Identification of Protor as a novel Rictor-binding component of mTOR complex-2 - PubMed (original) (raw)

Identification of Protor as a novel Rictor-binding component of mTOR complex-2

Laura R Pearce et al. Biochem J. 2007.

Abstract

The mTOR (mammalian target of rapamycin) protein kinase is an important regulator of cell growth. Two complexes of mTOR have been identified: complex 1, consisting of mTOR-Raptor (regulatory associated protein of mTOR)-mLST8 (termed mTORC1), and complex 2, comprising mTOR-Rictor (rapamycininsensitive companion of mTOR)-mLST8-Sin1 (termed mTORC2). mTORC1 phosphorylates the p70 ribosomal S6K (S6 kinase) at its hydrophobic motif (Thr389), whereas mTORC2 phosphorylates PKB (protein kinase B) at its hydrophobic motif (Ser473). In the present study, we report that widely expressed isoforms of unstudied proteins termed Protor-1 (protein observed with Rictor-1) and Protor-2 interact with Rictor and are components of mTORC2. We demonstrate that immunoprecipitation of Protor-1 or Protor-2 results in the co-immunoprecipitation of other mTORC2 subunits, but not Raptor, a specific component of mTORC1. We show that detergents such as Triton X-100 or n-octylglucoside dissociate mTOR and mLST8 from a complex of Protor-1, Sin1 and Rictor. We also provide evidence that Rictor regulates the expression of Protor-1, and that Protor-1 is not required for the assembly of other mTORC2 subunits into a complex. Protor-1 is a novel Rictor-binding subunit of mTORC2, but further work is required to establish its role.

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Figures

Figure 1

Figure 1. Identification of Protor-1

(A) HEK-293 Cells were deprived of serum overnight and left untreated or stimulated with IGF-1 (50 ng/ml) for 30 min. HEK-293 cell extracts were subjected to immunoprecipitation with an anti-Rictor or pre-immune IgG antibody. The immunoprecipitates were electrophoresed on a polyacrylamide gel and the protein bands were visualized following Colloidal Blue staining. Each band visualized in the immunoprecipitate was given the indicated number, excised together with the equivalent region of the pre-immune immunoprecipitate, digested with trypsin and analysed by MS. The results obtained are shown in Supplementary Table 1 at

http://www.BiochemJ.org/bj/405/bj4050513add.htm

. The bands identified as Protor-1α and/or Protor-1β and other mTORC2 components are indicated with an arrow and were not detected within the control immunoprecipitate undertaken with pre-immune IgG antibody. The molecular mass (in kDa) of the indicated proteins is shown in parentheses, and asterisks denote mTORC2 components. (B) HEK-293 cells stably expressing TAP–Rictor were treated in the absence or presence of IGF-1 as in (A). TAP–Rictor was affinity-purified, electrophoresed on a polyacrylamide gel, and the protein bands were visualized following Colloidal Blue staining, labelled and analysed as in (A). The results are shown in Supplementary Table 2 at

http://www.BiochemJ.org/bj/405/bj4050513add.htm

.

Figure 2

Figure 2. Protor-1 binds mTORC2

(A) HEK-293 cells were transfected with a DNA construct encoding FLAG–Protor-1α. At 36 h post-transfection, cells were lysed, and the FLAG–Protor-1α was immunoprecipitated (IP) and electrophoresed on a polyacrylamide gel. The major protein bands were visualized following Colloidal Blue staining and labelled as indicated. The molecular mass (in kDa) of the indicated proteins is shown in parentheses, and asterisks denote mTORC2 components. (B) The labelled Colloidal-Blue-stained bands identified in (A) were excised from the gel, digested with trypsin and their identities determined by tryptic peptide mass-spectral fingerprint, as described in the Materials and methods section. Accession numbers are for the NCBI Entrez Protein database. †Mascot protein score, where a value >67 is considered significant (P<0.05). N.P.D., no significant protein identity determined. (C) Schematic representation of the Protor-1 isoforms and Protor-2. The conserved region is shaded and numbering of residues is based on the human sequence. For a sequence alignment of these proteins, see Supplementary Figure 1 at

http://www.BiochemJ.org/bj/405/bj4050513add.htm

. (D) As in (A), except that HEK-293 cells were transfected with the indicated forms of Protor and a control FLAG-epitope-tagged protein (TD2; Genbank® accession number NM_014779). Immunoprecipitates were immunoblotted (IB) with the indicated antibodies. Similar results were obtained in three separate experiments.

Figure 3

Figure 3. Endogenous Protor-1 binds to mTORC2

(A) HEK-293 cell lysates were subjected to immunoprecipitation (IP) with the indicated antibodies raised against different mTORC2 components as well as the SPAK protein kinase as a control, which would not be expected to bind mTORC2 and Filamin-A detected within the FLAG–Protor-1α immunoprecipitate shown in Figure 2(A). Immunoprecipitates were immunoblotted with the indicated antibodies. Similar results were obtained in three separate experiments. The molecular mass (in kDa) of the indicated proteins is shown in parentheses. (B) As in (A), except that cells were lysed in a buffer containing either no detergents or the concentrations of the indicated detergents.

Figure 4

Figure 4. Rictor interacts with Protor-1 and regulates its expression

(A) HEK-293 cells were co-transfected with the indicated DNA constructs encoding the indicated mTORC2 components. At 36 h post-transfection, cells were lysed and Protor-1 and Sin1 were immunoprecipitated (IP). The cell lysates (upper panel), the Protor-1 immunoprecipitates (middle panel) and Sin1 immunoprecipitates (lower panel) were immunoblotted with the indicated antibodies. Similar results were obtained in two separate experiments. The molecular mass (in kDa) of the indicated proteins is shown in parentheses. (B) Cell lysates derived from control wild-type Rictor+/+ and knockout Rictor−/− mouse embryonic fibroblast cells [30] were subjected to immunoblot analysis with the indicated antibodies. To confirm the marked reduction in Protor-1 expression, endogenous Protor-1 was immunoprecipitated (IP) and immunoblotted (lower panel). (C) HeLa cells were transfected with the indicated siRNAs (sequences as described in the Materials and methods section). At 72 h post-transfection, cells were lysed and cell lysates (upper panel) and the immunoprecipitates (lower panel) were immunoblotted with the indicated antibodies. Similar results were obtained in two separate experiments. The molecular mass (in kDa) of the indicated proteins is shown in parentheses.

Figure 5

Figure 5. Model of the subunit organization of mTORC2

The dotted line indicates the detergent-sensitive interaction between mTOR/mLST8 and Rictor/Sin1/Protor. ? indicates that direct interaction of these subunits is uncertain.

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