting to note that neither T1135 on Rictor nor the specific inhibitory phosphorylation sites on IRS-1 are conserved in the Drosophila
orthologs of these proteins. However, Drosophila
TORC1 and S6K have been shown to exert negative regulatory effects on Akt, such that their knockdown increases Akt phosphorylation on its hydrophobic motif (45
). This suggests that there are alternative TORC1-dependent feedback mechanisms in Drosophila
or that yet another mechanism that is conserved in Drosophila
exists in mammalian cells. Given the complexity of signaling within the PI3K-mTOR signaling network, it would not be surprising if additional feedback mechanisms exist.
In cell lines lacking the tuberous sclerosis complex (TSC) tumor suppressors (TSC1 and TSC2), in which mechanisms of mTORC1-driven insulin resistance have been studied, chronic activation of mTORC1 causes a dramatic reduction in both IRS-1 transcript and protein levels, resulting in an inability of insulin to significantly stimulate PI3K activation (17
). However, these cells also exhibit a loss of mTORC2 kinase activity due to a role for the TSC1-TSC2 complex in activating mTORC2 (23
). Although we detect growth factor-independent phosphorylation of Rictor-T1135 in Tsc2
-deficient cells due to constitutive S6K1 activation, the T1135 site does not substantially affect mTORC2 kinase activity or signaling to SGK1 or PKCα, which are all attenuated by loss of the TSC1-TSC2 complex (23
). Furthermore, the effects of the TSC1-TSC2 complex on mTORC2 are largely independent of its effects on mTORC1 signaling. Therefore, while it might contribute to the overall block in Akt activation in TSC-deficient cells, Rictor-T1135 phosphorylation is unlikely to be a major mechanism underlying the severe attenuation of mTORC2 activity upon loss of the TSC1-TSC2 complex.
Our knowledge of mTORC2 regulation and function has lagged greatly behind that of mTORC1. Fundamental questions remain regarding mechanisms of mTORC2 assembly, activation, and interaction with its downstream substrates. Furthermore, it seems likely that we are just scratching the surface with regard to mTORC2 functions and, perhaps, mTORC2-independent functions of Rictor. It is possible that phosphorylation of T1135, and/or other sites identified in our study, will be important for the regulation of these other putative functions. Given the shear number of phosphorylation sites identified on just this one subunit of mTORC2, its regulation is likely to be quite complex and perhaps vary significantly dependent on specific stimuli and settings.