The present project was designed to investigate phosphorylation of p70S6K1 in an animal model of skeletal muscle overload. Within 24 h of male Sprague Dawley rats undergoing unilateral tenotomy to induce functional overloading of the plantaris muscle, phosphorylation of the Thr389 and Thr421/Ser424 sites on p70S6K1 was significantly elevated. Since the Thr421/Ser424 sites are purportedly mTORC1-independent, we sought to identify the kinase(s) responsible for their phosphorylation. Initially, we used IGF1 treatment of serum-deprived HEK293E cells as an in vitro model system, because IGF1 promotes phosphorylation of p70S6K1 on both the Thr389 and Thr421/Ser424 sites in skeletal muscle and in cells in culture. We found that, whereas the mTOR inhibitor TORIN2 prevented the IGF1-induced phosphorylation of the Thr421/Ser424 sites, it surprisingly enhanced phosphorylation of these sites during serum deprivation. JNK inhibition with SP600125 attenuated phosphorylation of the Thr421/Ser424 sites, and in combination with TORIN2 both the effect of IGF1 and the enhanced Thr421/Ser424 phosphorylation during serum deprivation were ablated. In contrast, both JNK activation with anisomycin and knockdown of the mTORC2 subunit rictor specifically stimulated phosphorylation of the Thr421/Ser424 sites, suggesting that mTORC2 represses JNK-mediated phosphorylation of these sites. The role of JNK in mediating p70S6K1 phosphorylation was confirmed in the animal model noted above, where rats treated with SP600125 exhibited attenuated Thr421/Ser424 phosphorylation. Overall, the results provide evidence that the mTORC1 and JNK signaling pathways coordinate the site-specific phosphorylation of p70S6K1. They also identify a novel role for mTORC1 and mTORC2 in the inhibition of JNK.