Figure 4.

cAMP agonists as well as insulin activate mTORC1 by stimulating the binding of 4E-BP1 to raptor and the phosphorylation of PRAS40. In panels A–C, PC Cl3 cells were treated without (−) or with (+) 10 μm forskolin (F) for 20 min. In panels D and E, they were treated without (c) or with 5 μg/ml insulin (i) and/or 10 μm forskolin (F) or 1 mU/ml TSH (T) for 20 min. A, Forskolin activates mTORC1. mTOR, raptor, and pS6K1 (T389) were immunodetected from whole-cell lysates (lys) (upper left panel). Endogenous mTOR complexes were immunoprecipitated (IP) with a raptor antibody (CRap) and a sample was taken for immunoblotting detection of mTOR and raptor (upper right panel). The immune complexes were mixed with [γ-³²P]ATP and recombinant 4E-BP1 fragment, and the in vitro kinase reaction (K) was performed without (c) or with mTOR inhibitors, 10 μm LY294002 (LY), 5 μm FKBP12 (FKB), or FKBP12 + 5 μm rapamycin (FKB + r), and phosphorylation of 4E-BP1 was visualized by immunoblotting of p4E-BP1 (T37/46) or phosphorimaging (³²P incorporation)(lower panel). B, Forskolin increases the T246 phosphorylation of PRAS40 and reduces the association of PRAS40 with mTORC1. mTOR, raptor, pS6K1 (T389), and pPRAS40 (T246) were immunodetected from whole-cell lysates (lys) (left panel). mTOR complexes were immunoprecipitated with the raptor antibody (CRap), and coimmunoprecipitated mTOR and PRAS40 were immunodetected (right panel). C, High-salt washing of mTORC1 increases its basal activity. mTOR complexes were immunoprecipitated with the raptor antibody (CRap) and washed with the same buffer containing 500 mm NaCl or 150 mm NaCl. Coimmunoprecipitated mTOR and raptor were immunodetected and the kinase activity (K) of these complexes was assayed as in panel A. D, Insulin, forskolin, and TSH activate mTORC1. mTORC1 complexes were immunoprecipitated with the raptor antibody (CRap) or nonimmune IgG (NI) as a control, and a sample was taken for immunoblotting detection of coimmunoprecipitated mTOR, raptor, and LST8. The kinase activity (K) of these complexes was assayed as in panel A. E, Insulin, forskolin, and TSH stimulate the binding of raptor to immobilized 4E-BP1. The same cell extracts as in panel D were incubated with 4E-BP1 or its mutant F113A (F/A) coupled to agarose beads. 4E-BP1-bound raptor was immunoblotted.