Specific blockade of Rictor-mTOR association inhibits mTORC2 activity and is cytotoxic in glioblastoma
Fig 5
JR-AB-000 and JR-AB-011 bind to Rictor and prevent Rictor-mTOR association.
(A) Surface plasmon resonance analysis of JR-AB-000 binding to immobilized Rictor, mSIN1 or mTOR as indicated (left panel). Binding sensorgrams of immobilized mTOR with Rictor over the indicated concentration range (right panel). The Kon, Koff and Kd were calculated by simultaneous non-linear regression using a 1:1 binding model and BIAevaluation 3.1 software. (B) Competitive binding curves of Rictor-mTOR association in the absence or presence of JR-AB2-011 or JR-AB2-000 as indicated (left panel). Analysis of selectivity of JR-AB2-011 (middle panel) or JR-AB2-000 (right panel) binding to Rictor, Raptor, mLST8 or Deptor as shown. Samples were preincubated with inhibitors and Rictor, Raptor, mLST8 or Deptor proteins as indicated and run over sensor chip containing immobilized mTOR. The IC50 values were calculated using the response units at the dissociation phase. (C) mTOR-Flag coupled beads binding to myc-Rictor in the presence of increasing JR-AB2-011 (top panels) or JR-AB2-000 (bottom panels). myc-Rictor was incubated with inhibitor for 1 h followed by incubation with FLAG agarose beads coupled to mTOR-Flag (mTOR-Flag beads). Binding of myc-Rictor to mTOR-Flag beads (Rictor-mTOR-Flag beads) was detected by immunoblotting with an anti-myc mAb. The amount of protein bound to FLAG agarose beads was detected with an anti-Flag mAb (loading control). Immunoblots were quantified via densitometric analyses and graphs are shown to the right of the blots. Three independent experiments were performed and one representative result is shown.