Yurina Hibi

AKIP1 Enhances NF-κB-dependent Gene Expression by Promoting the Nuclear Retention and Phosphorylation of p65

In this study, we have identified protein kinase A-interacting protein 1 (AKIP1) as a binding partner of NF-κB p65 subunit, and AKIP1 enhances the NF-κB-mediated gene expression. AKIP1 is a nuclear protein and known to interact with the catalytic subunit of PKA (PKAc). We identified AKIP1 by a yeast two-hybrid screen using the N terminus region of p65 as bait. The interaction between AKIP1 and p65 was confirmed by glutathione S-transferase pull-down assay in vitro and immunoprecipitation-Western blotting assay in vivo. We found that the PKAc was present in the AKIP1·p65 complex and enhanced the transcriptional activity of NF-κB by phosphorylating p65. In a transient luciferase assay, AKIP1 cotransfection efficiently increased the transcriptional activity of NF-κB induced by phorbol 12-myristate 13-acetate (PMA). When AKIP1 was knocked down by RNA interference, the PMA-mediated NF-κB-dependent gene expression was abolished, indicating a physiological role of AKIP1. We found that PKAc, which is maintained in an inactive form by binding to IκBα and NF-κB in resting cells, was activated by PMA-induced signaling and could phosphorylate p65. Overexpression of AKIP1 increased the PKAc binding to p65 and enhanced the PKAc-mediated phosphorylation of p65 at Ser-276. Interestingly, this p65 phosphorylation promoted nuclear translocation of p65 and enhanced NF-κB transcription. In fact, we observed that AKIP1 colocalized with p65 within the cells and appeared to retain p65 in nucleus. These findings indicate a positive role of AKIP1 in NF-κB signaling and suggest a novel mechanism by which AKIP1 augments the transcriptional competence of NF-κB.

A-kinase-interacting Protein 1 (AKIP1) Acts as a Molecular Determinant of PKA in NF-κB Signaling

The cAMP-dependent protein kinase (PKA) signaling pathway plays a crucial role in the pathogenesis of many NF-κB-related diseases. However, there have been controversial reports with regard to the PKA actions in the regulation of NF-κB activity. In this study, we have demonstrated the effect of PKA on NF-κB activity in view of AKIP1 action; and in 293 and HeLa cells, where the endogenous AKIP1 expression is minimal, PKA-activating agents inhibited the NF-κB-dependent reporter gene expression, blocked the interaction of PKAc and p65 subunit of NF-κB, and attenuated PKA-dependent phosphorylation of p65 on Ser-276. This inhibitory function of PKAc in NF-κB signaling was reversed by overexpression of AKIP1 in 293 cells. In the breast cancer cell line, MDA-MB231 cells and MCF7 cells, where the endogenous AKIP1 is abundant, the PKA signal was found to be synergized with NF-κB activation; PKA-activating agents enhanced NF-κB-dependent transcriptional activity and the interaction between p65 and PKAc and augmented the phosphorylation of p65 on Ser-276. After RNAi knockdown of AKIP1 in these breast cancer cells, we observed that PKA-activating agents antagonized NF-κB-dependent activation. Meanwhile, PKA inhibitor suppressed NF-κB-induced breast cancer cell proliferation and multiple NF-κB-dependent anti-apoptotic gene expression. It is likely that expression of AKIP1 determines the relationship between these two signal transduction pathways. These findings explained controversial results from various independent groups regarding the action of PKA signaling on the NF-κB activation cascade and suggested a possible therapeutic potential of PKA inhibitor in developing anti-cancer strategies.

A-kinase interacting protein 1 (AKIP1) acts as a molecular determinant of the role of PKA in NF-κB signaling

The cAMP-dependent protein kinase (PKA) signaling pathway plays a crucial role in the pathogenesis of many NF-κB-related diseases. However, there have been controversial reports with regard to the PKA actions in the regulation of NF-κB activity. In this study, we have demonstrated the effect of PKA on NF-κB activity in view of AKIP1 action; and in 293 and HeLa cells, where the endogenous AKIP1 expression is minimal, PKA-activating agents inhibited the NF-κB-dependent reporter gene expression, blocked the interaction of PKAc and p65 subunit of NF-κB, and attenuated PKA-dependent phosphorylation of p65 on Ser-276. This inhibitory function of PKAc in NF-κB signaling was reversed by overexpression of AKIP1 in 293 cells. In the breast cancer cell line, MDA-MB231 cells and MCF7 cells, where the endogenous AKIP1 is abundant, the PKA signal was found to be synergized with NF-κB activation; PKA-activating agents enhanced NF-κB-dependent transcriptional activity and the interaction between p65 and PKAc and augmented the phosphorylation of p65 on Ser-276. After RNAi knockdown of AKIP1 in these breast cancer cells, we observed that PKA-activating agents antagonized NF-κB-dependent activation. Meanwhile, PKA inhibitor suppressed NF-κB-induced breast cancer cell proliferation and multiple NF-κB-dependent anti-apoptotic gene expression. It is likely that expression of AKIP1 determines the relationship between these two signal transduction pathways. These findings explained controversial results from various independent groups regarding the action of PKA signaling on the NF-κB activation cascade and suggested a possible therapeutic potential of PKA inhibitor in developing anti-cancer strategies.

53BP2 induces apoptosis through the mitochondrial death pathway.

The p53 binding protein 2 (53BP2) has been identified as the interacting protein to p53, Bcl‐2, and p65 subunit of nuclear factor κB (NF‐κB). The TP53BP2 gene encodes two splicing variants, 53BP2S and 53BP2L, previously known as apoptosis stimulating protein 2 of p53 (ASPP2). We found that these 53BP2 proteins are located predominantly in the cytoplasm and induce apoptosis as demonstrated by cleavage of poly ADP ribose polymerase (PARP) and annexin V staining. Furthermore, we demonstrate that 53BP2 is located in the mitochondria and induces apoptosis associated with depression of the mitochondrial trans‐membrane potential (ΔΨm) and activation of caspase‐9. From these findings we conclude that 53BP2 induces apoptosis through the mitochondrial death pathway.

Inhibition of Human Immunodeficiency Virus Type 1 Replication in Latently Infected Cells by a Novel IκB Kinase Inhibitor

ABSTRACT In human immunodeficiency virus type 1 (HIV-1) latently infected cells, NF-κB plays a major role in the transcriptional induction of HIV-1 replication. Hence, downregulation of NF-κB activation has long been sought for effective anti-HIV therapy. Tumor necrosis factor alpha (TNF-α) stimulates IκB kinase (IKK) complex, a critical regulator in the NF-κB signaling pathway. A novel IKK inhibitor, ACHP {2-amino-6-[2-(cyclopropylmethoxy)-6-hydroxyphenyl]-4-piperidin-4-yl-nicotinonitrile}, was developed and evaluated as a potent and specific inhibitor for IKK-α and IKK-β. In this study, we examined the ability of this compound to inhibit HIV-1 replication in OM10.1 cells latently infected with HIV. When these cells were pretreated with ACHP, TNF-α-induced HIV-1 replication was dramatically inhibited, as measured by the HIV p24 antigen levels in the culture supernatants. Its 50% effective concentration was approximately 0.56 μM, whereas its 50% cytotoxic concentration was about 15 μM. Western blot analysis revealed inhibition of IκBα phosphorylation, IκBα degradation, p65 nuclear translocation, and p65 phosphorylation. ACHP was also found to suppress HIV-1 long terminal repeat (LTR)-driven gene expression through the inhibition of NF-κB activation. Furthermore, ACHP inhibited TNF-α-induced NF-κB (p65) recruitment to the HIV-1 LTR, as assessed by chromatin immunoprecipitation assay. These findings suggest that ACHP acts as a potent suppressor of TNF-α-induced HIV replication in latently infected cells and that this inhibition is mediated through suppression of IKK activity.

A Single-Nucleotide Synonymous Mutation in the gag Gene Controlling Human Immunodeficiency Virus Type 1 Virion Production

ABSTRACT Viral factors as well as host ones play major roles in the disease progression of human immunodeficiency virus type 1 (HIV-1) infection. We have examined cytotoxic T-lymphocyte activity and HIV-1 DNA PCR results of 312 high-risk seronegative drug users in northern Thailand and identified four seronegative cases positive for both assays. Furthermore, we have identified a synonymous mutation in nucleotide position 75 of the gag p17 gene (A426G) of HIV-1 that belongs to the CRF01_AE virus circulating in Thailand. The replication-competent HIV-1 clone containing the A426G mutation demonstrated a dramatic reduction of virion production and perturbation of viral morphogenesis without affecting viral protein synthesis in cells.

Inhibition of Human Immunodeficiency Virus Type 1 Replication by Blocking IκB Kinase with Noraristeromycin

Nuclear factor kappaB (NF-kappaB) is one of the critical transcription factors in inflammatory responses and replication of viruses such as human immunodeficiency virus (HIV). In fact, it has been demonstrated that various NF-kappaB inhibitors could block HIV replication. To explore more potent NF-kappaB inhibitors, we focused on carbocyclic adenine nucleosides that had been reported to have anti-inflammatory effects. We synthesized 15 carbocyclic adenine nucleoside compounds and examined their effects on the NF-kappaB-dependent gene expression using HEK293 cell line. Among these compounds, noraristeromycin (NAM) exhibited the most potent inhibitory effect on the NF-kappaB activity under the non-cytotoxic concentrations. NAM-inhibited IkappaBalpha phosphorylation and degradation upon stimulation of cells with tumour necrosis factor-alpha (TNF-alpha). In addition, NAM prevented p65 phoshorylation. These findings suggested that both IkappaB kinase-alpha (IKK-alpha) and -beta were targeted by NAM. Interestingly, in vitro kinase assay revealed that NAM inhibited the kinase activity of IKK-alpha more potently than that of IKK-beta. When we treated the cell lines, OM10.1 and Molt4/IIIB, in which HIV-1 is latently and chronically infected, we found a strong suppressive effect of NAM on HIV-1 viral replication upon stimulation with TNF-alpha.

Functional characterization of human cyclin T1 N-terminal region for human immunodeficiency virus-1 Tat transcriptional activation.

Transcription of the human immunodeficiency virus type 1 (HIV-1) requires the interaction of the cyclin T1 (CycT1) subunit of a host cellular factor, positive transcription elongation factor b, with the viral Tat protein at the transactivation response (TAR) element of nascent viral transcripts. The involvement of the interaction between Tat and CycT1 is known to be through the Tat-TAR recognition motif (TRM) on CycT1. Here, we have further characterized this molecular interaction and clarified the role of the CycT1 N-terminal region in Tat action. We found crucial and distinctive roles of Q46, Q50 and F176 of human CycT1 protein in Tat-mediated transcription by creating various Ala substitution mutants of CycT1 based on its three-dimensional structure. We confirmed the involvement of these amino acid residues in binding to Tat with Q46 and Q50, and to a lesser extent with F176, by in vitro pull-down assay. Relative transactivation activities of wild-type CycT1 chimeras and mutant derivatives on the HIV-1 long terminal repeat were determined by luciferase reporter assays. Whereas CycT1 Q46A alone had impaired transcriptional activity, the CycT1(Q46A)-Tat chimeric protein retained almost full activity of the wild-type CycT1. However, CycT1 mutants (C261Y, Q50A or F176A) or their chimeric counterparts had lost the transactivation capacity. Moreover, a triple-mutant chimera containing Q46A, Q50A and F176A mutations completely abolished the transcriptional activity, indicating that these amino acid residues are involved through distinct mechanisms. These findings provide new insights for the development of anti-HIV drugs.

Molecular Dynamics Simulation and Experimental Verification of the Interaction between Cyclin T1 and HIV-1 Tat Proteins

The viral encoded Tat protein is essential for the transcriptional activation of HIV proviral DNA. Interaction of Tat with a cellular transcription elongation factor P-TEFb containing CycT1 is critically required for its action. In this study, we performed MD simulation using the 3D data for wild-type and 4CycT1mutants3D data. We found that the dynamic structural change of CycT1 H2’ helix is indispensable for its activity for the Tat action. Moreover, we detected flexible structural changes of the Tat-recognition cavity in the WT CycT1 comprising of ten AAs that are in contact with Tat. These structural fluctuations in WT were lost in the CycT1 mutants. We also found the critical importance of the hydrogen bond network involving H1, H1’ and H2 helices of CycT1. Since similar AA substitutions of the Tat-CycT1 chimera retained the Tat-supporting activity, these interactions are considered primarily involved in interaction with Tat. These findings described in this paper should provide vital information for the development of effective anti-Tat compound.

Impaired plant growth and development caused by human immunodeficiency virus type 1 Tat

Previous attempts to express the human immunodeficiency virus 1 (HIV-1) Tat (trans-activator of transcription) protein in plants resulted in a number of physiological abnormalities, such as stunted growth and absence of seed formation, that could not be explained. In the study reported here, we expressed Tat in tomato and observed phenotypic abnormalities, including stunted growth, absence of root formation, chlorosis, and plant death, as a result of reduced cytokinin levels. These reduced levels were ascribed to a differentially expressed CKO35 in Tat-bombarded tomato. Of the two CKO isoforms that are naturally expressed in tomato, CKO43 and CKO37, only the expression of CKO37 was affected by Tat. Our analysis of the Tat confirmed that the Arg-rich and RGD motifs of Tat have functional relevance in tomato and that independent mutations at these motifs caused inhibition of the differentially expressed CKO isoform and the extracellular secretion of the Tat protein, respectively, in our Tat-bombarded tomato samples.