When statistically significant differences (< 0.05) were observed by ANOVA, a Student's and and = 7). 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 this 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. deficient in the S6 kinase gene present with delays in development and a reduction in body size due to reduced cell size compared with their wild-type counterparts (29). Thus, the coordinate activation of p70S6K1 likely plays a critical role in the growth-promoting processes associated with muscle mass hypertrophy. Regulation of p70S6K1 entails complex multisite phosphorylation of at least seven residues (19). Full activation of the kinase requires phosphorylation of three crucial sites: phosphorylation of the linker domain name at the change motif (Ser371) and hydrophobic motif (Thr389), as well as the catalytic domain name at the activation loop (Thr229) (28). It is L-Leucine well established that mTORC1 is the protein kinase responsible for phosphorylating p70S6K1 at Thr389, however, the hierarchy of phosphorylation events that precede phosphorylation of Thr389 remain somewhat controversial. Like other AGC kinases, p70S6K1 exhibits a bilobal L-Leucine fold structure with coordinating NH2- and COOH-terminal lobes (31). Activation of p70S6K1 entails priming phosphorylation of multiple proline-directed sites (Ser411, Ser418, Thr421, and Ser424) located in a basic pseudo-substrate domain name of the COOH-terminal lobe, which bears significant homology to the phosphorylated region of rpS6. In the inactive confirmation, this domain name serves an autoinhibitory function by folding over the NH2-terminal lobe (4). Mitogen-induced phosphorylation of the pseudo-substrate domain name relieves autoinhibition to induce a conformational switch in p70S6K1 that facilitates phosphorylation at Thr389 by mTORC1, phosphorylation of Thr229 by phosphoinositide-dependent kinase-1 (PDK1), and ultimately full activation (32). Thr421 and Ser424 represent the best-studied phosphorylation sites within the COOH-terminal autoinhibitory domain name of p70S6K1, and currently available phosphospecific L-Leucine antibodies do not distinguish between the two. Phosphorylation of the Thr421/Ser424 combined sites is usually both rapamycin insensitive and independent of the TOR signaling (TOS) motif that is required for mTORC1-mediated phosphorylation of other substrates, including p70S6K1 Thr389 (34). Thus, it is presumed that mTORC1 is not responsible for phosphorylation of these sites. Both Thr421 and Ser424 are followed by a proline in the primary sequence of p70S6K1. A number of kinases MAP2K2 phosphorylate serine or threonine residues when they are followed by a proline. Of the proline-directed kinases, the mitogen-activated protein kinases (MAPK) are of particular interest because they are present in skeletal muscle mass in multiple forms, including extracellular signal-regulated kinases (ERK)1 and -2, p38 MAPK, ERK5, big MAPK, and c-Jun NH2-terminal kinase (JNK) (24). Moreover, resistance exercise activates MAPKs and induces inflammatory-mediated responses that contribute to physiological processes such as muscle mass hypertrophy, lipid metabolism, insulin sensitivity, and glucose homeostasis (22, 24, 35). The goal of the present study was to assess p70S6K1 phosphorylation in a model of skeletal muscle mass hypertrophy and to identify the kinase(s) responsible for the observed changes. We found that phosphorylation of the Thr389 and Thr421/Ser424 sites was enhanced within 24 h following functional overloading of the plantaris muscle mass (37). Of significance, we present evidence that this mTOR and JNK signaling pathways coordinate in the site-specific phosphorylation of p70S6K during activation of cell growth. Specifically, both mTORC1 and JNK contribute to the phosphorylation of the p70S6K1 autoinhibitory domain name at Thr421/Ser424, whereas mTORC2 inhibits the phosphorylation of this domain name by JNK. MATERIALS AND METHODS Animal.