AMPK is a highly conserved sensor of cellular energy status that is activated under conditions of low intracellular ATP. AMPK responds to energy stress by suppressing cell growth and biosynthetic processes, in part through its inhibition of the rapamycin-sensitive mTOR (mTORC1) pathway. AMPK phosphorylation of the TSC2 tumor suppressor contributes to suppression of mTORC1; however, TSC2-deficient cells remain responsive to energy stress. Using a proteomic and bioinformatics approach, we sought to identify additional substrates of AMPK that mediate its effects on growth control. We report here that AMPK directly phosphorylates the mTOR binding partner raptor on two well-conserved serine residues, and this phosphorylation induces 14-3-3 binding to raptor. The phosphorylation of raptor by AMPK is required for the inhibition of mTORC1 and cell-cycle arrest induced by energy stress. These findings uncover a conserved effector of AMPK that mediates its role as a metabolic checkpoint coordinating cell growth with energy status.

Adenosine monophosphate-activated protein kinase (AMPK) is a conserved sensor of intracellular energy activated in response to low nutrient availability and environmental stress. In a screen for conserved substrates of AMPK, we identified ULK1 and ULK2, mammalian orthologs of the yeast protein kinase Atg1, which is required for autophagy. Genetic analysis of AMPK or ULK1 in mammalian liver and C. elegans revealed a requirement for these kinases in autophagy. In mammals, loss of AMPK or ULK1 resulted in aberrant accumulation of the autophagy adaptor p62 and defective mitophagy. Reconstitution of ULK1-deficient cells with a mutant ULK1 that cannot be phosphorylated by AMPK revealed that such phosphorylation is required for mitochondrial homeostasis and cell survival following starvation. These findings uncover a conserved biochemical mechanism coupling nutrient status with autophagy and cell survival.

Circadian clocks coordinate behavioral and physiological processes with daily light-dark cycles by driving rhythmic transcription of thousands of genes. Whereas the master clock in the brain is set by light, pacemakers in peripheral organs, such as the liver, are reset by food availability, although the setting, or "entrainment," mechanisms remain mysterious. Studying mouse fibroblasts, we demonstrated that the nutrient-responsive adenosine monophosphate-activated protein kinase (AMPK) phosphorylates and destabilizes the clock component cryptochrome 1 (CRY1). In mouse livers, AMPK activity and nuclear localization were rhythmic and inversely correlated with CRY1 nuclear protein abundance. Stimulation of AMPK destabilized cryptochromes and altered circadian rhythms, and mice in which the AMPK pathway was genetically disrupted showed alterations in peripheral clocks. Thus, phosphorylation by AMPK enables cryptochrome to transduce nutrient signals to circadian clocks in mammalian peripheral organs.The mammalian hypothalamic suprachiasmatic nucleus (SCN) acts as a master pacemaker, aligning behavioral and physiological rhythms to light-dark cycles (1). Initially, the SCN was thought to be the only site of self-sustaining molecular pacemakers in mammals, but subsequent reports have shown such clocks to be ubiquitous (2,3). Unlike those in the SCN, clocks in non-light-sensitive organs are entrained by daily feeding (2,4,5), which

Summary Many tumors become addicted to autophagy for survival, suggesting inhibition of autophagy as a potential broadly-applicable cancer therapy. ULK1/Atg1 is the only serine/threonine kinase in the core autophagy pathway and thus represents an excellent drug target. Despite recent advances in the understanding of ULK1 activation by nutrient deprivation, how ULK1 promotes autophagy remains poorly understood. Here, we screened degenerate peptide libraries to deduce the optimal ULK1 substrate motif and discovered fifteen phosphorylation sites in core autophagy proteins that were verified as in vivo ULK1 targets. We utilized these ULK1 substrates to perform a cell-based screen to identify and characterize a potent ULK1 small molecule inhibitor. The compound SBI-0206965 is a highly selective ULK1 kinase inhibitor in vitro and suppressed ULK1-mediated phosphorylation events in cells, regulating autophagy and cell survival. SBI-0206965 greatly synergized with mTOR inhibitors to kill tumor cells, providing a strong rationale for their combined use in the clinic.

The serine/threonine kinase ULK1 is a mammalian homolog of Atg1, part of the Atg1 kinase complex, which is the most upstream component of the core autophagy machinery conserved from yeast to mammals. In budding yeast, activity of the Atg1 kinase complex is inhibited by TORC1 (target of rapamycin complex 1), but how the counterpart ULK1 complex in mammalian cells is regulated has been unknown. Our laboratories recently discovered that AMPK associates with, and directly phosphorylates, ULK1 on several sites and this modification is required for ULK1 activation after glucose deprivation. In contrast, when nutrients are plentiful, the mTORC1 complex phosphorylates ULK1, preventing its association and activation by AMPK. These studies have revealed a molecular mechanism of ULK1 regulation by nutrient signals via the actions of AMPK and mTORC1.

Brown adipose tissue (BAT) mitochondria exhibit high oxidative capacity and abundant expression of both electron transport chain components and uncoupling protein 1 (UCP1). UCP1 dissipates the mitochondrial proton motive force (Δp) generated by the respiratory chain and increases thermogenesis. Here we find that in mice genetically lacking UCP1, cold-induced activation of metabolism triggers innate immune signaling and markers of cell death in BAT. Moreover, global proteomic analysis reveals that this cascade induced by UCP1 deletion is associated with a dramatic reduction in electron transport chain abundance. UCP1-deficient BAT mitochondria exhibit reduced mitochondrial calcium buffering capacity and are highly sensitive to mitochondrial permeability transition induced by reactive oxygen species (ROS) and calcium overload. This dysfunction depends on ROS production by reverse electron transport through mitochondrial complex I, and can be rescued by inhibition of electron transfer through complex I or pharmacologic depletion of ROS levels. Our findings indicate that the interscapular BAT of Ucp1 knockout mice exhibits mitochondrial disruptions that extend well beyond the deletion of UCP1 itself. This finding should be carefully considered when using this mouse model to examine the role of UCP1 in physiology.brown fat | mitochondria | ROS | UCP1 | electron transport chain U ncoupling protein 1 (UCP1) plays a role in acute adaptive thermogenesis in interscapular brown adipose tissue (BAT). UCP1 dissipates the mitochondrial protonmotive force (Δp) generated by the electron transport chain (ETC) and is important for thermal homeostasis in rodents and human infants (1, 2). Ucp1 orthologs are not limited to mammals, but are also expressed in ectothermic vertebrates (3) and protoendothermic mammals (4), suggesting that UCP1 may have an important role in biology beyond thermal control. For example, it is becoming increasingly evident that in specific respiratory states, UCP1 can reduce reactive oxygen species (ROS) levels in vitro (4-9). The mitochondrial ETC is a major source of ROS production in the cell, and ROS play important roles in physiology and pathophysiology (10-12). Reverse electron transport (RET) through mitochondrial complex I is a key mechanism by which ROS are generated in vivo (11, 13). Interestingly, RET relies critically on high Δp, whereas dissipation of Δp by UCP1 can lower ROS levels in isolated mitochondria (5-7).Thermogenic respiration in BAT is triggered by external stimuli that activate adrenergic signaling (14). Most notably, environmental cold induces the capacity for adrenergic-mediated BAT respiration in wild type (WT) animals, but only minimally in UCP1-KO animals (15, 16). It is understood that the respiratory response of BAT under these conditions is indicative of UCP1-mediated respiration; however, the rate of maximal chemically uncoupled oxygen consumption, an UCP1-independent parameter, is also lower in UCP1-KO adipocytes compared with WT (15, 16).Moreover, the basal respiratory rat...

Highlights d An upstream open reading frame represses translation of the PPARGC1A mRNA d uORF function is a conserved feature of human, fish, and fly PPARGC1A 5 0 UTRs d Atlantic bluefin tuna PPARGC1A 5 0 UTR lacks a uORF and supports elevated translation