Our previous data have demonstrated that TTP plays an important role in the regulation of mRNA stability in response to mTOR inhibition [24
]. Here we demonstrate that TTP function is linked to TOR activity via an mTORC2 component Protor-2. Protor-2 was identified as an interactor with TTP in a yeast two-hybrid screen and the two proteins were subsequently shown to be co-immunoprecipitatable in whole cell extracts. Binding of Protor-2 to TTP was shown to require sequences within the C-terminal half of both proteins and binding was induced in mTORC2 activated cells, consistent with previous reports describing the liberation of Protor-2 from the mTORC2 complex following hyperactivation [23
]. Forced ectopic expression of Protor-2 resulted in increased mRNA turnover of transcripts known to be regulated by TTP. Additionally, knockdown of Protor-2 reduced mRNA degradation of TTP-associated messages and inhibited TTP association with SGs and P-bodies following stress induction. These data are most consistent with a model of mTORC2 activity dependent TTP function in which, upon activation of mTORC2, Protor-2 is released from the complex and subsequently associates with TTP to promote decay of TTP-regulated mRNAs. In support of this model, we also tested the notion of whether the mitochondrial oxidative phosphorylation uncoupler FCCP would induce mTORC2 activity. As shown in supplementary figure 2
, FCCP exposure induced phospho-S473
-AKT accumulation in a rictor-dependent fashion. These data are consistent with reports demonstrating that several inhibitors of mitochondrial function can induce AKT activity via the accumulation of reactive oxygen species or inactivation of PTEN [35
]. Additionally, we have demonstrated that during other cellular stress responses such as heat shock, the mTORC2/AKT pathway is activated [37
TTP is known to play a role in the regulated mRNA turnover of several mRNA following T lymphocyte activation [2
]. TPA or LPS mediated activation of T cells results in induction of immediate, early genes the transcripts for several of which contain AREs which bind TTP. Current models suggest that upon cell stimulation, a class of TTP-regulatable mRNAs are induced coordinately with TTP expression. TTP then binds to these mRNAs and selectively mediates transcript destabilization resulting in a rapid cessation of expression of these factors [6
]. Many of these mRNAs encode pro-inflammatory proteins whose overexpression may be deleterious to the cell and thus, are regulated in this manner post-transcriptionally. The role of mTORC2 activity in the regulation of immediate, early gene expression is not known, however our data are indicative that it may play a role in the regulation of TTP-mediated mRNA destabilization. It is tempting to speculate that mTORC2 is activated following T cell stimulation and liberates Protor-2 which promotes TTP-mediated mRNA turnover.
The role of TTP in the regulation of mRNA turnover in response to cell stress is well known [6
]. However, the molecular events regulating TTP activity have only recently been described [12
]. The expression of ARE-containing mRNAs requires the activation of signaling cascades which ablate this decay program. The p38 MAPK/MK2 cascade phosphorylates TTP at two residues (serines 52 and 178) which induces adaptor 14-3-3 protein binding and sequesters TTP within the cytoplasm of the cell [12
]. 14-3-3 protein binding also results in the exclusion of TTP from cytoplasmic stress granules (SGs) and mRNA processing bodies (P-bodies), sites of storage of translationally arrested mRNAs and mRNA degradation, respectively [1
]. The observation that knockdown of Protor-2 inhibits TTP association with these subcellular structures, suggests a role for Protor-2 in trafficking TTP-bound mRNA substrates to SGs and/or P-bodies. The mechanism of Protor-2 promotion of TTP mediated decay may involve increased binding affinities of TTP to its mRNA substrates, regulating subcellular localization or controlling subsequent steps in the decay process such as mRNA deadenylation or decapping. It is also of interest whether other TIS11 family members such as BRF1 and BRF2, can bind Protor-2 and are potentially subject to mTORC2 regulation.
Recently Kedar et al., [39
] have reported TTP interactors derived from large-scale yeast two-hybrid screening efforts. As in our experiments, full-length TTP was found to be autoactivating, as were baits containing N-terminal fragments of the protein. While there was no overlap in the reported focused list of TTP interacting proteins with those identified in this study, the identification of proline-rich domain containing proteins was in common. In our screens, we also identified components of the mRNA decay machinery which appear to be able to interact with the C-terminal mRNA decay domain of TTP. Other studies have suggested that the N-terminal decay domain may function as a binding platform for mRNA decay enzymes [7
], however our results suggest that the C-terminal domain is also able to interact with components of the decay machinery. Additionally, it is also possible that the C-terminal domain may interact with other factors involved in mRNA remodeling or in the localization of mRNAs to cytoplasmic processing-bodies as previously suggested [7
]. Our finding that the RNA-helicase RHAU can bind the C-terminal domain of TTP supports this [40
The regulatory subunits of several multisubunit kinase complexes have been reported as having kinase-independent functions [41
]. These include the p85α regulatory subunit of PI3K, which has been shown to potentiate JNK signaling under certain conditions [42
]. It has also been proposed that the glucose regulatable yeast Snf1 kinase complex may be targeted to specific intracellular locations via interaction with regulatory subunits [43
]. There is also evidence that the regulatory subunits of casein kinase II have functions independent of the holoenzyme involving activation of c-Raf, c-Mos or Chk1 [41
]. It is certainly possible that many regulatory subunits or closely associated factors of specific kinase complexes have evolved kinase-independent functions and our studies with Protor-2 support this. In fact, the Rictor and Sin1 mTORC2 regulatory subunits are known to have TOR-independent functions [44
In conclusion, this work presents experimental evidence that mTORC2 activity may be functionally linked to regulated mRNA turnover. Recently Protor-2 has been implicated in the promotion of apoptosis [23
]. The regulated destabilization of antiapoptotic mRNAs known to contain AREs, such as Bcl-2, would be consistent with the function of Protor-2 we have observed and its role in apoptosis.
> The mTORC2 kinase component Protor-2 is found to interact with the mRNA destabilizing factor tristetraprolin. > Modulation of Protor-2 levels specifically alters the stability of mRNAs known to be degraded by tristetraprolin. > Inhibition of Protor-2 expression blocks the association of tristetraprolin with stress granules or processing bodies during stress. > We conclude that Protor-2 regulates tristetraprolin mediated mRNA turnover and may link mTORC2 signaling to regulated mRNA stability.