AKAP95 regulates ubiquitination and degradation of cyclin Ds/Es, influencing the G1/S transition of lung cancer cells.

A-kinase anchoring protein 95 (AKAP95) functions as a scaffold for protein kinase A. Prior work by our group has shown that AKAP95, in coordination with Connexin 43 (Cx43), modulates the expression of cyclin D and E proteins, thus affecting the cell cycle progression in lung cancer cells. In the current study, we confirmed that AKAP95 forms a complex with Cx43. Moreover, it associates with cyclins D1 and E1 during the G1 phase, leading to the formation of protein complexes that subsequently translocate to the nucleus. These findings indicate that AKAP95 might facilitate the nuclear transport of cyclins D1 and E1. Throughout this process, AKAP95 and Cx43 collectively regulate the expression of cyclin D, phosphorylate cyclin E1 proteins, and target their specific ubiquitin ligases, ultimately impacting cell cycle progression.

Hepatic neddylation targets and stabilizes electron transfer flavoproteins to facilitate fatty acid ß-oxidation.

Neddylation is a ubiquitination-like pathway that controls cell survival and proliferation by covalently conjugating NEDD8 to lysines in specific substrate proteins. However, the physiological role of neddylation in mammalian metabolism remains elusive, and no mitochondrial targets have been identified. Here, we report that mouse models with liver-specific deficiency of NEDD8 or ubiquitin-like modifier activating enzyme 3 (UBA3), the catalytic subunit of the NEDD8-activating enzyme, exhibit neonatal death with spontaneous fatty liver as well as hepatic cellular senescence. In particular, liver-specific UBA3 deficiency leads to systemic abnormalities similar to glutaric aciduria type II (GA-II), a rare autosomal recessive inherited fatty acid oxidation disorder resulting from defects in mitochondrial electron transfer flavoproteins (ETFs: ETFA and ETFB) or the corresponding ubiquinone oxidoreductase. Neddylation inhibition by various strategies results in decreased protein levels of ETFs in neonatal livers and embryonic hepatocytes. Hepatic neddylation also enhances ETF expression in adult mice and prevents fasting-induced steatosis and mortality. Interestingly, neddylation is active in hepatic mitochondria. ETFs are neddylation substrates, and neddylation stabilizes ETFs by inhibiting their ubiquitination and degradation. Moreover, certain mutations of ETFs found in GA-II patients hinder the neddylation of these substrates. Taken together, our results reveal substrates for neddylation and add insight into GA-II.

Arsenic induces bronchial epithelial carcinogenesis with mitochondrial dysfunction through AKAP95-mediated cell cycle alterations.

Arsenic is a widely existing pollutant in the environment, but the mechanism of occurrence and development of lung cancer by long-term arsenic exposure needs to be elucidated further. How the high and low doses of arsenic induce human bronchial epithelial cell transformation is yet to be elucidated. In the present study, human bronchial epithelial cells were exposed to varying high-dose sodium arsenite (NaAsO2) for the short-term or treated with low dose for long-term. The data showed that both short- and long-term treatment promoted G1/S transition of Beas-2B cells, inducing a significant increase in the expression of AKAP95, cyclin D1, cyclin D2, and cyclin E1. However, silencing AKAP95 by treating cells with siAKAP95 exerted a protective function that inhibited G1/S transition, suggesting a regulatory mechanism of AKAP95 on the cell cycle during cell malignant transformation induced by NaAsO2. In addition, mitochondrial dysfunctions occurred during NaAsO2 exposure. Beas-2B cells exposed to low-dose NaAsO2 for long-term were subcultured for 20 generations, and the exposure time was positively proportional to the growth and migration rate of the cells. The exposed cells were used in a tumor-bearing transplantation experiment (mice), and the results showed that the longer the exposure time, the faster the tumor volume growth rate of As-Beas-2B cells. Tumor tissues were excised for hematoxylin-eosin staining, which showed altered cell morphology and increased volume.

Platforms Formed from a Three-Dimensional Cu-Based Zwitterionic Metal-Organic Framework and Probe ss-DNA: Selective Fluorescent Biosensors for Human Immunodeficiency Virus 1 ds-DNA and Sudan Virus RNA Sequences.

We herein report a water-stable three-dimensional Cu-based metal-organic framework (MOF) 1 supported by a tritopic quaternized carboxylate and 4,4'-dipyridyl sulfide as an ancillary ligand. This MOF exhibits unique pore shapes with aromatic rings, positively charged pyridinium and unsaturated Cu(II) cation centers, free carboxylates, tessellating H2O, and coordinating SO4(2-) on the pore surface. Compound 1 can interact with two carboxyfluorescein (FAM)-labeled single-stranded DNA sequences (probe ss-DNA, delineated as P-DNA) through electrostatic, π-stacking, and/or hydrogen-bonding interactions to form two P-DNA@1 systems, and thus quench the fluorescence of FAM via a photoinduced electron-transfer process. These P-DNA@1 systems can be used as effective fluorescent sensors for human immunodeficiency virus 1 double-stranded DNA and Sudan virus RNA sequences, respectively, with detection limits of 196 and 73 pM, respectively.

A nano-integrated diagnostic and therapeutic platform with oxidation-reduction reactions in tumor microenvironments.

In the present study, we developed a nano-integrated diagnostic and therapeutic platform with oxidation-reduction reactions in tumor microenvironments (TMEs). The proposed platform resolved the contradiction of particle size between the enhanced permeability and retention (EPR) effect and tumor interstitial penetration, as well as poor circulation and low drug-loading efficiency. Flower-like MnO2 NPs were used as the core and modified with hyaluronate (HA) and H2PtCl6 to obtain MnO2-HA@H2PtCl6 (MHP). The maximum drug-loading efficiency rate of H2PtCl6 reached 35% due to its chelation with HA. MHP showed satisfactory integrity and stability during circulation and can also be used as a magnetic resonance imaging (MRI) contrast agent. In addition, MHP as a radiosensitizer achieved an excellent tumor inhibition effect in combination with radiotherapy. Importantly, MHP released ultra-small nanoparticles, USNPs, (∼20 nm) through the supramolecular self-assembly abilities of Mn2+, HA, and H2PtCl6 in TMEs, leading to the increase of penetration into multicellular spheres and solid tumors (Scheme), as well as prolonging its retention in tumors.

Ultrasonically initiated emulsion polymerization of styrene in the presence of Fe2+.

Ultrasonically initiated emulsion polymerization of styrene was carried out in the presence of Fe(2+). The addition of a small amount of Fe(2+) markedly enhanced the polymerization rate of styrene. In the presence of 50 microM Fe(2+), the conversion of monomer in the reaction time of 60 min was 2.4 times as high as that in the absence of Fe(2+). The increase in the polymerization rate was due to higher concentration of hydroxyl (*OH) radicals generated via Fenton reaction of Fe(2+) with hydrogen peroxide (H(2)O(2)), which was proved by a lower amount of H(2)O(2) in Fe(2+) aqueous solution compared with that in pure water during ultrasonic irradiation. However, the addition of excessive Fe(2+) had no further accelerating effect on the polymerization rate due to the reduction of *OH radicals by Fe(2+). So it is an effective way to add an appropriate amount of Fe(2+) to accelerate ultrasonically initiated emulsion polymerization of styrene.

Enhancement of ultrasonically initiated emulsion polymerization rate using aliphatic alcohols as hydroxyl radical scavengers.

Ultrasonically initiated emulsion polymerization of styrene was carried out in the presence of aliphatic alcohols, e.g. methanol, ethanol, n-pronanol and n-butanol, as volatile hydroxyl radical scavengers. With the addition of methanol, the polymerization rate of styrene increased, while the molecular weight and the average particle size of the produced polystyrene decreased because more radicals were produced in the presence of methanol. This is true also for the other polymerization system using other aliphatic alcohols, such as ethanol, n-pronanol and n-butanol, suggesting that the alcohols enter into cavitation bubbles and further react with hydroxyl radicals (*OH) from the sonolysis of water to produce hydroxyalkyl radicals, so as to reduce the recombination of *H and *OH radicals, therefore more radicals will be present in the systems for initiating polymerization. Obviously, it is an effective way to enhance ultrasonically initiated emulsion polymerization rate of styrene by adding volatile hydroxyl radical scavenger.

Synchronous detection of ebolavirus conserved RNA sequences and ebolavirus-encoded miRNA-like fragment based on a zwitterionic copper (II) metal-organic framework.

From a three-dimensional (3D) metal-organic framework (MOF) of {[Cu(Cmdcp)(phen)(H2O)]2·9H2O}n (1, H3CmdcpBr = N-carboxymethyl-(3,5-dicarboxyl)pyridinium bromide, phen = phenanthroline), a sensitive and selective fluorescence sensor has been developed for the simultaneous detection of ebolavirus conserved RNA sequences and ebolavirus-encoded microRNA-like (miRNA-like) fragment. The results from molecular dynamics simulation confirmed that MOF 1 absorbs carboxyfluorescein (FAM)-tagged and 5(6)-carboxyrhodamine, triethylammonium salt (ROX)-tagged probe ss-DNA (probe DNA, P-DNA) by π…π stacking and hydrogen bonding, as well as additional electrostatic interactions to form a sensing platform of P-DNAs@1 with quenched FAM and ROX fluorescence. In the presence of targeted ebolavirus conserved RNA sequences or ebolavirus-encoded miRNA-like fragment, the fluorophore-labeled P-DNA hybridizes with the analyte to give a P-DNA@RNA duplex and released from MOF 1, triggering a fluorescence recovery. Simultaneous detection of two target RNAs has also been realized by single and synchronous fluorescence analysis. The formed sensing platform shows high sensitivity for ebolavirus conserved RNA sequences and ebolavirus-encoded miRNA-like fragment with detection limits at the picomolar level and high selectivity without cross-reaction between the two probes. MOF 1 thus shows the potential as an effective fluorescent sensing platform for the synchronous detection of two ebolavirus-related sequences, and offer improved diagnostic accuracy of Ebola virus disease.

All-trans retinoic acid induces autophagic degradation of ubiquitin-like modifier activating enzyme 3 in acute promyelocytic leukemia cells.

All-trans retinoic acid (ATRA) has demonstrated notable success in the treatment of acute promyelocytic leukemia (APL) by inducing granulocytic differentiation. The underlying mechanisms of ATRA therapeutic effects have not been entirely clarified. Here, we reported that the regulation of neddylation, a ubiquitination-like post-translational modification, was involved in the treatment of ATRA on APL. Treating APL cells with ATRA led to the degradation of UBA3, a subunit of neddylation E1. Lysosome-autophagy pathway but not proteasome pathway was responsible for the degradation of UBA3. Neddylation suppression in APL cells was capable of inducing apoptosis, differentiation and proliferation inhibition, suggesting a pivotal role of neddylation in APL cells. ATRA treatment also led to UBA3 degradation in primary APL cells. Taken together, our findings indicated that neddylation was important to maintain the malignant features of APL cells, and suppression of neddylation was involved in the effects of ATRA on APL cells.

Polystyrene/Fe3O4 magnetic emulsion and nanocomposite prepared by ultrasonically initiated miniemulsion polymerization.

Ultrasonically initiated miniemulsion polymerization of styrene in the presence of Fe3O4 nanoparticles was successfully employed to prepare polystyrene (PS)/Fe3O4 magnetic emulsion and nanocomposite. The effects of Fe3O4 nanoparticles on miniemulsion polymerization process, the structure, morphology and properties of PS/Fe3O4 nanocomposite were investigated. The increase in the amount of Fe3O4 nanoparticles drastically increases the polymerization rate due to that Fe3O4 nanoparticles increase the number of radicals and the cavitation bubbles. Polymerization kinetics of ultrasonically initiated miniemulsion polymerization is similar to that of conventional miniemulsion polymerization. PS/Fe3O4 magnetic emulsion consists of two types of particles: latex particles with Fe3O4 nanoparticles and latex particles with no encapsulated Fe3O4 nanoparticles. Fe3O4 nanoparticles lower the molecular weight of PS and broaden the molecular weight and particle size distribution. Thermal stability of PS/Fe3O4 nanocomposite increases with the increase in Fe3O4 content. PS/Fe3O4 emulsion and nanocomposite exhibit magnetic properties. PS/Fe3O4 magnetic particles can be separated from the magnetic emulsion by an external magnetic field and redispersed into the emulsion with agitation.

A metal-organic framework based PCR-free biosensor for the detection of gastric cancer associated microRNAs.

We report herein five sensing platforms for the detection of five gastric cancer associated microRNAs (miRNAs). The sensing platforms are hybrids formed from a water-stable metal organic framework (MOF) {[Cu(dcbb)2(H2O)2]·10H2O}n (1, H2dcbbBr=1-(3,5-dicarboxybenzyl)-4,4'-bipyridinium bromide), respectively with five carboxyfluorescein (FAM) labeled probe single-stranded DNA (probe ss-DNA, denoted as P-DNA). Within the hybrid, MOF 1 tightly interacts with the P-DNA through electrostatic and/or π-stacking interactions and results in fluorescence quenching of FAM via a photo-induced electron transfer (PET) process. In the presence of the complementary target miRNAs miR-185, miR-20a, miR-92b, miR-25 and miR-210, which are expressed abnormally in the plasma of gastric carcinoma patients, P-DNA is released from the surface of MOF 1 ascribed to the stronger base pair matching, leading to the FAM fluorescence recovery. Each P-DNA@1 system is effective and reliable for the detection of its complementary target miRNA with the detection limits from 91 to 559pM, and is not interfered by other four miRNA sequences.

Impaired Autophagy and Defective T Cell Homeostasis in Mice with T Cell-Specific Deletion of Receptor for Activated C Kinase 1.

Autophagy plays a central role in maintaining T cell homeostasis. Our previous study has shown that hepatocyte-specific deficiency of receptor for activated C kinase 1 (RACK1) leads to lipid accumulation in the liver, accompanied by impaired autophagy, but its in vivo role in T cells remains unclear. Here, we report that mice with T cell-specific deletion of RACK1 exhibit normal intrathymic development of conventional T cells and regulatory T (Treg) cells but reduced numbers of peripheral CD4+ and CD8+ T cells. Such defects are cell intrinsic with impaired mitochondrial clearance, increased sensitivity to cell death, and decreased proliferation that could be explained by impaired autophagy. Furthermore, RACK1 is essential for invariant natural T cell development. In vivo, T cell-specific loss of RACK1 dampens concanavalin A-induced acute liver injury. Our data suggest that RACK1 is a key regulator of T cell homeostasis.

Neddylation is required for herpes simplex virus type I (HSV-1)-induced early phase interferon-beta production.

Type I interferons such as interferon-beta (IFN-ß) play essential roles in the host innate immune response to herpes simplex virus type I (HSV-1) infection. The transcription of type I interferon genes is controlled by nuclear factor-κB (NF-κB) and interferon regulatory factor (IRF) family members including IRF3. NF-κB activation depends on the phosphorylation of inhibitor of κB (IκB), which triggers its ubiqitination and degradation. It has been reported that neddylation inhibition by a pharmacological agent MLN4924 potently suppresses lipopolysaccharide (LPS)-induced proinflammatory cytokine production with the accumulation of phosphorylated IκBα. However, the role of neddylation in type I interferon expression remains unknown. Here, we report that neddylation inhibition with MLN4924 or upon UBA3 deficiency led to accumulation of phosphorylated IκBα, impaired IκBα degradation, and impaired NF-κB nuclear translocation in the early phase of HSV-1 infection even though phosphorylation and nuclear translocation of IRF3 were not affected. The blockade of NF-κB nuclear translocation by neddylation inhibition becomes less efficient at the later time points of HSV-1 infection. Consequently, HSV-1-induced early phase IFN-ß production significantly decreased upon MLN4924 treatment and UBA3 deficiency. NF-κB inhibitor JSH-23 mimicked the effects of neddylation inhibition in the early phase of HSV-1 infection. Moreover, the effects of neddylation inhibition on HSV-1-induced early phase IFN-ß production diminished in the presence of NF-κB inhibitor JSH-23. Thus, neddylation contributes to HSV-1-induced early phase IFN-ß production through, at least partially, promoting NF-κB activation.

A water-stable metal-organic framework of a zwitterionic carboxylate with dysprosium: a sensing platform for Ebolavirus RNA sequences.

We herein report a water-stable 3D dysprosium-based metal-organic framework (MOF) that can non-covalently interact with probe ss-DNA. The formed system can serve as an effective fluorescence sensing platform for the detection of complementary Ebolavirus RNA sequences with the detection limit of 160 pM.

A zwitterionic 1D/2D polymer co-crystal and its polymorphic sub-components: a highly selective sensing platform for HIV ds-DNA sequences.

Polymorphic compounds {[Cu(dcbb)2(H2O)2]·10H2O}n (2, 1D chain), [Cu(dcbb)2]n (3, 2D layer) and their co-crystal {[Cu(dcbb)2(H2O)][Cu(dcbb)2]2}n (4) have been prepared from the coordination reaction of a 2D polymer [Na(dcbb)(H2O)]n (1, H2dcbbBr = 1-(3,5-dicarboxybenzyl)-4,4'-bipyridinium bromide) with Cu(NO3)2·3H2O at different temperatures in water. Compounds 2-4 have an identical metal-to-ligand stoichiometric ratio of 1 : 2, but absolutely differ in structure. Compound 3 features a 2D layer structure with aromatic rings, positively charged pyridinium and free carboxylates on its surface, promoting electrostatic, π-stacking and/or hydrogen-bonding interactions with the carboxyfluorescein (FAM) labeled probe single-stranded DNA (probe ss-DNA, delineates as P-DNA). The resultant P-DNA@3 system facilitated fluorescence quenching of FAM via a photoinduced electron transfer process. The P-DNA@3 system functions as an efficient fluorescent sensor selective for HIV double-stranded DNA (HIV ds-DNA) due to the formation of a rigid triplex structure with the recovery of FAM fluorescence. The system reported herein also distinguishes complementary HIV ds-DNA from mismatched target DNA sequences with the detection limit of 1.42 nM.

A zinc(II)-based two-dimensional MOF for sensitive and selective sensing of HIV-1 ds-DNA sequences.

Coordination reaction of a known three-dimensional (3D) polymer precursor {Na3[Na9(Cbdcp)6(H2O)18]}n (A, Cbdcp = N-(4-carboxybenzyl)-(3,5-dicarboxyl)pyridinium) with Zn(NO3)2·6H2O in H2O or H2O/DMF at 100 °C and in the presence of aspirin, 5-fluorouracil (5-FU) as modulators, trans-1,2-bis(4-pyridyl)ethylene (bpe) or 1,2-bis(4-pyridyl)ethane (bpea) as ancillary ligands afforded six novel Zn(II)-based metal-organic frameworks (MOFs), that is, {[Zn(Cbdcp)(H2O)3]·H2O}n (1, 1D zigzag chain), {[Zn(HCbdcp)2]·H2O}n (2, 2D sheet), {[Zn(Cbdcp)(bpe)1/2]·2H2O}n (3, 3D polymer), {[Zn(Cbdcp)(bpe)1/2]·2H2O}n (4, 2D network), {[Zn(Cbdcp)(bpea)1/2]·2H2O}n (5, 3D polymer) and {[Zn(Cbdcp)(bpea)1/2]·2H2O}n (6, 2D network). Among them, compound 2 contains aromatic rings, positively charged pyridinium, Zn(2+) cation centers and carboxylic acid groups lined up on the 2D sheet structure with a certain extended surface exposure. The unique structure of 2 facilitates effective association with carboxyfluorescein (FAM) labeled probe single stranded DNA (probe ss-DNA, delineates as P-DNA) to yield a P-DNA@2 system, and leads to fluorescence quenching of FAM via a photoinduced electron transfer process. The P-DNA@2 system is effective and reliable for the detection of human immunodeficiency virus 1 ds-DNA (HIV ds-DNA) sequences and capable of distinguishing complementary HIV ds-DNA from mismatched target sequences with the detection limit as low as 10 pM (S/N = 3).

The NEDD8-activating enzyme inhibitor MLN4924 induces G2 arrest and apoptosis in T-cell acute lymphoblastic leukemia.

The first-in-class compound MLN4924 is a small molecule inhibitor that selectively inactivates NEDD8-activating enzyme (NAE). The anticancer effects of MLN4924 have been attributed to impaired neddylation of Cullin proteins. Here, we show that treatment of T-cell acute lymphoblastic leukemia (T-ALL) cells with MLN4924 potently suppressed the neddylation of Cullins and the oncogenic growth of T-ALL cells in-vitro. Moreover, MLN4924 induced disease regression in an in vivo xenograft model. MLN4924 also induced cell cycle arrest at G2 phase and apoptosis in T-ALL cells. However, inhibition of the neddylation of Cullins alone could not explain the effects of MLN4924 in T-ALL cells. Gene expression profiling indicated ribosome function, steroid biosynthesis, and hematopoietic cell lineage pathways were affected by MLN4924 treatment. MLN4924 also induced nucleolar disruption, suggesting nucleolar stress signaling might contribute to the anticancer effects of MLN4924 in T-ALL cells. In addition, MLN4924 treatment reduced 14-3-3ξ\δ protein levels in T-ALL cells. Thus, MLN4924 may inhibit T-ALL cell proliferation via several pathways.

Oridonin triggers apoptosis in colorectal carcinoma cells and suppression of microRNA-32 expression augments oridonin-mediated apoptotic effects.

Oridonin, a bioactive diterpenoid isolated from Rabdosia rubescens, has been found to exhibit various anti-tumor effects. In this work, to investigate its pharmacological effects on human colorectal carcinoma HCT-116 and LoVo cells, cell proliferation and apoptosis were respectively evaluated by 3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) assay, annexin V-FITC, and propidium iodide (PI) staining. Western blotting was used to detect the expression levels of Bim, Bax, Bcl-2, cytosolic cytochrome c, procaspase-9, cleaved caspase-9, procaspase-3, and caspase-3 proteins. Caspase-Glo-9 and Caspase-Glo-3 assays were applied to determine caspase-9 and caspase-3 activity. MicroRNA-32 (miR-32) expression level was detected by real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The in vivo anti-tumor effects of oridonin were evaluated using cell lines HCT-116 and LoVo xenograft model. The results indicated that oridonin effectively inhibited cell proliferation and induced apoptosis in HCT-116 and LoVo cells in a concentration-dependent manner. Oridonin treatment upregulated the expression levels of Bim, Bax, cytosolic cytochrome c, cleaved caspase-9 and cleaved caspase-3 proteins, downregulated the expression levels of Bcl-2, procaspase-9 and procaspase-3 proteins, and meanwhile obviously activated caspase-9 and caspase-3 in a dose-dependent manner in HCT-116 and LoVo cells. The results of qRT-PCR demonstrated that oridonin treatment significantly decreased miR-32 expression, and furthermore, suppression of miR-32 expression by miR-32 inhibitors augmented oridonin-mediated inhibitory and apoptotic effects in HCT-116 and LoVo cells. In vivo results indicated that oridonin administration through intraperitoneal injection suppressed tumor growth in nude mice. Therefore, these findings suggest that oridonin maybe is a potential candidate for colorectal cancer treatment.

RACK1 Promotes Autophagy by Enhancing the Atg14L-Beclin 1-Vps34-Vps15 Complex Formation upon Phosphorylation by AMPK.

Autophagy is essential for maintaining tissue homeostasis. Although adaptors have been demonstrated to facilitate the assembly of the Atg14L-Beclin 1-Vps34-Vps15 complex, which functions in autophagosome formation, it remains unknown whether the autophagy machinery actively recruits such adaptors. WD40-repeat proteins are a large, highly conserved family of adaptors implicated in various cellular activities. However, the role of WD40-repeat-only proteins, such as RACK1, in postnatal mammalian physiology remains unknown. Here, we report that hepatocyte-specific RACK1 deficiency leads to lipid accumulation in the liver, accompanied by impaired Atg14L-linked Vps34 activity and autophagy. Further exploration indicates that RACK1 participates in the formation of autophagosome biogenesis complex upon its phosphorylation by AMPK at Thr50. Thr50 phosphorylation of RACK1 enhances its direct binding to Vps15, Atg14L, and Beclin 1, thereby promoting the assembly of the autophagy-initiation complex. These observations provide insight into autophagy induction and establish a pivotal role for RACK1 in postnatal mammalian physiology.

RACK1 antagonizes TNF-α-induced cell death by promoting p38 activation.

p38 mitogen-activated protein kinase (MAPK) activity has been reported to either promote or suppress cell death, which depends on cell type and stimulus. Our previous report indicates that p38 exerts a protective role in tumor necrosis factor (TNF)-α-induced cell death in L929 fibroblastoma cells. However, key molecules regulating p38 activation remain unclear. Here, we show that ectopic expression of scaffold protein receptor for activated C kinase 1 (RACK1) suppressed TNF-α-induced cell death in L929 cells, which was associated with enhanced p38 activation. Knockdown of endogenous RACK1 expression exhibited opposite effects. The protective role of RACK1 in TNF-α-induced cell death diminished upon blockade of p38 activation. Therefore, RACK1 antagonizes TNF-α-induced cell death through, at least partially, augmenting p38 activation. Further exploration revealed that RACK1 directly bound to MKK3/6 and enhanced the kinase activity of MKK3/6 without affecting MKK3/6 phosphorylation. Similar effects of RACK1 were also observed in primary murine hepatocytes, another cell type sensitive to TNF-α-induced cell death. Taken together, our data suggest that RACK1 is a key factor involved in p38 activation as well as TNF-α-induced cell death.