While HM site phosphorylation is strongly linked to ERK activation and could occur via autophosphorylation or ERK, the role of other kinases has not been excluded [26,31,32]. at this site requires an intact mTORC2. We also found that RSK is robustly phosphorylated at Ser380 upon nutrient withdrawal or inhibition of glycolysis, conditions that increase mTORC2 activation. However, pharmacological inhibition of mTOR did not abolish RSK phosphorylation at Ser380, indicating that mTOR catalytic activity is not required for this phosphorylation. Since RSK and SIN1 colocalize at the membrane during serum restimulation and acute glutamine withdrawal, mTORC2 could act as a scaffold to enhance RSK HM site phosphorylation. Among the known RSK substrates, the CCT subunit of the chaperonin containing TCP-1 (CCT) complex had defective phosphorylation in the absence of mTORC2. Our findings indicate that the mTORC2-mediated phosphorylation of the RSK HM site could confer RSK substrate specificity and reveal that RSK responds to nutrient fluctuations. strong class=”kwd-title” Keywords: RSK, mTORC2, p90 ribosomal s6 kinase, nutrients, AGC kinases, MAPK/ERK, CCT, CCT/TRiC, chaperonin, starvation, metabolism 1. Introduction mTOR orchestrates metabolic processes in response to levels of nutrients in order to promote cell growth or survival [1,2,3]. It forms two distinct signaling complexes; mTOR complex 1 (mTORC1) and complex 2 (mTORC2). mTORC1 is composed of the evolutionarily conserved components mTOR, raptor, and mLST8 while mTORC2 contains mTOR, rictor, SIN1, and mLST8. In higher eukaryotes mTOR also associates with other proteins distinct from mTORC1 and mTORC2 [4,5]. mTOR is a serine/threonine protein kinase and its activity is modulated by its protein partners. The best-characterized substrate of mTORC2 is AKT which is a member of the AGC family of protein kinases [6]. Members of this family including AKT are phosphorylated at the kinase activation loop by PDK1 (phosphoinositide-dependent kinase 1) [7]. They are also Rabbit polyclonal to USP37 phosphorylated at one or more sites at the two conserved motifs turn motif (TM) and hydrophobic motif (HM), which are adjacent to the Immethridine hydrobromide kinase domain. There is accumulating evidence supporting that mTOR either as part of mTORC1 or mTORC2 phosphorylates directly or indirectly the TM and Immethridine hydrobromide HM of AGC kinases [8,9,10,11,12,13,14,15]. mTORC2 phosphorylates the HM site (Ser473) of AKT in response to growth factors [10]. Recently we and others have also shown that this phosphorylation is enhanced upon nutrient withdrawal [16,17,18]. On the other hand, mTORC2 mediates Immethridine hydrobromide phosphorylation of the TM of AKT as well as the HM/TM of PKCs constitutively [9,11,13,19,20]. These observations suggest that specificity of mTORC2 activity towards these targets is likely to be modulated compartmentally in response to levels of growth signals or intracellular metabolites. Indeed we found that the TM phosphorylation of AKT occurs during translation when nascent AKT is associated with translating ribosomes [19]. Identification of Immethridine hydrobromide other downstream targets or effectors of mTORC2 should help unravel the precise mechanisms involved in mTORC2 signaling The p90 ribosomal S6 kinase (RSK), another member of the AGC kinase family functions in translation, metabolism, cell adhesion/migration and becomes deregulated in diseases such as cancer [21,22,23,24,25,26]. RSK has different isoforms, RSK1C4, with distinct as well as overlapping functions. RSK1C4 consists of two kinase domains, the N-Terminal kinase domain (NTKD), which is homologous to the catalytic domain of AGC kinase family and another at the carboxyl terminus (CTKD), which is homologous to the calcium/calmodulin-dependent protein kinase (CaMK) family (Figure 1A). The CTKD and NTKD promote autophosphorylation and substrate phosphorylation, respectively [27,28]. The MAPK family member, ERK1/2, facilitates the activation of RSK. It docks at the C-terminal end and phosphorylates Thr573 of the CTKD activation loop [29]. ERK1/2 is also linked to phosphorylation of Ser363 at the TM, which is located at the linker region between the two kinase domains. This linker region harbors the conserved TM and HM of AGC kinases. Phosphorylation of Ser380 at the HM serves as a docking site for PDK1 that then phosphorylates Ser221 of the Immethridine hydrobromide NTKD, resulting in full activation of RSK [30]. While HM site.