We have investigated the effects of insulin, amino acids, and the degree of muscle loading on the phosphorylation of Ser2448, a site in the mammalian target of rapamycin (mTOR) phosphorylated by protein kinase B (PKB) in vitro. Phosphorylation was assessed by immunoblotting with a phosphospecific antibody (anti-Ser(P)2448) and with mTAb1, an activating antibody whose binding is inhibited by phosphorylation in the region of mTOR that contains Ser2448. Incubating rat diaphragm muscles with insulin increased Ser2448 phosphorylation but did not change the total amount of mTOR. Insulin, but not amino acids, activated PKB, as evidenced by increased phosphorylation of both Ser308 and Thr473 in the kinase. Ser2448 phosphorylation was also modulated by muscle-loading. Overloading the rat plantaris muscle by synergist muscle ablation, which promotes hypertrophy of the plantaris muscle, increased Ser2448 phosphorylation. In contrast, unloading the gastrocnemius muscle by hindlimb suspension, which promotes atrophy of the muscle, decreased Ser2448 phosphorylation, an effect that was fully reversible. Neither overloading nor hindlimb suspension significantly changed the total amount of mTOR. In summary, our results demonstrate that atrophy and hypertrophy of skeletal muscle are associated with decreases and increases in Ser2448 phosphorylation, suggesting that modulation of this site may have an important role in the control of protein synthesis.
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