07-888 Sigma-AldrichAnti-phospho-PRAS40 (Thr246) (Proline-Rich AKT substrate) Antibody

Activation of mammalian target of rapamycin complex 1 (mTORC1) by amino acids is mediated in part by the Rag GTPases, which bind the raptor subunit of mTORC1 in an amino acid-stimulated manner and promote mTORC1 interaction with Rheb-GTP, the immediate activator. Here we examine whether the ability of amino acids to regulate mTORC1 binding to Rag and mTORC1 activation is due to the regulation of Rag guanyl nucleotide charging. Rag heterodimers in vitro exhibit a very rapid, spontaneous exchange of guanyl nucleotides and an inability to hydrolyze GTP. Mutation of the Rag P-loop corresponding to Ras(Ser-17) abolishes guanyl nucleotide binding. Such a mutation in RagA or RagB inhibits, whereas in RagC or RagD it enhances, Rag heterodimer binding to mTORC1. The binding of wild-type and mutant Rag heterodimers to mTORC1 in vitro parallels that seen with transient expression, but binding to mTORC1 in vitro is entirely independent of Rag guanyl nucleotide charging. HeLa cells stably overexpressing wild-type or P-loop mutant RagC exhibit unaltered amino acid regulation of mTORC1. Despite amino acid-independent raptor binding to Rag, mTORC1 is inhibited by amino acid withdrawal as in parental cells. Rag heterodimers extracted from (32)P-labeled whole cells, or just from the pool associated with the lysosomal membrane, exhibit constitutive [(32)P]GTP charging that is unaltered by amino acid withdrawal. Thus, amino acids promote mTORC1 activation without altering Rag GTP charging. Raptor binding to Rag, although necessary, is not sufficient for mTORC1 activation. Additional amino acid-dependent steps couple Rag-mTORC1 to Rheb-GTP.

The second messenger phosphatidylinositol (3,4,5)-trisphosphate (PIP(3)), formed by the p110 family of PI3-kinases, promotes cellular growth, proliferation, and survival, in large part by activating the protein kinase Akt/PKB. We show that inositol polyphosphate multikinase (IPMK) physiologically generates PIP(3) as well as water soluble inositol phosphates. IPMK deletion reduces growth factor-elicited Akt signaling and cell proliferation caused uniquely by loss of its PI3-kinase activity. Inhibition of p110 PI3-kinases by wortmannin prevents IPMK phosphorylation and activation. Thus, growth factor stimulation of Akt signaling involves PIP(3) generation through the sequential activations of the p110 PI3-kinases and IPMK. As inositol phosphates inhibit Akt signaling, IPMK appears to act as a molecular switch, inhibiting or stimulating Akt via its inositol phosphate kinase or PI3-kinase activities, respectively. Drugs regulating IPMK may have therapeutic relevance in influencing cell proliferation.

The 13-lined ground squirrel (Ictidomys tridecemlineatus) is capable of entering into extended periods of torpor during winter hibernation. The state of torpor represents a hypometabolic shift wherein the rate of oxygen consuming processes are strongly repressed in an effort to maintain cellular homeostasis as the availability of food energy becomes limited. We are interested in studying hibernation/torpor because of the robust state of tolerance to constrained oxygen delivery, oligemia, and hypothermia achieved by the tissues of hibernating mammals. The role of the serine/threonine kinase Akt (also known as PKB) has been examined in torpor in previous studies. However, this is the first study that examines the level of Akt phosphorylation in the liver during the two transition phases of the hibernation cycle: entrance into torpor, and the subsequent arousal from torpor. Our results indicate that Akt is activated in the squirrel liver by phosphorylation of two key residues (Thr(308) and Ser(473)) during entrance into torpor and arousal from torpor. Moreover, we observed increased phosphorylation of key substrates of Akt during the two transition stages of torpor. Finally, this study reports the novel finding that PRAS40, a component of the TORC1 multi-protein complex and a potentially important modulator of metabolism, is regulated during torpor.