Jiaxue Wu

Nuclear Export of NF90 to Stabilize IL-2 mRNA Is Mediated by AKT-Dependent Phosphorylation at Ser647 in Response to CD28 Costimulation

IL-2 is one of the most important cytokines required for T cell-mediated immune responses. Costimulation of CD28 in T cells up-regulates IL-2 mRNA levels via transcription activation and mRNA stabilization. Upon T cell activation, NF90, an AU-rich element (ARE)-binding protein, translocates from the nucleus into the cytoplasm, where it binds to the ARE-containing 3′ untranslated regions of IL-2 mRNA and slows down degradation of IL-2 mRNA. The translocation of NF90 is mediated through a nuclear export signal at its N terminus, but how it is triggered is still unclear. Phosphorylation of ARE-binding proteins has been reported as a signal transduction pathway to stabilize ARE-containing transcripts. In this study, we demonstrate that AKT phosphorylates NF90 on Ser647 upon CD28 costimulation. This phosphorylation is necessary for nuclear export of NF90 and IL-2 mRNA stabilization by this protein, because a mutation at Ser647 abolished both functions. We observed that treatment of cells with CD28 costimulation induced distinct increase in phosphorylation of AKT and NF90 at Ser647 concomitantly. Phosphorylation at Ser647 of NF90 up-regulated IL-2 production in response to CD28 costimulation. In vivo and in vitro data support a model in which CD28 costimulation activates AKT to phosphorylate NF90 at Ser647 and phosphorylation triggers NF90 to relocate to the cytoplasm and stabilize IL-2 mRNA.

Arginine68 is an essential residue for the C-terminal cleavage of human Atg8 family proteins

BackgroundAutophagy is a conserved cellular process that degrades and recycles cytoplasmic components via a lysosomal pathway. The phosphatidylethanolamine (PE)-conjugation of the Atg8 protein plays an important role in the yeast autophagy process. In humans, six Atg8 homologs, including MAP1LC3A, MAP1LC3B, MAP1LC3C (refer to LC3A, LC3B, and LC3C hereafter), GABARAP, GABARAPL1, and GABARAPL2 have been reported. All of them can be conjugated to PE through a ubiquitin-like conjugation system, and be located to autophagosomes.ResultsIn this study, we found 3 new alternative splicing isoforms in LC3B, GABARAP, and GABARAPL1, (designated as LC3B-a, GABARAP-a and GABARAPL1-a, respectively). None of them can go through the PE-conjugation process and be located to autophagosomes. Interestingly, compared with LC3B, LC3B-a has a single amino acid (Arg68) deletion due to the NAGNAG alternative splicing in intron 3. Through structural simulations, we found that the C-terminal tail of LC3B-a is less mobile than that of LC3B, thus affecting its C-terminal cleavage by human ATG4 family proteins. Furthermore, we found that Arg68 is an essential residue facilitating the interaction between human Atg8 family proteins and ATG4B by forming a salt bridge with Asp171 of ATG4B. Depletion of this salt bridge reduces autophagosomes formation and autophagic flux under both normal and nutrition starvation conditions.ConclusionsThese results suggest Arg68 is an essential residue for the C-terminal cleavage of Atg8 family proteins during the autophagy process.

Membrane palmitoylated protein 3 promotes hepatocellular carcinoma cell migration and invasion via up-regulating matrix metalloproteinase 1

Membrane associated guanylate kinase (MAGUK) family, has been extensively studied in cellular adhesion and signal transduction at sites of cell–cell contact. Recently, growing attention has been paid to its role in the initiation and progression of various cancers. However, its role in hepatocellular carcinoma (HCC) has been rarely investigated. In this study, we found that membrane palmitoylated protein 3 (MPP3), a member of MAGUK family, was significantly up-regulated in both high metastatic potential cell lines and clinical tissue samples of HCC, and the most significant increase was observed in the tumors invading the portal veins. Higher level of MPP3 correlated with poorer survival of patients with HCC. Forced expression of MPP3 significantly enhanced HCC cell migration and invasion, whereas knockdown of this gene inhibited this oncogenic effect. Mechanismly, we found that MPP3 promoted HCC cell migration and invasion via up-regulating matrix metalloproteinase 1 (MMP1). These findings indicate that MPP3 play an important role in HCC metastasis by promoting cell migration and invasion, suggesting that it may serve as a novel prognostic marker and molecular target for therapy of HCC.

Epigallocatechin-3-gallate enhances ER stress-induced cancer cell apoptosis by directly targeting PARP16 activity

Poly(ADP-ribose) polymerases (PARPs) are ADP-ribosylating enzymes and play important roles in a variety of cellular processes. Most small-molecule PARP inhibitors developed to date have been against PARP1, a poly-ADP-ribose transferase, and suffer from poor selectivity. PARP16, a mono-ADP-ribose transferase, has recently emerged as a potential therapeutic target, but its inhibitor development has trailed behind. Here we newly characterized epigallocatechin-3-gallate (EGCG) as a potential inhibitor of PARP16. We found that EGCG was associated with PARP16 and dramatically inhibited its activity in vitro. Moreover, EGCG suppressed the ER stress-induced phosphorylation of PERK and the transcription of unfolded protein response-related genes, leading to dramatically increase of cancer cells apoptosis under ER stress conditions, which was dependent on PARP16. These findings newly characterized EGCG as a potential inhibitor of PARP16, which can enhance the ER stress-induced cancer cell apoptosis, suggesting that a combination of EGCG and ER stress-induced agents might represent a novel approach for cancer therapy or chemoprevention.

Molecular chaperone CCT3 supports proper mitotic progression and cell proliferation in hepatocellular carcinoma cells

CCT3 was one of the subunits of molecular chaperone CCT/TRiC complex, which plays a central role in maintaining cellular proteostasis. We demonstrated that expressions of CCT3 mRNA and protein are highly up-regulated in hepatocellular carcinoma (HCC) tissues, and high level of CCT3 is correlated with poor survival in cancer patients. In HCC cell lines, CCT3 depletion suppresses cell proliferation by inducing mitotic arrest at prometaphase and apoptosis eventually. We also identified CCT3 as a novel regulator of spindle integrity and as a requirement for proper kinetochore-microtubule attachment during mitosis. Moreover, we found that CCT3 depletion sensitizes HCC cells to microtubule destabilizing drug Vincristine. Collectively, our study suggests that CCT3 is indispensible for HCC cell proliferation, and provides a potential drug target for treatment of HCC.

A novel mechanism for activation of Aurora-A kinase by Ajuba

Aurora-A is a centrosome-localized serine/threonine kinase, which plays a critical role in mitotic and meiotic cell division processes. However, the regulation of Aurora-A is still not fully understood. Previously, we have found an intramolecular inhibitory regulation mechanism of Aurora-A: the N-terminal regulatory domain (aa 1-128, Nt) can interact with the C-terminal catalytic domain (aa 129-403, Cd) and inhibit the kinase activity of Aurora-A. In this study, we found that the PreLIM domain of Ajuba, another important activator of Aurora-A, induces the autophosphorylation of the C-terminal kinase domain of Aurora-A, and is phosphorylated by the C-terminal. Moreover, the LIM domain of Ajuba can competitively bind to the N-terminal of Aurora-A, and inhibited the interaction between N-terminal and C-terminal of Aurora A. Taken together, these results suggest a novel mechanism for regulation of Aurora-A by Ajuba.

PARP10 suppresses tumor metastasis through regulation of Aurora A activity

ADP-ribosylation, including poly-ADP-ribosylation (PARylation) and mono-ADP-ribosylation (MARylation), is a multifunctional post-translational modification catalyzed by intracellular ADP-ribosyltransferases (ARTDs or PARPs). Although PARylation has been investigated most thoroughly, the function of MARylation is currently largely undefined. Here, we provide evidences that deficiency of PARP10, a mono-ADP-ribosyltransferase, markedly increased the migration and invasion of tumor cells through regulation of epithelial–mesenchymal transition (EMT), and PARP10 inhibited tumor metastasis in vivo, which was dependent on its enzyme activity. Mechanistically, we found that PARP10 interacted with and mono-ADP-ribosylated Aurora A, and inhibited its kinase activity, thereby regulating its downstream signaling. Moreover, the expression level of PARP10 was downregulated in intrahepatic metastatic hepatocellular carcinoma (HCC) compared with its corresponding primary HCC and adjacent non-tumorous tissues. Taken together, our results indicated that PARP10 has an important role in tumor metastasis suppression via negatively regulation of Aurora A activity.

Two newly identified sites in the N -terminal regulatory domain of Aurora-A are essential for auto-inhibition

Aurora-A, a centrosome-localized serine/threonine kinase, is over-expressed in multiple human cancers. We previously reported Zhang et al. (Biochem Biophys Res Commun 2007, 357:347–352) intramolecular inhibitory regulation of Aurora-A between its N-terminal (Nt) regulatory domain (amino acids 1–128, Nt) and C-terminal catalytic domain (aa 129–403, Cd). Here, we identified two essential sites located on the Nt of Aurora-A (Lys 99 and Lys 119) and demonstrate that mutation of either residue to Gly could cause the Nt and C-terminal lobes of the catalytic domain in Aurora-A to form a closed conformation, resulting in a loss of kinase activity. This inactive conformation was reversed by adding C26 peptide (274–299) or Ajuba, which is a required activator of Aurora-A. Over-expression of either mutant induced G2/M arrest. These results provide a basis for future anti-cancer studies targeting Aurora-A.

Aurora-A kinase-inactive mutants disrupt the interaction with Ajuba and cause defects in mitotic spindle formation and G2/M phase arrest in HeLa cells.

Aurora-A is a centrosome-localized serine/threonine kinase that is overexpressed in multiple human cancers. We previously reported an intramolecular inhibitory regulation of Aurora-A between its N-terminal regulatory domain (Nt, amino acids [aa] 1-128) and the C-terminal catalytic domain (Cd, aa 129-403). Here, we demonstrate that although both Aurora-A mutants (AurA-K250G and AurA-D294G/Y295G) lacked interactions between the Nt and Cd, they also failed to interact with Ajuba, an essential activator of Aurora-A, leading to loss of kinase activity. Additionally, overexpression of either of the mutants resulted in centrosome amplification and mitotic spindle formation defects. Both mutants were also able to cause G2/M arrest and apoptosis. These results indicate that both K250 and D294/Y295 are critical for direct interaction between Aurora-A and Ajuba and the function of the Aurora-A complex in cell cycle progression. [BMB Reports 2014; 47(11): 631-636]

Nogo‐B promotes tumor angiogenesis and provides a potential therapeutic target in hepatocellular carcinoma

Tumor angiogenesis is one of the hallmarks of cancer as well as an attractive target for cancer therapy. Characterization of novel pathways that act in parallel with the VEGF/VEGFR axis to promote tumor angiogenesis may provide insights into novel anti‐angiogenic therapeutic targets. We found that the expression level of Nogo‐B is positively correlated with tumor vessel density in hepatocellular carcinoma (HCC). While Nogo‐B depletion inhibited tumor angiogenesis, Nogo‐B overexpression promoted tumor angiogenesis in a tumor xenograft subcutaneous model of the human HCC cell line. Mechanically, Nogo‐B regulates tumor angiogenesis based on its association with integrin αvβ3 and activation of focal adhesion kinase. Moreover, Nogo‐B antibody successfully abolished the function of Nogo‐B in tumor angiogenesis in vitro and in vivo. Collectively, our results strongly suggest that Nogo‐B is an important tumor angiogenic factor and blocking Nogo‐B selectively inhibits tumor angiogenesis.