Substrate specificity of TOR complex 2 is determined by a ubiquitin-fold domain of the Sin1 subunit

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Summary:

The authors show that Sin1 in S. pombe, unlike mammals, is not required for the catalytic activity of TORC2. They further demonstrate that the Conserved Region In the Middle (Sin1CRIM) forms a unique domain that specifically binds the substrate Gad8. They solved the 3-D structure of Sin1CRIM, which exhibits a ubiquitin-like fold with a characteristic acidic loop. The specific substrate recognition function is conserved in human Sin1CRIM. Importantly, the latter binds Akt.

Essential revisions:

The paper presents very strong data to support the authors' conclusions and clarifies discrepancies in the literature. Most of the revisions that are requested by the reviewers deal with explanations as to why certain experiments were not performed, and some control experiments that can be dealt with in a relatively short time. One comment that is of particular importance relates to mutations in the negatively charged loop that are drastic and might well disturb overall folding. Interactions of these residues in the structure should be shown. The reviewer asks whether milder variants have been tested? NMR would be suited to address this concern.

Reviewer #1:

The two complexes of TOR kinase, TORC1 & 2 are central regulators of cell growth, proliferation and dynamics. Tatebe et al. provide novel functional and structural insights to the role of Sin1 in TORC2 substrate recognition. They conclude that Sin1 in S. pombe (in contrast to mammals) is not required for TORC2 integrity and show that the CRIM rather than the PH/RBD domain of Sin1 is essential for TORC2-mediated phosphorylation of Gad8. A specific substrate recognition was also demonstrated for the CRIM domain of human Sin1. In a random mutagenesis screen the authors identify a series of mutations in S. pombe Sin1, which abolish Gad8, but not Tor1 interaction. The authors then present an NMR structure of S. pombe mSin1-CRIM, which reveals that the mutations identified in the mutagenesis screen affect buried residues and thus rather disturb the fold than the recognition mechanisms. In the structure, the authors identify a negatively charged loop as a potential interaction element, and vary this loop by mutating either all hydrophobic or all negatively charged residues; these drastic variations indeed diminish Gad8 interaction.

The role of Sin1-CRIM in substrate recognition is further demonstrated by fusing it to the Rictor homologue Ste20 in a Δsin1 background resulting in productive phosphorylation of Gad8 by TORC2. The fact that binding of Gad8 is sufficient for phosphorylation is indicated by the fact that Gad8 is phosphorylated even by TORC1 when fused to the Raptor homologue mip1.

The manuscript is well written and provides important novel insights to the role of Sin1 in TORC2 substrate recognition. The integration between the different parts of the manuscripts, in particular functional and structural studies, could be improved.

1) Results of random mutagenesis: The identified residues should be labelled in Figure 3—figure supplement 1, and implications of perturbed folding for different modes of TORC2 interaction should be discussed.

2) NMR studies: The NMR studies carried out by the authors would be optimally suited to monitor the folding state of all SIN1-CRIM variants used here and the authors should discuss why corresponding experiments have not been carried out.

3) The mutations in the negatively charged loop are drastic and might well disturb overall folding. Interactions of these residues in the structure should be shown. Have milder variants been tested? NMR would be suited to address this concern.

4) The minimal fragment of Sin1 necessary for the interaction with Gad8 is aa. 281-400. Further truncation from either end abolishes Gad8 interaction. The two flanking regions are distant in the folded domain. The authors should discuss whether this indicates multiple interactions sites.

5) In later experiments, the authors again used mutations L348S and L364S (Figure 3B), which presumably affect the fold of Sin1-CRIM. Why weren't the variants in the acidic loop tested here?

Reviewer #2:

Tatebe et al. found that the TORC2 component, SIN1, in fission yeast is not required for the integrity of TORC2, but determines the substrate specificity of TORC2. They identified the conserved CRIM domain in SIN1 as the region that is required for binding to the substrate Gad8. Using truncation and point mutants, they also showed that human SIN1 could perform a similar function towards its substrate Akt. Structural analysis of the CRIM domain further identified an acidic loop that is essential for interaction with the TORC2 substrates. Fusion of Gad8 with Ste20, in the absence of SIN1, enabled Gad8 phosphorylation, suggesting that SIN1 is dispensable for TORC2 catalytic activity.

Using a combination of biochemical, genetic, and structural analyses, the study provides new insights on how TORC2 phosphorylates its substrates and elucidates the importance of SIN1. The results are quite robust and convincing and the fusion studies are particularly interesting. Some comments below could further support the conclusion and extend relevance to mammalian TORC2.

1) Do the truncation mutants in Figure 1 still bind TORC2? They should also test some of their mutants for binding to mammalian mTOR and rictor.

2) In Figure 2B, binding of GAD8 to the CRIM-containing mutants should be compared with the RBD/PH-only containing mutants as negative control.

3. Phosphorylation of other mTORC2 target sites (e.g. Akt T450) and substrates (e.g. PKC) should be examined in Figure 4G. Do the mutants also bind mTOR and rictor?

4) Where was the analysis on sin1-CRM alone on Figure 5A? This was stated in the text but not included in the figure. The effect of ste20-sin1CRM on growth upon stress was also rather weak yet Gad8 was strongly phosphorylated by this mutant. Could this mutant be less efficient for phosphorylation of other AGC targets of TORC2?

Reviewer #3:

The mTORC2 kinase plays an important role in conveying growth factor signals to downstream AGC family protein kinases by phosphorylating their hydrophobic motif, an essential step in their activation. Unlike the related mTORC1 kinase, for which a substrate recruitment motif named TOS has been established, how mTORC2 recruits its substrates has not been well understood. While some studies have implicated the mTORC2 subunit Sin1 in substrate recruitment, other studies have reached contradictory conclusions.

In this manuscript, Tatebe et al. provide a wealth of experimental data conclusively showing that Sin1 recruits the Gad8 kinase and its human homologue AKT, and that this interaction is necessary for substrate phosphorylation. The data include yeast two hybrid assays, IPs and mutagenesis, which map the interaction to the Sin1 CRIM domain, as well as heterologous fusion of the CRIM domain to other mTORC2 subunits. They also report the solution structure of the CRIM domain, which adopts a ubiquitin fold and has an acidic loop to which most of the mutations that abrogate Gad8 binding map. The authors then go on to identify similar interactions between human Sin1 CRIM and AKT. Overall this manuscript is well written and the experiments are well executed with proper controls. The data supports the authors' conclusions.

The only drawback is that it has been previously reported (Cameron et al. 2011) that human SIN1 interacts with the mTORC2 substrates PKC epsilon and AKT through essentially the same CRIM acidic patch, although I agree with the authors that this has been somewhat controversial in the field (discussed by the authors in the second paragraph of the subsection “Sin1 CRIM domain binds the AGC kinases that are phosphorylated by TORC2”). As it settles this question, and also provides detailed structural and mutational data, I believe the manuscript merits publication in eLife.