TY - JOUR
T1 - mTORC1 and JNK coordinate phosphorylation of the p70S6K1 autoinhibitory domain in skeletal muscle following functional overloading
AU - Martin, Tony D.
AU - Dennis, Michael D.
AU - Gordon, Bradley S.
AU - Kimball, Scot R.
AU - Jefferson, Leonard S.
PY - 2014/6/15
Y1 - 2014/6/15
N2 - 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 mammalian target of rapamycin complex 1 (mTORC1) independent, we sought to identify the kinase(s) responsible for their phosphorylation. Initially, we used IGF-I treatment of serum-deprived HEK-293E cells as an in vitro model system, because IGF-I 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 IGF-I-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 IGF-I 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.
AB - 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 mammalian target of rapamycin complex 1 (mTORC1) independent, we sought to identify the kinase(s) responsible for their phosphorylation. Initially, we used IGF-I treatment of serum-deprived HEK-293E cells as an in vitro model system, because IGF-I 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 IGF-I-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 IGF-I 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.
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U2 - 10.1152/ajpendo.00064.2014
DO - 10.1152/ajpendo.00064.2014
M3 - Article
C2 - 24801387
AN - SCOPUS:84902657333
SN - 0193-1849
VL - 306
SP - E1397-E1405
JO - American Journal of Physiology - Endocrinology and Metabolism
JF - American Journal of Physiology - Endocrinology and Metabolism
IS - 12
ER -