SUMMARY AMP-activated protein kinase (AMPK) is a master sensor and regulator of cellular energy status. Upon metabolic stress, AMPK suppresses anabolic and promotes catabolic processes to regain energy homeostasis. Cancer cells can occasionally suppress the growth restrictive AMPK pathway by mutation of an upstream regulatory kinase. Here, we describe a widespread mechanism to suppress AMPK through its ubiquitination and degradation by the cancer-specific MAGE-A3/6-TRIM28 ubiquitin ligase. MAGE-A3 and MAGE-A6 are highly similar proteins normally expressed only in the male germline, but frequently re-activated in human cancers. MAGE-A3/6 are necessary for cancer cell viability and sufficient to drive tumorigenic properties of non-cancerous cells. Screening for targets of MAGE-A3/6-TRIM28 revealed that it ubiquitinates and degrades AMPKα1. This leads to inhibition of autophagy, activation of mTOR signaling, and hypersensitization to AMPK agonists, such as metformin. These findings elucidate a germline mechanism commonly hijacked in cancer to suppress AMPK.

The study of macroautophagy in mammalian cells has described induction, vesicle nucleation, and membrane elongation complexes as key signaling intermediates driving autophagosome biogenesis. How these components are recruited to nascent autophagosomes is poorly understood, and although much is known about signaling mechanisms that restrain autophagy, the nature of positive inductive signals that can promote autophagy remain cryptic. We find that the Ras-like small G-protein, RalB, is localized to nascent autophagosomes and is activated upon nutrient deprivation. RalB and its effector Exo84 are required for nutrient starvation-induced autophagocytosis, and RalB activation is sufficient to promote autophagosome formation. Through direct binding to Exo84, RalB induces the assembly of catalytically active ULK1 and Beclin1-VPS34 complexes on the exocyst, which are required for isolation membrane formation and maturation. Thus, RalB signaling is a primary adaptive response to nutrient limitation that directly engages autophagocytosis through mobilization of the core vesicle nucleation machinery.

The innate immune signaling kinase, TBK1, couples pathogen surveillance to induction of host defense mechanisms. Pathological activation of TBK1 in cancer can overcome programmed cell death cues, enabling cells to survive oncogenic stress. The mechanistic basis of TBK1 prosurvival signaling, however, has been enigmatic. Here we show that TBK1 directly activates AKT by phosphorylation of the canonical activation loop and hydrophobic motif sites independently of PDK1 and mTORC2. Upon mitogen stimulation, triggering of the innate immune response, re-exposure to glucose, or oncogene activation, TBK1 is recruited to the exocyst, where it activates AKT. In cells lacking TBK1, insulin activates AKT normally, but AKT activation by exocyst-dependent mechanisms is impaired. Discovery and characterization of a 6-aminopyrazolopyrimidine derivative, as a selective low nanomolar TBK1 inhibitor, indicates this regulatory arm can be pharmacologically perturbed independently of canonical PI3K/PDK1 signaling. Thus, AKT is a direct TBK1 substrate that connects TBK1 to prosurvival signaling.

Two unrelated children with HSE carry distinct heterozygous mutations in the gene encoding TANK-binding kinase 1.

The tumor suppressor protein p53 mediates stress-induced growth arrest or apoptosis and plays a major role in safeguarding genome integrity. In response to DNA damage, p53 can be modified at multiple sites by phosphorylation and acetylation. We report on the characterization of p53 C-terminal phosphorylation by CHK1 and CHK2, two serine/ threonine (Ser/Thr) protein kinases, previously implicated in the phosphorylation of the p53 N terminus. Using tryptic phosphopeptide mapping, we have identified six additional CHK1 and CHK2 sites residing in the final 100 amino acids of p53. Phosphorylation of at least three of these sites, Ser366, Ser378, and Thr387, was induced by DNA damage, and the induction at Ser366 and Thr387 was abrogated by small interfering RNA targeting chk1 and chk2. Furthermore, mutation of these phosphorylation sites has a different impact on p53 C-terminal acetylation and on the activation of p53-targeted promoters. Our results demonstrate a possible interplay between p53 C-terminal phosphorylation and acetylation, and they provide an additional mechanism for the control of the activity of p53 by CHK1 and CHK2. INTRODUCTIONThe protein p53 is often referred to as a "tumor suppressor," because it is frequently mutated in Ͼ50% of human cancers (Olivier et al., 2002). In response to stress, p53 undergoes extensive posttranslational modification, including phosphorylation and acetylation (Appella and Anderson, 2001;Brooks and Gu, 2003; Xu, 2003). Consequently, p53 is stabilized and activated (Vousden, 2002). As a transcription factor, p53 is composed of an N-terminal activation domain, a central specific DNA binding domain, and a C-terminal tetramerization domain, followed by a regulatory domain rich in basic amino acids (Ko and Prives, 1996). On activation, p53 binds to an array of gene promoters, some of which, such as p21/waf1, are responsible for stress-induced cell cycle arrest, whereas others, such as bax and puma, are responsible for driving cells into apoptosis (Vousden and Lu, 2002;Harms et al., 2004).Phosphorylation of p53 mostly occurs in the N-terminal activation domain at the Ser6, Ser9, Ser15, Thr18, Ser20, Ser33, Ser37, Ser46, Thr55, and Thr81 residues, with some phosphorylation occurring in the C-terminal linker and basic regions at Ser315, Ser371, Ser376, Ser378, and Ser392. Phosphorylation on most of these sites is induced by DNA damage, with some, such as Thr55 and Ser376, being repressed upon genotoxic stress (Appella and Anderson, 2001;Holmberg et al., 2002). How these individual residues contribute to p53 stabilization and activation is still not fully understood, and, at times, has been the subject of debate.Many protein kinases have been implicated in phosphorylating p53 (Holmberg et al., 2002). Notably, phosphorylation at Ser15 by ATM/ATR, either directly or through CHK1/ CHK2, or at Ser20 by CHK1/CHK2 has been shown to alleviate the inhibition or degradation of p53 by Mdm2, leading to p53 stabilization and activation (Shieh et al., 1997(Shieh et al., , 2000Banin et al., 1998;...

SUMMARY Generating a balanced network of inhibitory and excitatory neurons during development requires precise transcriptional control. In the dorsal spinal cord, Ptf1a, a basic helix-loop-helix (bHLH) transcription activator, maintains this delicate balance by inducing homeodomain (HD) transcription factors such as Pax2 to specify the inhibitory lineage, while suppressing HD factors such as Tlx1/3 that specify the excitatory lineage. We uncover the mechanism by which Ptf1a represses excitatory cell fate in the inhibitory lineage. We identify Prdm13 as a direct target of Ptf1a and reveal that Prdm13 actively represses excitatory cell fate by binding to regulatory sequences near the Tlx1 and Tlx3 genes to silence their expression. Prdm13 acts through multiple mechanisms including interactions with the bHLH factor Ascl1 to repress Ascl1 activation of Tlx3. Thus, Prdm13 is a key component of a highly coordinated transcriptional network that determines the balance of inhibitory versus excitatory neurons in the dorsal spinal cord.

Proper regulation of cell cycle progression is pivotal for maintaining genome stability. In a search for DNA damage-inducible, CHK1-modulated genes, we have identified BTG3 (B-cell translocation gene 3) as a direct p53 target. The p53 transcription factor binds to a consensus sequence located in intron 2 of the gene both in vitro and in vivo, and depletion of p53 by small interfering RNA (siRNA) abolishes DNA damage-induced expression of the gene. Furthermore, ablation of BTG3 by siRNA in cancer cells results in accelerated exit from the DNA damage-induced G2/M block. In vitro, BTG3 binds to and inhibits E2F1 through an N-terminal domain including the conserved box A. Deletion of the interaction domain in BTG3 abrogates not only its growth suppression activity, but also its repression on E2F1-mediated transactivation. We also present evidence that by disrupting the DNA binding activity of E2F1, BTG3 participates in the regulation of E2F1 target gene expression. Therefore, our studies have revealed a previously unidentified pathway through which the activity of E2F1 may be guarded by activated p53.

A challenge for biomedical research is the development of pharmaceuticals that appropriately target disease mechanisms. Natural products can be a rich source of bioactive chemicals for medicinal applications but can act through unknown mechanisms and can be difficult to produce or obtain. To address these challenges, we developed a new marine-derived, renewable natural products resource and a method for linking bioactive derivatives of this library to the proteins and biological processes that they target in cells. We used cell-based screening and computational analysis to match gene expression signatures produced by natural products to those produced by siRNA and synthetic microRNA libraries. With this strategy, we matched proteins and microRNAs with diverse biological processes and also identified putative protein targets and mechanisms of action for several previously undescribed marine-derived natural products. We confirmed mechanistic relationships for selected short-interfering RNAs, microRNAs, and compounds with functional roles in autophagy, chemotaxis mediated by discoidin domain receptor 2, or activation of the kinase AKT. Thus, this approach may be an effective method for screening new drugs while simultaneously identifying their targets.