(a) Constitutively active RagA and RagB stimulate S6K phosphorylation. 200 ng of each mammalian RagA, RagB, RagC, or RagD construct was co-transfected with HA-S6K (20 ng) into HEK293 cells. Their corresponding dominant negative mutants (RagA T21N, RagB T54N, RagC S75N, RagD S76N), and constitutively active mutants (RagA Q66L, RagB Q99L, RagC Q120L, RagD Q121L), were also tested. Phosphorylation and protein levels were determined by immunoblotting with appropriate antibodies, as indicated. Molecular weights of markers are indicated on the right.
(b) RagA has a dominant role over RagC in regulating S6K phosphorylation. 200 ng of each indicated Rag constructs were co-transfected with 20 ng of HA-S6K. The different Rag mutants used in the transfection are indicated on the top of each lane.
(c) Rag regulates TORC1 but not TORC2 activity. 200 ng of each indicated Rag constructs were co-transfected with 20 ng of HA-S6K, 20 ng of Myc-4EBP1, or 100 ng of GST-AKT. Phosphorylation and protein levels were determined by immunoblotting with appropriate antibodies, as indicated.