FIG. 6.

PKC-mediated phosphorylation of S6KβII at Ser486 is insensitive to specific TOR/FRAP and PI3-K inhibitors and does not effect S6K activity. HEK 293 cells were transiently transfected with wild-type EE-S6KβII or EE-S6KβII S486A and incubated in the presence (+) or absence (−) of 1 μM PMA for 30 min after 24 h of starvation. Rapamycin (R) or wortmannin (W) was added for 30 min before cell stimulation. Recombinant S6KβII was immunoprecipitated with anti-EE-tag antibody and used for the in vitro S6K assay or analyzed by immunoblotting with anti-pS486 antibody. IgG, immunoglobulin G; PVDF, polyvinylidene difluoride; WB, Western blot.