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Mitochondrial copper metabolism and delivery to cytochrome c oxidase and mitochondrially localized CuZn-superoxide dismutase (Sod1) requires a growing number of intermembrane space proteins containing a twin Cx9C motif. Among them, Cmc1 was recently identified by our group. Here we describe another conserved mitochondrial metallochaperone-like protein, Cmc2, a close homologue of Cmc1, whose function affects both cytochrome c oxidase and Sod1. In the yeast Saccharomyces cerevisiae, Cmc2 localizes to the mitochondrial inner membrane facing the intermembrane space. In the absence of Cmc2, cytochrome c oxidase activity measured spectrophoto-metrically and cellular respiration measured polarographically are undetectable. Additionally, mutant cmc2 cells display 2-fold increased mitochondrial Sod1 activity, whereas CMC2 overexpression results in Sod1 activity decreased to 60% of wild-type levels. CMC1 overexpression does not rescue the respiratory defect of cmc2 mutants or vice versa. However, Cmc2 physically interacts with Cmc1 and the absence of Cmc2 induces a 5-fold increase in Cmc1 accumulation in the mitochondrial membranes. Cmc2 function is conserved from yeast to humans. Human CMC2 localizes to the mitochondria and CMC2 expression knockdown produces cytochrome c oxidase deficiency in Caenorhabditis elegans. We conclude that Cmc1 and Cmc2 have cooperative but nonoverlapping functions in cytochrome c oxidase biogenesis. [ABSTRACT FROM AUTHOR]

Previously, a signaling pathway was described [Oak, Zhou, and Jarrett (2003) J. Biol. Chem. 278, 39287-39295] that links matrix laminin binding on the outside of the sarcolemma to Grb2 binding to syntrophin on the inside surface of the sarcolemma and by way of Grb2-Sos1-Rac1-PAK1-JNK ultimately results in the phosphorylation of c-jun on Ser65. How this signaling is initiated was investigated. Grb2- binding to syntrophin is increased by the addition of either laminin-1 or the isolated laminin α1 globular domain modules LG4-5, a protein referred to as E3. This identifies the LG4-5 sequences as the region of laminin responsible for signaling. Since laminin α1 LG4 is known to bind α-dystroglycan, this directly implicates α-dystroglycan as the laminin-signaling receptor. E3 or laminin-1 increase Grb2-binding and Rac1 activation. In the presence of E3 or laminin-1, syntrophin is phosphorylated on a tyrosine residue, and this increases and alters Grb2 binding. The α-dystroglycan antibody, IIH6, which blocks binding of laminins to α-dystroglycan, blocks both the laminin-induced Sos1/2 recruitment and syntrophin phosphorylation, showing that it is α-dystroglycan binding the LG4-5 region of laminin that is responsible. The C-terminal SH3 domain of Grb2 (C-SH3) binds only to nonphosphorylated syntrophin, and phosphorylation causes the Grb2 SH2 domain to bind and prevents SH3 binding. Syntrophin, tyrosine phosphate, β-dystroglycan, and Rac1 all co-localize to the sarcolemma of rat muscle sections. A model for how this phosphorylation may initiate downstream events in laminin signaling is presented. [ABSTRACT FROM AUTHOR]

Histone deacetylase (HDAC) inhibitors have been shown to induce cell cycle arrest and apoptosis in cancer cells. However, the mechanisms of HDAC inhibitor induced apoptosis are incompletely understood. In this study, depsipeptide, a novel HDAC inhibitor, was shown to be able to induce significant apoptotic cell death in human lung cancer cells. Further study showed that Bim, a BH3-only proapoptotic protein, was significantly upregulated by depsipeptide in cancer cells, and Bim's function in depsipeptide-induced apoptosis was confirmed by knockdown of Bim with RNAi. In addition, we found that depsipeptide-induced expression of Bim was directly dependent on acetylation of forkhead box class O1 (FoxO1) that is catalyzed by cyclic adenosine monophosphate-responsive element-binding protein-binding protein, and indirectly induced by a decreased four-and-a-half LIM-domain protein 2. Moreover, our results demonstrated that FoxO1 acetylation is required for the depsipeptide-induced activation of Bim and apoptosis, using transfection with a plasmid containing FoxO1 mutated at lysine sites and a luciferase reporter assay. These data show for the first time that an HDAC inhibitor induces apoptosis through the FoxO1 acetylation-Bim pathway. [ABSTRACT FROM AUTHOR]

Most agents that damage DNA act through posttranslational modifications of p53 and activate its downstream targets. However, whether cellular responses to nucleoside analogue-induced DNA damage also operate through p53 posttranslational modification has not been reported. In this study, the relationship between p53 activation and its posttranslational modifications was investigated in the human cancer cell lines A549 and HCT116 in response to 5-aza-2′-deoxycytidine (5-aza-CdR) or cytarabine treatment. 5-Aza-CdR induces p53 posttranslational modifications through activation of an ATM- and Rad3-related (AIR) signaling pathway, and 5-aza-CdR-induced association of replication protein A with chromatin is required for the binding of AIR to chromatin. Upon treatment with 5-aza-CdR, AIR activation is clearly associated with p53 phosphorylation at Ser15, but not at Thr18, Ser20, or Ser37. This specific p53 phosphorylation at Ser15 in turn results in acetylation of p53 at Lys320 and Lys373/Lys382 through transcriptional cofactors p300/CBP-associated factor and p300, respectively. These p53 posttranslational modifications are directly responsible for 5-aza-CdR induced p21Waf1/Cip1 expression because the binding activity of acetylated p53 at Lys320/Lys373/Lys382 to the p21Waf1/Cip1 promoter, as well as p21Waf1/Cip1 expression itself are significantly increased after 5-aza-CdR treatment. It is of interest that p53 phosphorylation at Ser15 and acetylations at Lys320/ Lys373/Lys382 mutually interact in the 5-aza-CdR induced p21Waf1/Cip1 expression shown by transfection of artificially mutated p53 expression vectors including S15A, K320R, and K373R/K382R into p53-null H1299 cells. These data taken together show for the first time that 5-aza-CdR activates the ATR signaling pathway, which elicits a specific p53 phosphorylation-acetylation cascade to induce p21Waf1/Cip1 expression. [ABSTRACT FROM AUTHOR]

J. Neurochem. (2011) 118, 864-878. Abstract Glycogen synthase kinase-3β (GSK-3β) plays a crucial role in memory deficits and tau hyperphosphorylation as seen in Alzheimer's disease, the most common dementia in the aged population. We reported that ventricular co-injection of wortmannin and GF-109203X (WT/GFX) can induce tau hyperphosophorylation and memory impairment of rats through activation of GSK-3 [Liu S. J., Zhang A. H., Li H. L., Wang Q., Deng H. M., Netzer W. J., Xu H. X. and Wang J. Z. (2003) J. Neurochem. 87, 1333]. In the present study, we found that feeding the rats with Acetyl-L-Carnitine (ALCAR, 50 mg/day·rat, per os) for 2 weeks rescued the WT/GFX-induced spatial memory retention impairment of the rats by antagonizing GSK-3β activation independent of Akt, PKCζ and Erk1/2. We also found that ALCAR arrested microtubule-associated protein tau hyperphosphorylation at multiple Alzheimer's disease sites in vivo and in vitro. Moreover, ALCAR enhanced the expression of several memory-associated proteins including c-Fos, synapsin I in rat hippocampus. These results suggest that ALCAR could ameliorate WT/GFX-induced spatial memory deficits through inhibition tau hyperphosphorylation and modulation of memory-associated proteins. [ABSTRACT FROM AUTHOR]