Fig. 3. mTORC2 Binds and Phosphorylates a Novel TOR-Interaction Motif.
(A) Autoradiograph (35S) and Western blot of HA or Myc immunoprecipitates from a pulse-chase experiment of COS7 cells co-expressing HA-PKCβII and Myc-mTOR. The double asterisk (**) denotes the position of mature, fully phosphorylated PKC and the dash (−) indicates the position of unphosphorylated PKC. Blots are representative of three independent experiments.
(B) (top) Immunoblot analysis of 1-step, 15-mer peptide arrays spanning residues 615-643 (left) and residues 642-669 (right) of the PKCβII C-tail overlaid with Triton-solubilized lysate from WT MEFs expressing HA-Sin1 or Myc-mTOR and probed with antibodies for Sin1 (HA) or mTOR (Myc). The positions of the turn motif Thr641 and hydrophobic motif Ser660 are indicated. (far right) Peptide arrays of the indicated hydrophobic motif sequences were generated with phospho-Ser (pS) or unphosphorylated Ser (S) at the hydrophobic motif site and probed for mTOR as in panels on left. (bottom) Ala-scans of the indicated C-tail peptides were probed for Sin1 (HA) and mTOR; first spot is the wild-type peptide (WT). Blots are representative of three independent experiments.
(C) Docking of the Sin1 CRIM domain (NMR structure, PDB ID: 2RVK) to the PKCβII catalytic domain (X-ray structure, PDB ID: 2I0E). (inset) Interactions of the CRIM domain acidic loop with the PKCβII TOR-interaction motif helix are shown.
(D) Sequence alignment of the active-site tether and turn motif regions in the PKC C-tail, indicating the novel TOR-interaction motif Thr conserved in mTORC2-dependent PKC isozymes.
(E) Western blot of FLAG immunoprecipitates (IP:FLAG) from WT (+/+) or Sin1 KO (Sin1−/−) MEFs expressing FLAG-PKCβII alone or with HA-Sin1 and probed with an antibody to pThr634 or FLAG. Tubulin blot represents 10% whole-cell lysate input prior to immunoprecipitation.
(F) Western blot of whole-cell lysates (WCL) from HEK-293t cells expressing FLAG-PKCβII, treated with Torin (250 nM) for 36 h co-transfection, and probed with the indicated antibodies. (right) Quantification reflects the TIM phospho-signal (pThr634) relative to total PKC.
(G) Western blot from in vitro mTORC2 kinase assay, performed by incubation of HA-Sin1 or HA empty vector control (Vec) immunoprecipitated from HEK-293t cells transfected with these HA constructs, and GST-tagged PKCβII C-tail (a.a.601-673) WT or T634A purified by GST pulldown, in the presence or absence of Torin (200 nM) and probed with antibodies to mTORC2 components, GST, or an antibody to phospho-Thr. The asterisk (*) represents phosphorylated GST-PKCβII C-tail peptide and the dash (−) represents unphosphorylated peptide.
(H) Relative abundance of PKCβII C-tail peptide or phosphorylation at Thr634 for the in vitro kinase assay shown in (H) as determined by LC-MS. Data were obtained from two independent kinase assays.
(I) Representative spectrum of in vivo PKCβII Thr634 phosphorylation from analysis of a mouse brain phospho-proteomic dataset. Blue color denotes y ions and red color denotes b ions. (ppm-parts per million, Xcorr- spectral match correlation score).
**p < 0.01 by One-way ANOVA and Tukey HSD Test or Student’s t-test. Error bars represent SEM from at least three independent experiments.