Choline is an essential anabolic substrate for the synthesis of phospholipids. Choline kinase phosphorylates choline to phosphocholine that serves as a precursor for the production of phosphatidylcholine, the major phospholipid constituent of membranes and substrate for the synthesis of lipid signaling molecules. Nuclear magnetic resonance (NMR)-based metabolomic studies of human tumors have identified a marked increase in the intracellular concentration of phosphocholine relative to normal tissues. We postulated that the observed intracellular pooling of phosphocholine may be required to sustain the production of the pleiotropic lipid second messenger, phosphatidic acid. Phosphatidic acid is generated from the cleavage of phosphatidylcholine by phospholipase D2 and is a key activator of the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K)/AKT survival signaling pathways. In this study we show that the steady-state concentration of phosphocholine is increased by the ectopic expression of oncogenic H-Ras V12 in immortalized human bronchial epithelial cells. We then find that small interfering RNA (siRNA) silencing of choline kinase expression in transformed HeLa cells completely abrogates the high concentration of phosphocholine, which in turn decreases phosphatidylcholine, phosphatidic acid and signaling through the MAPK and PI3K/AKT pathways. This simultaneous reduction in survival signaling markedly decreases the anchorage-independent survival of HeLa cells in soft agar and in athymic mice. Last, we confirm the relative importance of phosphatidic acid for this pro-survival effect as phosphatidic acid supplementation fully restores MAPK signaling and partially rescues HeLa cells from choline kinase inhibition. Taken together, these data indicate that the pooling of phosphocholine in cancer cells may be required to provide a ready supply of phosphatidic acid necessary for the feedforward amplification of cancer survival signaling pathways.

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The tumor suppressor p53 is induced by genotoxic stress in both normal and transformed cells and serves to transcriptionally coordinate cell cycle checkpoint control and programmed cell death responses. Macrophage migration inhibitory factor (MIF) is an autocrine and paracrine acting cytokine/growth factor that promotes lung adenocarcinoma cell motility, anchorage-independence and neo-angiogenic potential. Several recent studies indicate that the only known homolog of MIF, D-dopachrome tautomerase (D-DT - also referred to as MIF-2), has functionally redundant activities with MIF and cooperatively promotes MIF-dependent pro-tumorigenic phenotypes. We now report that MIF and D-DT synergistically inhibit steady state p53 phosphorylation, stabilization and transcriptional activity in human lung adenocarcinoma cell lines. The combined loss of MIF and D-DT by siRNA leads to dramatically reduced cell cycle progression, anchorage independence, focus formation and increased programmed cell death when compared to individual loss of MIF or D-DT. Importantly, p53 mutant and p53 null lung adenocarcinoma cell lines were only nominally rescued from the cell growth effects of MIF/D-DT combined deficiency suggesting only a minor role for p53 in these transformed cell growth phenotypes. Finally, increased p53 activation was found to be independent of aberrantly activated AMP-activated protein kinase (AMPK) that occurs in response to MIF/D-DT-deficiency but is dependent on reactive oxygen species (ROS) that mediate aberrant AMPK activation in these cells. Combined, these findings suggest that both p53 wildtype and mutant human lung adenocarcinoma tumors rely on MIF family members for maximal cell growth and survival.

Tail-anchored proteins are a group of membrane proteins oriented with their amino terminus in the cytoplasm and their carboxy terminus embedded in intracellular membranes. This group includes the apoptosis-mediating proteins of the Bcl-2 family as well as the vesicle targeting proteins of the SNARE group, among others. A stretch of hydrophobic amino acids at the extreme carboxy terminus of these proteins serves both as a membrane anchor and as a targeting signal. Tail-anchored proteins are differentially targeted to either the endoplasmic reticulum or the mitochondrial outer membrane and the mechanism which accomplishes this selective targeting is poorly understood. Here we define important characteristics of the signal/anchor region which directs proteins to the mitochondrial outer membrane. We have created an artificial sequence consisting of a stretch of 16 leucines bounded by positively charged amino acids. Using this template we demonstrate that moderate hydrophobicity distinguishes the mitochondrial tail-anchor sequence from that of the endoplasmic reticulum tail-anchor sequence. A change as small as introduction of a single polar residue into a sequence that otherwise targets to the endoplasmic reticulum can substantially switch targeting to the mitochondrial outer membrane. Further we show that a mitochondrially targeted tail-anchor has a higher propensity for the formation of alpha-helical structure than a sequence directing tail-anchored proteins to the endoplasmic reticulum.

Here we address the function of the hydrophobic carboxy-terminal tail of the pro-apoptotic protein Bax. The tail is tucked into a hydrophobic pocket within the closed/inactive conformation of Bax. Apoptotic stimulation changes the Bax conformation, exposing a mitochondrial-targeting signal. We confirmed that the Bax tail alone can specifically target and anchor a passenger protein to the mitochondria. Surprisingly, we determined that the Bax tail does not play the primary targeting role in Bax mitochondrial translocation. Mutating the Bax tail to produce an ER-targeting signal had no effect on Bax mitochondrial targeting. Additionally, we demonstrated that the Bax tail has a negative regulatory effect on Bax activation. Mutations that disrupt the tail interactions with the hydrophobic pocket resulted in constitutive activation and mitochondrial targeting. Deletion of the Bax tail also resulted in an active conformation of Bax, however, mitochondrial targeting was abolished. Thus, the Bax tail is required for mitochondrial translocation. By generating a mutant-tail that cannot insert into membrane, we determined that insertion of the Bax tail is required for Bax mitochondrial targeting. Our data support a model whereby the Bax tail must be released from the pocket for activation of Bax, then functions as an anchor to stabilize Bax at the mitochondrial membrane after the initial addressing step.

Here, I address the function of the carboxy-terminal hydrophobic helix of the pro-apoptotic protein Bax. There has been considerable controversy as to whether this sequence is required for the targeting and insertion of Bax into the mitochondrial outer membrane. The Bax carboxy-terminal tail is tucked into a hydrophobic pocket within the closed/inactive conformation of Bax. Apoptotic stimulation results in an opening of the Bax conformation, exposing a mitochondrial-targeting signal and subsequent insertion of Bax into the mitochondrial outer membrane. Here, I confirm that the Bax tail alone can specifically target and anchor a cytosolic passenger protein to the mitochondria. Surprisingly, however, I find that the carboxy-terminal tail is not responsible for the specific targeting of Bax to the mitochondria rather than other cellular membranes. Specifically, replacing the Bax tail with an ER-targeting tail-anchor had no effect on Bax mitochondrial targeting, in the context of full-length Bax. This contrasts to the targeting function of tail-anchor signals in other tailanchored proteins. In addition, I demonstrated that the Bax tail has a negative IV regulatory effect on Sax activation. Mutations that disrupt the interaction of the Sax tail with the hydrophobic pocket resulted in an open/active conformation of Sax and constitutive mitochondrial targeting. Deletion of the Sax tail also resulted in an open/active conformation of Sax, however the anchor-deleted form of Sax was not associated with mitochondria. This indicates a requirement of the Sax tail for mitochondrial translocation. Sy introducing charged residues into the tail sequence to block insertion of the sequence into the hydrophobic bilayer, I show that insertion of the Sax tail is required for Sax mitochondrial targeting. My data support a model whereby the Sax tail must be released from its hydrophobic pocket to initiate the change into an open/active conformation. The tail then functions as an anchor to stabilize Sax at the mitochondrion after the initial addressing step. v

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AMP-activated protein kinase (AMPK) is a nutrient and metabolic stress sensing enzyme activated by the tumor suppressor kinase, LKB1. Because macrophage migration inhibitory factor (MIF) and its functional homolog, D-dopachrome tautomerase (D-DT), have pro-tumorigenic functions in non-small cell lung carcinoma (NSCLC) but have AMPK activating properties in nonmalignant cell types, we set out to investigate this apparent paradox. Our data now suggest that, in contrast to MIF and D-DT's AMPK activating properties in non-transformed cells, MIF and D-DT cooperatively act to inhibit steady state phosphorylation and activation of AMPK in LKB1 wildtype and LKB1 mutant human NSCLC cell lines. Our data further indicate that MIF and D-DT - acting through their shared cell surface receptor, CD74 - antagonize NSCLC AMPK activation by maintaining glucose uptake, ATP production and redox balance resulting in reduced Ca2+/calmodulin-dependent kinase kinase [[Unsupported Character - Symbol Font β]] (CaMKK[[Unsupported Character - Symbol Font β]])-dependent AMPK activation. Combined, these studies indicate that MIF and D-DT cooperate to inhibit AMPK activation in an LKB1-independent manner. This suggests that MIF and D-DT promote a unique and well conserved adaptation pathway that is usurped by the malignant cells in order to counter metabolic stress-induced, AMPK-dependent, tumor suppression. Importantly, because the LKB1 tumor suppressor locus is mutated in over 30% of NSCLC lesions rendering these tumors insensitive to AMPK-dependent growth suppression, the identification of unique AMPK agonists would be expected to have a tremendous impact on NSCLC disease management for clinicians. Taken together, simultaneous therapeutic targeting of both MIF and D-DT represents an innovative and clinically efficacious approach to re-activating metabolic stress-induced tumor suppression in NSCLC malignant lesions. Citation Format: Stephanie Brock, Beatriz Rendon, Kavitha Yaddanapudi, Robert Mitchell. Negative regulation of AMPK-dependent metabolic-stress pathways by MIF and D-DT in non-small cell lung carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5264. doi:10.1158/1538-7445.AM2013-5264