Mammalian cells respond to nutrient deprivation by inhibiting energy consuming processes, such as proliferation and protein synthesis, and by stimulating catabolic processes, such as autophagy. p70 S6 kinase (S6K1) plays a central role during nutritional regulation of translation. S6K1 is activated by growth factors such as insulin, and by mammalian target of rapamycin (mTOR), which is itself regulated by amino acids. The Class IA phosphatidylinositol (PI) 3-kinase plays a well recognized role in the regulation of S6K1. We now present evidence that the Class III PI 3-kinase, hVps34, also regulates S6K1, and is a critical component of the nutrient sensing apparatus. Overexpression of hVps34 or the associated hVps15 kinase activates S6K1, and insulin stimulation of S6K1 is blocked by microinjection of inhibitory anti-hVps34 antibodies, overexpression of a FYVE domain construct that sequesters the hVps34 product PI3P, or small interfering RNA-mediated knock-down of hVps34. hVps34 is not part of the insulin input to S6K1, as it is not stimulated by insulin, and inhibition of hVps34 has no effect on phosphorylation of Akt or TSC2 in insulin-stimulated cells. However, hVps34 is inhibited by amino acid or glucose starvation, suggesting that it lies on the nutrient-regulated pathway to S6K1. Consistent with this, hVps34 is also inhibited by activation of the AMP-activated kinase, which inhibits mTOR/S6K1 in glucose-starved cells. hVps34 appears to lie upstream of mTOR, as small interfering RNA knock-down of hVps34 inhibits the phosphorylation of another mTOR substrate, eIF4E-binding protein-1 (4EBP1). Our data suggest that hVps34 is a nutrient-regulated lipid kinase that integrates amino acid and glucose inputs to mTOR and S6K1.

We detected a protein in rabbit skeletal muscle extracts that was phosphorylated rapidly by SGK1 (serum- and glucocorticoid-induced kinase 1), but not by protein kinase Bα, and identified it as NDRG2 (N-myc downstream-regulated gene 2). SGK1 phosphorylated NDRG2 at Thr³³⁰, Ser³³² and Thr³⁴⁸ in vitro. All three residues were phosphorylated in skeletal muscle from wild-type mice, but not from mice that do not express SGK1. SGK1 also phosphorylated the related NDRG1 isoform at Thr³²⁸, Ser³³⁰ and Thr³⁴⁶ (equivalent to Thr³³⁰, Ser³³² and Thr³⁴⁸ of NDRG2), as well as Thr³⁵⁶ and Thr³⁶⁶. Residues Thr³⁴⁶, Thr³⁵⁶ and Thr³⁶⁶ are located within identical decapeptide sequences GTRSRSHTSE, repeated three times in NDRG1. These threonines were phosphorylated in NDRG1 in the liver, lung, spleen and skeletal muscle of wild-type mice, but not in SGK1^−/− mice. Knock-down of SGK1 in HeLa cells using small interfering RNA also suppressed phosphorylation of the threonine residues in the repeat region of NDRG1. The phosphorylation of NDRG1 by SGK1 transformed it into an excellent substrate for GSK3 (glycogen synthase kinase 3), which could then phosphorylate Ser³⁴², Ser³⁵² and Ser³⁶² in the repeat region. Incubation of HeLa cells with the specific GSK3 inhibitor CT 99021 increased the electrophoretic mobility of NDRG1 in HeLa cells, demonstrating that this protein is phosphorylated by GSK3 in cells. Our results identify NDRG1 and NDRG2 as physiological substrates for SGK1, and demonstrate that phosphorylation of NDRG1 by SGK1 primes it for phosphorylation by GSK3.

PI 3‐kinases are important regulators of endocytic trafficking. We have previously proposed a model in which the Rab5 GTPase recruits EEA1 to the early endosome both directly, by binding to EEA1, and indirectly, through the recruitment of the p150/hVps34 PI 3‐kinase and the production of PI[3]P in the endosomal membrane. In this study we have examined this model in vivo. We find that both endogenous hVps34 and p150 are targeted to enlarged endosomal structures in cells expressing constitutively activated Rab5, where they are significantly colocalized with EEA1. Recombinant fragments of p150 disrupt the endosomal localization of EEA1, showing that p150 is required for EEA1 targeting. We further analyzed the mechanism of GTP‐dependent Rab5‐p150 binding, and showed the p150 HEAT and WD40 domains are required for binding, whereas deletion of the protein kinase domain increases binding to Rab5. Overexpression of constitutively active Rab5 caused a redistribution of epitope‐tagged hVps34 and p150 to Rab5‐positive endosomes. However, subcellular fractionation showed that this was not due to a significant recruitment of hVps34 or p150 from the cytosolic to the particulate fraction. These data suggest that the binding of Rab5 to the HEAT/WD40 domains of p150 is important in regulating the localization of hVps34/p150. However, Rab5 does not appear to act by directly recruiting p150/hVps34 complexes from the cytosol to the endosomal membrane.

Glucose and glycolysis are important for the proinflammatory functions of many immune cells, and depletion of glucose in pathological microenvironments is associated with defective immune responses. Here we show a contrasting function for glucose in dendritic cells (DCs), as glucose represses the proinflammatory output of LPS-stimulated DCs and inhibits DC-induced T-cell responses. A glucose-sensitive signal transduction circuit involving the mTOR complex 1 (mTORC1), HIF1α and inducible nitric oxide synthase (iNOS) coordinates DC metabolism and function to limit DC-stimulated T-cell responses. When multiple T cells interact with a DC, they compete for nutrients, which can limit glucose availability to the DCs. In such DCs, glucose-dependent signalling is inhibited, altering DC outputs and enhancing T-cell responses. These data reveal a mechanism by which T cells regulate the DC microenvironment to control DC-induced T-cell responses and indicate that glucose is an important signal for shaping immune responses.

Hypoxia in the microenvironment of many solid tumours is an important determinant of malignant progression. The ISR (integrated stress response) protects cells from the ER (endoplasmic reticulum) stress caused by severe hypoxia. Likewise, autophagy is a mechanism by which cancer cells can evade hypoxic cell death. In the present paper we report that the autophagy-initiating kinase ULK1 (UNC51-like kinase 1) is a direct transcriptional target of ATF4 (activating transcription factor 4), which drives the expression of ULK1 mRNA and protein in severe hypoxia and ER stress. We demonstrate that ULK1 is required for autophagy in severe hypoxia and that ablation of ULK1 causes caspase-3/7-independent cell death. Furthermore, we report that ULK1 expression is associated with a poor prognosis in breast cancer. Collectively, the findings of the present study identify transcriptional up-regulation of ULK1 as a novel arm of the ISR, and suggest ULK1 as a potentially effective target for cancer therapy.

Direct pharmacological targeting of the anti-apoptotic B-cell lymphoma-2 (BCL-2) family is an attractive therapeutic strategy for treating cancer. Obatoclax is a pan-BCL-2 family inhibitor currently in clinical development. Here we show that, although obatoclax can induce mitochondrial apoptosis dependent on BCL-2 associated x protein/BCL-2 antagonist killer (BAX/BAK) consistent with its on-target pharmacodynamics, simultaneous silencing of both BAX and BAK did not abolish acute toxicity or loss of clonogenicity. This is despite complete inhibition of apoptosis. Obatoclax dramatically reduced viability without inducing loss of plasma membrane integrity. This was associated with rapid processing of light chain-3 (LC3) and reduction of S6 kinase phosphorylation, consistent with autophagy. Dramatic ultrastructural vacuolation, not typical of autophagy, was also induced. Silencing of beclin-1 failed to prevent LC3 processing, whereas knockout of autophagy-related (Atg)7 abolished LC3 processing but failed to prevent obatoclax-induced loss of clonogenicity or ultrastructural changes. siRNA silencing of Atg7 in BAX/BAK knockout mouse embryonic fibroblasts did not prevent obatoclax-induced loss of viability. Cells selected for obatoclax resistance evaded apoptosis independent of changes in BCL-2 family expression and displayed reduced LC3 processing. In summary, obatoclax exhibits BAX- and BAK-dependent and -independent mechanisms of toxicity and activation of autophagy. Mechanisms other than autophagy and apoptosis are blocked in obatoclax resistant cells and contribute significantly to obatoclax's anticancer efficacy.