Mechanistic insights into the cytotoxic and apoptotic effects of 1-butyl-3-methylimidazolium bromide and 1-butylpyridinium bromide on human cell lines

The serine/threonine kinase B-Raf is the second most frequently occurring human oncogene after Ras. Mutations of B-Raf occur with the highest incidences in melanoma, and the most common mutant, V600E, renders B-Raf constitutively active. The sodium proton exchanger isoform 1 (NHE1) is a ubiquitously expressed plasma membrane protein responsible for regulating intracellular pH, cell volume, cell migration, and proliferation. A screen of protein kinases that bind to NHE1 revealed that B-Raf bound to the cytosolic regulatory tail of NHE1. Immunoprecipitation of NHE1 from HeLa and HEK cells confirmed the association of B-Raf with NHE1 in vivo. The expressed and purified C-terminal 182 amino acids of the NHE1 protein were also shown to associate with B-Raf protein in vitro. Because treatment with the kinase inhibitor sorafenib decreased NHE1 activity in HeLa and HEK cells, we examined the role of B-Raf in regulating NHE1 in malignant melanoma cells. Melanoma cells with the B-Raf(V600E) mutation demonstrated increased resting intracellular pH that was dependent on elevated NHE1 activity. NHE1 activity after an acute acid load was also elevated in these cell lines. Moreover, inhibition of B-Raf activity by either sorafenib, PLX4720, or siRNA reduction of B-Raf levels abolished ERK phosphorylation and decreased NHE1 activity. These results demonstrate that B-Raf associates with and stimulates NHE1 activity and that B-Raf(V600E) also increases NHE1 activity that raises intracellular pH.

SARAF Inactivates the Store Operated Calcium Entry Machinery to Prevent Excess Calcium Refilling

The human mixed lineage leukemia-5 (MLL5) gene is frequently deleted in myeloid malignancies. Emerging evidence suggests that MLL5 has important functions in adult hematopoiesis and the chromatin regulatory network, and it participates in regulating the cell cycle machinery. Here, we demonstrate that MLL5 is tightly regulated through phosphorylation on its central domain at the G(2)/M phase of the cell cycle. Upon entry into mitosis, the phosphorylated MLL5 delocalizes from condensed chromosomes, whereas after mitotic exit, MLL5 becomes dephosphorylated and re-associates with the relaxed chromatin. We further identify that the mitotic phosphorylation and subcellular localization of MLL5 are dependent on Cdc2 kinase activity, and Thr-912 is the Cdc2-targeting site. Overexpression of the Cdc2-targeting MLL5 fragment obstructs mitotic entry by competitive inhibition of the phosphorylation of endogenous MLL5. In addition, G(2) phase arrest caused by depletion of endogenous MLL5 can be compensated by exogenously overexpressed full-length MLL5 but not the phosphodomain deletion or MLL5-T912A mutant. Our data provide evidence that MLL5 is a novel cellular target of Cdc2, and the phosphorylation of MLL5 may have an indispensable role in the mitotic progression.

It is widely acknowledged that E2F1 and GSK3beta are both involved in the process of cell differentiation. However, the relationship between E2F1 and GSK3beta in cell differentiation has yet to be discovered. Here, we provide evidence that in the differentiation of PC12 cells induced by nerve growth factor (NGF), GSK3beta was increased at both the mRNA and protein levels, whereas E2F1 at these two levels was decreased. Both wild-type GSK3beta and its kinase-defective mutant GSK3beta KM can inhibit E2F1 by promoting its ubiquitination through physical interaction. In addition, the colocalization of GSK3beta and E2F1 and their subcellular distribution, regulated by NGF, were observed in the process of PC12 differentiation. At the tissue level, GSK3beta colocalized and interacted with E2F1 in mouse hippocampus. Furthermore, GSK3beta facilitated neurite outgrowth by rescuing the promoter activities of Cdk inhibitors p21 and p15 from the inhibition caused by E2F1. To summarize, our findings suggest that GSK3beta can promote the ubiquitination of E2F1 via physical interaction and thus inhibit its transcription activity in a kinase activity independent manner, which plays an important role in the NGF-induced PC12 differentiation.

Post-transcriptionally Regulated Expression System in Human Xenogeneic Transplantation Models

Sterol regulatory element-binding proteins (SREBPs) are a family of transcription factors that regulates cholesterol and lipid metabolism. The active forms of these transcription factors are targeted by a number of post-translational modifications, including phosphorylation. Phosphorylation of Thr-426 and Ser-430 in SREBP1a creates a docking site for the ubiquitin ligase Fbw7, resulting in the degradation of the transcription factor. Here, we identify a novel phosphorylation site in SREBP1a, Ser-434, which regulates the Fbw7-dependent degradation of SREBP1. We demonstrate that both SREBP1a and SREBP1c are phosphorylated on this residue (Ser-410 in SREBP1c). Importantly, we demonstrate that the mature form of endogenous SREBP1 is phosphorylated on Ser-434. Glycogen synthase kinase-3 phosphorylates Ser-434, and the phosphorylation of this residue is attenuated in response to insulin signaling. Interestingly, phosphorylation of Ser-434 promotes the glycogen synthase kinase-3-dependent phosphorylation of Thr-426 and Ser-430 and destabilizes SREBP1. Consequently, mutation of Ser-434 blocks the interaction between SREBP1 and Fbw7 and attenuates Fbw7-dependent degradation of SREBP1. Importantly, insulin fails to enhance the levels of mature SREBP1 in cells lacking Fbw7. Thus, the degradation of mature SREBP1 is controlled by cross-talk between multiple phosphorylated residues in its C-terminal domain and the phosphorylation of Ser-434 could function as a molecular switch to control these processes.

High expression levels of SLFN11 correlate with the sensitivity of human cancer cells to DNA-damaging agents. However, little is known about the underlying mechanism. Here, we show that SLFN11 interacts directly with RPA1 and is recruited to sites of DNA damage in an RPA1-dependent manner. Furthermore, we establish that SLFN11 inhibits checkpoint maintenance and homologous recombination repair by promoting the destabilization of the RPA-ssDNA complex, thereby sensitizing cancer cell lines expressing high endogenous levels of SLFN11 to DNA-damaging agents. Finally, we demonstrate that the RPA1-binding ability of SLFN11 is required for its function in the DNA damage response. Our findings not only provide novel insight into the molecular mechanisms underlying the drug sensitivity of cancer cell lines expressing SLFN11 at high levels, but also suggest that SLFN11 expression can serve as a biomarker to predict responses to DNA-damaging therapeutic agents.

Aurora-A is a centrosome kinase and plays a pivotal role in G(2)/M cell cycle progression. Expression of Aurora-A is cell cycle-dependent. Levels of Aurora-A mRNA and protein are low in G(1)/S, accumulate during G(2)/M, and decrease rapidly after mitosis. Previous studies have shown regulation of the Aurora-A protein level during the cell cycle through the ubiquitin-proteasome pathway. However, the mechanism of transcriptional regulation of Aurora-A remains largely unknown. Here, we demonstrated that E2F3 modulates Aurora-A mRNA expression during the cell cycle. Ectopic expression of E2F3 induces Aurora-A expression. Stable knockdown of E2F3 decreases mRNA and protein levels of Aurora-A and delays G(2)/M entry. Further, E2F3 directly binds to Aurora-A promoter and stimulates the promoter activity. Deletion and mutation analyses of the Aurora-A promoter revealed that a region located 96-bp upstream of the transcription initiation site is critical for the activation of the promoter by E2F3. In addition, expression of E2F3 positively correlates with the protein level of Aurora-A in human ovarian cancer examined. These results indicate for the first time that Aurora-A is transcriptionally regulated by E2F3 during the cell cycle and that E2F3 is a causal factor for up-regulation of Aurora-A in a subset of human ovarian cancer. Thus, the E2F3-Aurora-A axis could be an important target for cancer intervention.

Directed Engineering of a High-expression Chimeric Transgene as a Strategy for Gene Therapy of Hemophilia A

Iron regulatory protein 2 (IRP2) is a key iron sensor that post-transcriptionally regulates mammalian iron homeostasis by binding to iron-responsive elements (IREs) in mRNAs that encode proteins involved in iron metabolism (e.g. ferritin and transferrin receptor 1). During iron deficiency, IRP2 binds IREs to regulate mRNA translation or stability, whereas during iron sufficiency IRP2 is degraded by the proteasome. Here, we identify an iron-independent IRP2 phosphorylation site that is regulated by the cell cycle. IRP2 Ser-157 is phosphorylated by Cdk1/cyclin B1 during G(2)/M and is dephosphorylated during mitotic exit by the phosphatase Cdc14A. Ser-157 phosphorylation during G(2)/M reduces IRP2 RNA-binding activity and increases ferritin synthesis, whereas Ser-157 dephosphorylation during mitotic exit restores IRP2 RNA-binding activity and represses ferritin synthesis. These data show that reversible phosphorylation of IRP2 during G(2)/M has a role in modulating the iron-independent expression of ferritin and other IRE-containing mRNAs during the cell cycle.

The role of heat shock protein 70 (Hsp70) in virus replication has been discussed for many viruses. The known suppressive role of Hsp70 in influenza virus replication is based on studies conducted in cells with various Hsp70 expression levels. In this study, we determined the role of Hsp70 in influenza virus replication in HeLa and HEK293T cells, which express Hsp70 constitutively. Co-immunoprecipitation and immunofluorescence studies revealed that Hsp70 interacted with PB2 or PB1 monomers and PB2/PB1 heterodimer but not with the PB1/PA heterodimer or PB2/PB1/PA heterotrimer and translocated into the nucleus with PB2 monomers or PB2/PB1 heterodimers. Knocking down Hsp70 resulted in reduced virus transcription and replication activities. Reporter gene assay, immunofluorescence assay, and Western blot analysis of nuclear and cytoplasmic fractions from infected cells demonstrated that the increase in viral polymerase activity during the heat shock phase was accompanied with an increase in Hsp70 and viral polymerases levels in the nuclei, where influenza virus replication takes place, whereas a reduction in viral polymerase activity was accompanied with an increase in cytoplasmic relocation of Hsp70 along with viral polymerases. Moreover, significantly higher levels of viral genomic RNA (vRNA) were observed during the heat shock phase than during the recovery phase. Overall, for the first time, these findings suggest that Hsp70 may act as a chaperone for influenza virus polymerase, and the modulatory effect of Hsp70 appears to be a sequel of shuttling of Hsp70 between nuclear and cytoplasmic compartments.

Axin is a negative regulator of Wnt/β-catenin signaling via regulating the level of β-catenin, which is a key effector molecule. Therefore, controlling the level of Axin is a critical step for the regulation of Wnt/β-catenin signaling. It has been shown that ubiquitination-mediated proteasomal degradation may play a critical role in the regulation of Axin; however, the E3 ubiquitin ligase(s), which attaches ubiquitin to a target protein in combination with an E2 ubiquitin-conjugating enzyme, for Axin has not yet been identified. Here, we show that Smurf2 is an E3 ubiquitin ligase for Axin. Transient expression of Smurf2 down-regulated the level of Axin and increased the ubiquitination of Axin. Conversely, shRNA specific to Smurf2 blocked Axin ubiquitination. Essential domains of Axin responsible for Smurf2 interaction as well as Smurf2-mediated down-regulation and ubiquitination were identified. In vitro ubiquitination assays followed by analysis using mass spectroscopy revealed that Smurf2 specifically ubiquitinylated Lys(505) of Axin and that the Axin(K505R) mutant resisted degradation. Knockdown of endogenous Smurf2 increased the level of endogenous Axin and resulted in reduced β-catenin/Tcf reporter activity. Overall, our data strongly suggest that Smurf2 is a genuine E3 ligase for Axin.

Toll-like receptors (TLRs) are proteins involved in recognition of foreign pathogen-associated molecular patterns and activation of processes leading to innate immune recognition. We show that stimulation of fibroblasts with a TLR5 ligand, flagellin, can induce proliferation of serum-starved cells or prevent cell cycle exit upon serum withdrawal independently of autologous growth factor secretion. Other TLR ligands, such as poly(I:C) and lipopolysaccharide, can have a similar effect only if the action of type I interferons is blocked. Flagellin stimulation can prevent cell cycle arrest induced by overexpression of exogenous cyclin-dependent kinase inhibitor p27. Stimulation of TLR5 and overexpression of MyD88, but not TRIF, TIRAP, or TRAM, result in p27 degradation, which can be suppressed by dominant negative Akt and mutation of the p27 C-terminal Thr(187) site. These data provide evidence for a nonimmune and cell autonomous role of TLR signaling, whereby TLR stimulation provides a positive signal for cell division.

The ligand-mediated down-regulation of the growth factor receptors is preceded by the involvement of various other factors. In particular, a ubiquitin ligase, Cbl, plays a central role in this event. Several candidates that have potential effects on the negative control of the epidermal growth factor (EGF) receptor have now been identified by our recent studies in phospho-proteomics. Among these molecules, we focus on characterizing a novel protein, Ymer, which is a tyrosine-phosphorylated and ubiquitinated protein. Ymer is found to be phosphorylated at tyrosine 145 and 146 upon EGF stimulation, and lysine 129 of Ymer has been identified as a ubiquitination site. Ymer has two motifs interacting with the ubiquitin (MIU) domains that might function as a binding site for the ubiquitinated EGF receptor. Although Ymer and EGF receptors are associated in an EGF-dependent manner, their interaction is required not only for MIU domains but also for the tyrosine phosphorylation of Ymer. Phosphorylated Ymer is mainly located at the plasma membrane with EGF receptor and functions in its endocytosis and degradation. Furthermore, EGF-mediated secondary modifications of an activated-EGF receptor are inhibited by overexpressing Ymer in COS7 cells. Therefore, Ymer may have competitive effects on the activation of the EGF receptor. Our findings suggest that Ymer functions as a novel inhibitor for the down-regulation of the EGF receptor and plays a crucial role for regulating the amount of the EGF receptor on the cell surface membrane.

Epstein-Barr virus (EBV) productive replication occurs in an S-phase-like cellular environment with high cyclin-dependent kinase (CDK) activity. The EBV protein kinase (PK), encoded by the viral BGLF4 gene, is a Ser/Thr protein kinase, which phosphorylates both viral and cellular proteins, modifying the cellular environment for efficient viral productive replication. We here provide evidence that the EBV PK phosphorylates the CDK inhibitor p27(Kip1), resulting in ubiquitination and degradation in a proteasome-dependent manner during EBV productive replication. Experiments with BGLF4 knockdown by small interfering RNA and BGLF4 knock-out viruses clarified that EBV PK is involved in p27(Kip1) degradation upon lytic replication. Transfection of the BGLF4 expression vector revealed that EBV PK alone could phosphorylate the Thr-187 residue of p27(Kip1) and that the ubiquitination and degradation of p27(Kip1) occurred in an SCF(Skp2) ubiquitin ligase-dependent manner. In vitro, EBV PK proved capable of phosphorylating p27(Kip1) at Thr-187. Unlike cyclin E-CDK2 activity, the EBV PK activity was not inhibited by p27(Kip1). Overall, EBV PK enhances p27(Kip1) degradation effectively upon EBV productive replication, contributing to establishment of an S-phase-like cellular environment with high CDK activity.