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.

A novel light-driven chloride-pumping rhodopsin (ClR) containing an ‘NTQ motif' in its putative ion conduction pathway has been discovered and functionally characterized in a genomic analysis study of a marine bacterium. Here we report the crystal structure of ClR from the flavobacterium Nonlabens marinus S1-08T determined under two conditions at 2.0 and 1.56 Å resolutions. The structures reveal two chloride-binding sites, one around the protonated Schiff base and the other on a cytoplasmic loop. We identify a ‘3 omega motif' formed by three non-consecutive aromatic amino acids that is correlated with the B–C loop orientation. Detailed ClR structural analyses with functional studies in E. coli reveal the chloride ion transduction pathway. Our results help understand the molecular mechanism and physiological role of ClR and provide a structural basis for optogenetic applications.

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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]

Induction of endoplasmic reticulum (ER)-to-Golgi blockade or ER stress induces Golgi reassembly stacking protein (GRASP)-mediated, Golgi-independent unconventional cell-surface trafficking of the folding-deficient ΔF508-cystic fibrosis transmembrane conductance regulator (CFTR). However, molecular mechanisms underlying this process remain elusive. Here, we show that phosphorylation-dependent dissociation of GRASP homotypic complexes and subsequent relocalization of GRASP to the ER play a critical role in the unconventional secretion of CFTR. Immunolocalization analyses of mammalian cells revealed that the Golgi protein GRASP55 was redistributed to the ER by stimuli that induce unconventional secretion of ΔF508-CFTR, such as induction of ER-to-Golgi blockade by the Arf1 mutant. Notably, the same stimuli also induced phosphorylation of regions near the C-terminus of GRASP55 and dissociation of GRASP homomultimer complexes. Furthermore, phosphorylation-mimicking mutations of GRASP55 induced the monomerization and ER relocalization of GRASP55, and these changes were nullified by phosphorylation-inhibiting mutations. These results provide mechanistic insights into how GRASP accesses the ER-retained ΔF508-CFTR and mediates the ER stress-induced unconventional secretion pathway.

The lobster skeletal muscle Ca2+ release channel, known also as the ryanodine receptor, is composed of four polypeptides of approximately 5000 amino acids each, like its mammalian counterparts. Clones encoding the carboxy-terminal region of the lobster ryanodine receptor were isolated from a lobster skeletal muscle cDNA library. Analysis of the deduced 1513 carboxy-terminal amino acid sequence suggests a cytoplasmic Ca2+ binding domain consisting of two EF-hand Ca2+ binding motifs (amino acid residues 594-656). The Ca2+ binding properties of this domain were assessed by preparing bacterial fusion proteins with sequences from the lobster Ca2+ binding domain and the corresponding sequences of the rabbit cardiac and skeletal muscle ryanodine receptors. The lobster skeletal muscle fusion protein bound 45Ca2+ in Ca2+ overlays, and bound two Ca2+ under equilibrium binding conditions with a Hill dissociation constant (KH) of 0.9 mM and coefficient (nH) of 1.4. Rabbit skeletal and cardiac fusion proteins bound two Ca2+ with KHs of 3.7 and 3.8 mM and nHs of 1.1 and 1.3, respectively. Similar to results previously reported for the mammalian RyRs, the lobster RyR was activated by micromolar Ca2+ and inhibited by millimolar Ca2+, as determined in single-channel and [3H]ryanodine binding measurements. These results suggest that the two EF-hand Ca2+ binding domain of the lobster Ca2+ release channel as well as the corresponding regions of the mammalian channels may play a role in Ca2+ inactivation of sarcoplasmic reticulum Ca2+ release.

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...

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