Several ring between ring fingers (RBR) -domain proteins, such as Parkin and Parc, have been shown to be E3 ligases involved in important biological processes. Here, we identify a poorly characterized RBR protein, Ring Finger protein 144A (RNF144A), as the first, to our knowledge, mammalian E3 ubiquitin ligase for DNAPKcs. We show that DNA damage induces RNF144A expression in a p53-dependent manner. RNF144A is mainly localized in the cytoplasmic vesicles and plasma membrane and interacts with cytoplasmic DNA-dependent protein kinase, catalytic subunit (DNA-PKcs). DNA-PKcs plays a critical role in the nonhomologous end-joining DNA repair pathway and provides prosurvival signaling during DNA damage. We show that RNF144A induces ubiquitination of DNA-PKcs in vitro and in vivo and promotes its degradation. Depletion of RNF144A leads to an increased level of DNA-PKcs and resistance to DNA damaging agents, which is reversed by a DNA-PK inhibitor. Taken together, our data suggest that RNF144A may be involved in p53-mediated apoptosis through down-regulation of DNA-PKcs when cells suffer from persistent or severe DNA damage insults.endosome | DDR | transmembrane domain R ing Finger protein 144A (RNF144A) and RNF144B belong to the RNF144 family and are very conserved in higher eukaryotes. Both proteins contain an RING1-in between rings (IBR) -RING2 [termed the ring between ring fingers (RBR)] domain in the N terminus and a potential single-transmembrane (TM) domain in the C terminus. RBR domain-containing proteins usually possess an E3 ubiquitin ligase activity and are involved in regulation of the cell cycle and apoptosis (1-3). These proteins function like RING (Really Interesting New Gene)/HECT (Homologous to the E6AP Carboxyl Terminus) hybrids (i.e., they use their RING1 to bind E2s) but then transfer ubiquitin onto a conserved cysteine residue in the RING2 domain through a thioester linkage similar to the E3 thioester-linked ubiquitin intermediates in HECT-type E3 ligases (4). It has been shown that DNA damage induces RNF144B expression to promote cell apoptosis through regulation of the stability of p21 (5), BAX (1), and p73 (6). RNF144B shares 71% homology of amino acids with RNF144A.

HUANG, KUO-CHIN, BEE-HORNG LUE, RUOH-FANG YEN, CHRISTOPHER G. SHEN, SHIUH-RONG HO, TONG-YUAN TAI, AND WEI-SHIUNG YANG. Plasma adiponectin levels and metabolic factors in nondiabetic adolescents. Obes Res. 2004;12:119 -124. Objectives: The relationship of plasma adiponectin levels with various anthropometric and metabolic factors has been surveyed extensively in adults. However, how plasma adiponectin levels are related to various anthropometric indices and cardiovascular risk factors in adolescents is not as vigorously studied. In this study, we investigated this among healthy nondiabetic adolescents. Research Methods and Procedures: Two hundred thirty nondiabetic subjects (125 boys and 105 girls, ϳ10 to 19 years old) were included. The plasma adiponectin, fasting plasma glucose, insulin, lipids and anthropometric indices including body height, weight, waist circumference, and hip circumference were examined. Body fat mass (FM) and percentage were obtained from DXA scan. The homeostasis model assessment was applied to estimate the degree of insulin resistance. Results: The plasma adiponectin levels were significantly higher in girls (30.79 Ϯ 14.48 g/mL) than boys (22.87 Ϯ 11.41 g/mL). The plasma adiponectin levels were negatively related to BMI, FM, FM percentage, waist circumference, waist-to-hip ratio, insulin resistance, plasma insulin, triglycerides, and uric acid levels, but positively with high-density lipoprotein cholesterol (HDL-C) with the adjustment for age and gender. Using different multivariate linear regression models, only age and HDL-C were consistently related to the plasma adiponectin levels after adjustment for the other variables. Discussion: The relationship between plasma adiponectin and various anthropometric indices and metabolic factors, especially HDL-C, previously reported in adults was present in the healthy nondiabetic adolescents. Whether variation of plasma adiponectin levels in healthy nondiabetic adolescents may influence their future coronary artery disease risk warrants further investigation.

This study investigated the mechanism by which the transcription factor Sp1 is degraded in prostate cancer cells. We recently developed a thiazolidinedione derivative, (Z)-5-(4-hydroxy-3-trifluoromethylbenzylidene)-3-(1-methylcyclohexyl)-thiazolidine-2,4-dione (OSU-CG12), that induces Sp1 degradation in a manner paralleling that of glucose starvation. Based on our finding that thiazolidinediones suppress ␤-catenin and cyclin D1 by up-regulating the E3 ligase SCF ␤-TrCP , we hypothesized that ␤-transducin repeat-containing protein (␤-TrCP) targets Sp1 for proteasomal degradation in response to glucose starvation or OSU-CG12. Here we show that either treatment of LNCaP cells increased specific binding of Sp1 with ␤-TrCP. This direct binding was confirmed by in vitro pull-down analysis with bacterially expressed ␤-TrCP. Although ectopic expression of ␤-TrCP enhanced the ability of OSU-CG12 to facilitate Sp1 degradation, suppression of endogenous ␤-TrCP function by a dominant-negative mutant or small interfering RNA-mediated knockdown blocked OSU-CG12-facilitated Sp1 ubiquitination and/or degradation. Sp1 contains a C-terminal conventional DSG destruction box ( 727 DSGAGS 732 ) that mediates ␤-TrCP recognition and encompasses a glycogen synthase kinase 3␤ (GSK3␤) phosphorylation motif (SXXXS). Pharmacological and molecular genetic approaches and mutational analyses indicate that extracellular signal-regulated kinasemediated phosphorylation of Thr739 and GSK3␤-mediated phosphorylation of Ser728 and Ser732 were critical for Sp1 degradation. The ability of OSU-CG12 to mimic glucose starvation to activate ␤-TrCP-mediated Sp1 degradation has translational potential to foster novel strategies for cancer therapy.In addition to maintaining the basal transcription of housekeeping genes, increasing evidence indicates that the transcription factor Sp1 also plays an important role in regulating the expression of a host of key effectors of signaling pathways governing cell cycle progression, cell proliferation, angiogenesis, apoptosis, and metastasis (Wierstra, 2008). These target proteins include receptor tyrosine kinases and their growth factor ligands, cyclin-dependent kinase inhibitors, c-Myc, Mdm2, Mcl-1, survivin, XIAP, Fas ligand, PUMA, and death receptor 5 (Wierstra, 2008). Moreover, Sp1

Adiponectin is an adipose-derived plasma protein. Recently the plasma adiponectin levels have been linked to most variables of metabolic syndrome (MS) and risk factors of coronary artery disease (CAD). However, its relation with blood pressure is yet unclear. Here we report the relationship between the plasma adiponectin levels and blood pressures in 68 female adolescents (age 16.1 +/- 1.8 yr). We found that the plasma adiponectin levels correlated significantly with both the systolic blood pressure (SBP) (gamma = -0.47, P = 0.000) and diastolic blood pressure (gamma = -0.30, P = 0.014). In linear regression models with adjustment for age and the other anthropometric or metabolic factors, only the SBP, but not the diastolic blood pressure, was independently related to the plasma adiponectin levels. The mean plasma adiponectin levels between the subjects in the lowest quartile of SBP (SBP < or = 100 mm Hg) and those in the highest quartile (SBP > or = 118 mm Hg) were significantly different (P = 0.035). In conclusion, the SBP is inversely related to the plasma adiponectin independent of the other variables of the MS and other risk factors of CAD in healthy adolescent females. This further strengthens the potential roles of adiponectin in the pathophysiology of MS and CAD.

Background: RNF144A is a membrane-associated E3 ubiquitin ligase. Results: The GXXXG motif within the TM domain of RNF144A mediates its self-association and activation of E3 ligase activity. Conclusion: The TM domain regulates RNF144A through two independent steps: membrane localization and GXXXG motifmediated self-association. Significance: The GXXXG motif is conserved among all RBR-TM proteins, suggesting a similar regulation for other RBR-TM proteins.

The protein O-linked β-N-acetylglucosamine (O-GlcNAc) modification plays an important role in skeletal muscle development and physiological function. In this study, bitransgenic mice were generated that overexpressed NCOAT(GK), an O-GlcNAcase-inactive spliced variant of the O-GlcNAcase gene, specifically in skeletal muscle using the muscle creatine kinase promoter. Expression of the chimeric enhanced green fluorescent protein-NCOAT(GK) transgene caused an increase of cellular O-GlcNAc levels, along with the accumulation and activation of proapoptotic factors in muscles of bitransgenic mice. The consequence of overexpressing the transgene for a 2-wk period was muscle atrophy and, in some cases, resulted in the death of male mice. Muscle atrophy is a common complication of many diseases, some of which correlate markedly with high cellular O-GlcNAc levels, such as diabetes. Our study provides direct evidence linking muscle atrophy and the disruption of O-GlcNAcase activity.

RNF144A is a single-pass transmembrane RBR E3 ligase that interacts with and degrades cytoplasmic DNA-PKcs, which is an epidermal growth factor receptor (EGFR)-interacting partner. Interestingly, RNF144A expression is positively correlated with EGFR mRNA and protein levels in several types of cancer. However, the relationship between RNF144A and EGFR is poorly understood. This study reports an unexpected role for RNF144A in the regulation of EGF/EGFR signaling and EGF-dependent cell proliferation. EGFR ligands, but not DNA-damaging agents, induce a DNA-PKcs-independent interaction between RNF144A and EGFR. RNF144A promotes EGFR ubiquitination, maintains EGFR protein, and prolongs EGF/EGFR signaling during EGF stimulation. Moreover, depletion of RNF144A by multiple independent approaches results in a decrease in EGFR expression and EGF/EGFR signaling. RNF144A knockout cells also fail to mount an immediate response to EGF for activation of G/S progression genes. Consequently, depletion of RNF144A reduces EGF-dependent cell proliferation. These defects may be at least in part due to a role for RNF144A in regulating EGFR transport in the intracellular vesicles during EGF treatment.

a b s t r a c tThe FOXO4 transcription factor plays an important role in cell survival in response to oxidative stress. The regulation of FOXO4 is orchestrated by post-translational modifications including phosphorylation, acetylation, and ubiquitination. Here, we demonstrate that O-GlcNAcylation also contributes to the FOXO4-dependent oxidative stress response. We show that hydrogen peroxide treatment of HEK293 cells increases FOXO4 association with OGT, the enzyme that adds O-GlcNAc to proteins, causing FOXO4 O-GlcNAcylation and enhanced transcriptional activity under acute oxidative stress. O-GlcNAcylation is known to be protective for cells under stress conditions, including oxidative stress. Our data provide a mechanism of FOXO4 anti-oxidative protection through O-GlcNAcylation. Structured summary:MINT-7299700, MINT-7299716: Foxo4 (uniprotkb:Q9WVH3) physically interacts (MI:0915) with Ogt (uniprotkb:P56558) by anti tag coimmunoprecipitation (MI:0007) MINT-7299691: Ogt (uniprotkb:O15294) physically interacts (MI:0915) with Foxo4 (uniprotkb:P98177) by anti bait coimmunoprecipitation (MI:0006)