NH2-terminal kinase; Baf-A 1 , bafilomycin A 1 ; 3-MA, 3-methyladenine; WM, wortmannin; NAC, N-acetylcysteine; BHA, butylated hydroxyanisole; Mito-TEMPO, (2-(2,2,6,6-Tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl)triphenylphosphonium chloride; DAPI, (4',6-diamidino-2-phenylindole; PARP, poly(ADP-ribose)polymerase; RET/PTC, rearranged in transformation/papillary thyroid carcinomas; LAMP-1, lysosomal-associated membrane protein 1; MNNG, N-methyl-N'-nitro-N-nitrosoguanidine; IM-54, 2-(1H-Indol-3-yl)-3-pentylamino-maleimide; t-BHP, tert-butyl hydroperoxide; siRNA, small interfering RNATargeted disruption of STAT3 function has proven to be a useful cancer therapeutic approach by inducing apoptotic cell death. Cucurbitacin is currently under development as a small molecule of STAT3 inhibitor to trigger cell death in many cancers. Here, we systematically studied the molecular mechanisms underlying cucurbitacin-induced cell death, in particular the involvement of autophagy. Treatment with cucurbitacin resulted in non-apoptotic cell death in a caspaseindependent manner. Notably, cucurbitacin enhanced excessive conversion of lipidated LC3 (LC3-II) and accumulation of autophagosomes in many cell types. Such autophagy and cell death induced by cucurbitacin were independent of its ability to inhibit STAT3 function, but mainly mediated by enhanced production of mitochondrial-derived reactive oxygen species (ROS), and subsequently activation of extracellular signal-regulated kinase (ERK) and c-jun NH2-terminal kinase (JNK). Interestingly, both the autophagy inhibitor wortmannin and knockdown of Atg5 or Beclin 1 failed to rescue the cells from cucurbitacin-induced cell death, as suppression of autophagy induced the mode of cell death to shift from autophagic cell death to caspase-dependent apoptosis. Thus the present study provides new insights into the molecular mechanisms underlying cucurbitacin-mediated cell death and supports cucurbitacin as a potential anti-cancer drug through modulating the balance between autophagic and apoptotic modes of cell death.

Leucine zipper/EF hand-containing transmembrane-1 (LETM1) is a mitochondrial inner membrane protein that was first identified in Wolf-Hirschhorn syndrome, and was deleted in nearly all patients with the syndrome. LETM1 encodes for the human homologue of yeast Mdm38p, which is a mitochondria-shaping protein of unclear function. Here, we describe LETM1-mediated regulation of mitochondrial ATP production and biogenesis. We show that LETM1 overexpression can induce necrotic cell death in HeLa cells, in which LETM1 reduces mitochondrial biogenesis and ATP production. LETM1 acts as an anchor protein and associates with mitochondrial ribosome protein L36. Adenovirus-mediated overexpression of LETM1 reduced mitochondrial mass and expression of many mitochondrial proteins. LETM1-mediated inhibition of mitochondrial biogenesis enhanced glycolytic ATP supply and activated protein kinase B activity and cell survival signaling. The expression levels of LETM1 were significantly increased in multiple human cancer tissues compared with normals. These data suggest that LETM1 serves as an anchor protein for complex formation with the mitochondrial ribosome and regulates mitochondrial biogenesis. The increased expression of LETM1 in human cancer suggests that dysregulation of LETM1 is a key feature of tumorigenesis. [Cancer Res 2009;69(8):3397-404]

The zinc-finger protein A20 has crucial physiological functions as a dual inhibitor of nuclear factor-jB (NF-jB) activation and apoptosis in tumor necrosis factor (TNF) receptor 1 signaling pathway. Although the molecular basis for the anti-NF-jB function of A20 has been well elucidated, the anti-apoptotic function of A20 is largely unknown. Here, we report a novel mechanism underlying the anti-apoptotic function of A20: A20 blocks TNF-induced apoptosis through suppression of c-jun N-terminal kinase (JNK) by targeting apoptosis signal-regulating kinase1 (ASK1). First, the ectopic expression of A20 drastically inhibits TNF-induced JNK activation and apoptosis in multiple cell types including those deficient of NF-jB activation. Unexpectedly, the blunting effect of A20 on TNF-induced JNK activation is not mediated by affecting the TNFR1 signaling complex formation. Instead, A20 interacts with ASK1, an important MAPKK kinase in the JNK signaling cascade. More importantly, overexpression of wild-type A20, but not of mutant A20 (ZnF4; C624A, C627A), promotes degradation of the ASK1 through the ubiquitin-proteasome system. Taken together, the results from this study reveal a novel anti-apoptotic mechanism of A20 in TNF signaling pathway: A20 binds to ASK1 and mediates ASK1 degradation, leading to suppression of JNK activation and eventually blockage of apoptosis.

TNF receptor 1 can activate signaling pathways leading to the activation of NF-κB. A20, an NF-κB-inducible protein, negatively regulates these signaling pathways and acts as an anti-inflammatory mediator. Therefore, A20 is viewed as a potential therapeutic target for inflammatory disease. In this study, we examined the effect of A20 on an OVA-induced allergic airway inflammation model in mice. We used an adenovirus containing A20 cDNA (Ad-A20) that was delivered intratracheally before OVA challenge. Single administration of Ad-A20 reduced airway inflammatory cell recruitment and peribronchiolar inflammation and suppressed the production of various cytokines in bronchoalveolar fluid. In addition, Ad-A20 suppressed mucus production and prevented the development of airway hyperresponsiveness. The protective effect of Ad-A20 was mediated by the inhibition of the NF-κB signaling pathway. Taken together, our results suggest that the development of an immunoregulatory strategy based on A20 may have therapeutic potential for the treatment of allergic asthma.

3-Phosphoinositide-dependent protein kinase-1 (PDK1) appears to play a central regulatory role in many cell signalings between phosphoinositide-3 kinase and various intracellular serine/threonine kinases. In resting cells, PDK1 is known to be constitutively active and is further activated by tyrosine phosphorylation (Tyr 9 and Tyr 373/376 ) following the treatment of the cell with insulin or pervanadate. However, little is known about the mechanisms for this additional activation of PDK1. Here, we report that the SH2 domain of Src, Crk, and GAP recognized tyrosine-phosphorylated PDK1 in vitro. Destabilization of PDK1 induced by geldanamycin (a Hsp90 inhibitor) was partially blocked in HEK 293 cells expressing PDK1-Y9F. Co-expression of Hsp90 enhanced PDK1-Src complex formation and led to further increased PDK1 activity toward PKB and SGK. Immunohistochemical analysis with anti-phospho-Tyr 9 antibodies showed that the level of Tyr 9 phosphorylation was markedly increased in tumor samples compared with normal. Taken together, these data suggest that phosphorylation of PDK1 on Tyr 9 , distinct from Tyr 373/376 , is important for PDK1/Src complex formation, leading to PDK1 activation. Furthermore, Tyr 9 phosphorylation is critical for the stabilization of both PDK1 and the PDK1/Src complex via Hsp90-mediated protection of PDK1 degradation.One of the key features of multicellular organisms is that all cells are able to adjust to changes in the surrounding environment. A diverse set of environmental cues utilize intracellular protein phosphorylation-dephosphorylation cascades to rapidly and reversibly transduce their signals from their plasma membrane receptors to the cytoplasm and the nucleus. 3-Phosphoinositide-dependent protein kinase-1 (PDK1) 3 was originally identified as an upstream kinase for protein kinase B (PKB/Akt) (1) and is recognized as a master protein kinase for regulating in many cell-signaling pathways (2-5).Targets of PDK1 include many of the AGC family of protein kinases, including protein kinase B (PKB/Akt), p70 ribosomal protein S6 kinase (p70 S6K ), cyclic AMP-dependent protein kinase, protein kinase C, serum and glucocorticoid-inducible kinase (SGK), p90 ribosomal protein S6 kinase (RSK), and p21-activated kinase-1 (PAK1) (4). However, the generation of PDK1-ablated or PDK1-hypomorphic (ϳ10% of PDK1 expression) mice revealed that most of the PDK1 substrates identified in vitro were not physiological targets for PDK1 in vivo, with the exception of PKB, p70 S6K , and RSK (6, 7). PDK1(Ϫ/Ϫ) mice die at embryonic day 9.5 with multiple abnormalities, whereas hypomorphic PDK1 mice are viable (6). Nevertheless, these mice are 40 -50% smaller than control animals due to small cell size, but not cell number, providing genetic evidence that PDK1 is essential for mouse embryonic development and regulates cell size (6).PDK1 possesses an N-terminal kinase domain and a C-terminal pleckstrin homology domain (8, 9). Phosphorylation of PKB by PDK1 is dependent upon prior activation by phosphoinositide 3-kinase a...