RPS6 phosphorylation occurs to a greater extent in the periphery of human skeletal muscle fibers, near focal adhesions, after anabolic stimuli

Abstract

Following anabolic stimuli (mechanical loading and/or amino acid provision), the mechanistic target of rapamycin complex 1 (mTORC1), a master regulator of protein synthesis, translocates toward the cell periphery. However, it is unknown if mTORC1-mediated phosphorylation events occur in these peripheral regions or before translocation (i.e., in central regions). We therefore aimed to determine the cellular location of a mTORC1-mediated phosphorylation event, RPS6^Ser240/244, in human skeletal muscle following anabolic stimuli. Fourteen young, healthy males either ingested a protein-carbohydrate beverage (0.25 g/kg protein and 0.75 g/kg carbohydrate) alone [<i>n</i> = 7; 23 ± 5 yr; 76.8 ± 3.6 kg; and 13.6 ± 3.8% body fat (BF), FED] or following a whole body resistance exercise bout (<i>n</i> = 7; 22 ± 2 yr; 78.1 ± 3.6 kg; and 12.2 ± 4.9%BF, EXFED). Vastus lateralis muscle biopsies were obtained at rest (PRE) and 120 and 300 min following anabolic stimuli. RPS6^Ser240/244 phosphorylation measured by immunofluorescent staining or immunoblot was positively correlated (<i>r</i> = 0.76, <i>P</i> < 0.001). Peripheral staining intensity of p-RPS6^Ser240/244 increased above PRE in both FED and EXFED at 120 min (∼54% and ∼138%, respectively, <i>P</i> < 0.05) but was greater in EXFED at both poststimuli time points (<i>P</i> < 0.05). The peripheral-to-central ratio of p-RPS6^240/244 staining displayed a similar pattern, even when corrected for total RPS6 distribution, suggesting RPS6 phosphorylation occurs to a greater extent in the periphery of fibers. Moreover, p-RPS6^Ser240/244 intensity within paxillin-positive regions, a marker of focal adhesion complexes, was elevated at 120 min irrespective of stimulus (<i>P</i> = 0.006) before returning to PRE at 300 min. These data confirm that RPS6^Ser240/244 phosphorylation occurs in the region of human muscle fibers to which mTOR translocates following anabolic stimuli and identifies focal adhesion complexes as a potential site of mTORC1 regulation in vivo.