Toru Natsume

Acetylation regulates subcellular localization of eukaryotic translation initiation factor 5A (eIF5A)

Eukaryotic translation initiation factor 5A (eIF5A) is a protein subject to hypusination, which is essential for its function. eIF5A is also acetylated, but the role of that modification is unknown. Here, we report that acetylation regulates the subcellular localization of eIF5A. We identified PCAF as the major cellular acetyltransferase of eIF5A, and HDAC6 and SIRT2 as its major deacetylases. Inhibition of the deacetylases or impaired hypusination increased acetylation of eIF5A, leading to nuclear accumulation. As eIF5A is constitutively hypusinated under physiological conditions, we suggest that reversible acetylation plays a major role in controlling the subcellular localization of eIF5A.

The PX-RICS/14-3-3ζ/θ complex couples N-cadherin/β-catenin with dynein/dynactin to mediate its export from the endoplasmic reticulum

We have recently shown that β-catenin-facilitated export of cadherins from the endoplasmic reticulum requires PX-RICS, a β-catenin-interacting GTPase-activating protein for Cdc42. Here we show that PX-RICS interacts with isoforms of 14-3-3 and couples the N-cadherin-β-catenin complex to the microtubule-based molecular motor dynein-dynactin. Similar to knockdown of PX-RICS, knockdown of either 14-3-3ζ or -θ resulted in the disappearance of N-cadherin and β-catenin from the cell-cell boundaries. Furthermore, we found that PX-RICS and 14-3-3ζ/θ are present in a large multiprotein complex that contains dynein-dynactin components as well as N-cadherin and β-catenin. Both RNAi- and dynamitin-mediated inhibition of dynein-dynactin function also led to the absence of N-cadherin and β-catenin at the cell-cell contact sites. Our results suggest that the PX-RICS-14-3-3ζ/θ complex links the N-cadherin-β-catenin cargo with the dynein-dynactin motor and thereby mediates its endoplasmic reticulum export.

The subcellular localization and activity of cortactin is regulated by acetylation and interaction with Keap1

Promoting acetylation and thus nuclear localization of a cytoskeletal remodeling protein may limit cancer metastasis. Acetylation against cell migration The actin-binding protein cortactin promotes cell migration through cytoskeletal remodeling in the cell cortex and is abundant in certain types of aggressive cancers. Ito et al. found that the cytosolic protein Keap1 promoted the localization of cortactin to the cell cortex and thus cell migration. Cortactin shuttled between the cytoplasm and the nucleus; however, upon acetylation, cortactin no longer bound to Keap1 and became predominantly localized in the nucleus. Thus, increasing the acetylation of cortactin or preventing it from binding to Keap1 may suppress the metastasis of cancer cells. Cortactin is an F-actin–binding protein that localizes to the cell cortex, where the actin remodeling that is required for cell migration occurs. We found that cortactin shuttled between the cytoplasm and the nucleus under basal conditions. We identified Kelch-like ECH-associated protein 1 (Keap1), a cytosolic protein that is involved in oxidant stress responses, as a binding partner of cortactin that promoted the cortical localization of cortactin and cell migration. The ability of cortactin to promote cell migration is regulated by various posttranslational modifications, including acetylation. We showed that the acetylated form of cortactin was mainly localized to the nucleus and that acetylation of cortactin decreased cell migration by inhibiting the binding of cortactin to Keap1. Our findings reveal that Keap1 regulates cell migration by affecting the subcellular localization and activity of cortactin independently of its role in oxidant stress responses.

Identification and functional analysis of Zranb2 as a novel Smad-binding protein that suppresses BMP signaling.

Smads 1/5/8 transduce the major intracellular signaling of bone morphogenetic proteins (BMPs). In the present study, we analyzed Smad1‐binding proteins in HEK293T cells using a proteomic technique and identified the protein, zinc‐finger, RAN‐binding domain‐containing protein 2 (ZRANB2). Zranb2 interacted strongly with Smad1, Smad5, and Smad8 and weakly with Smad4. The overexpression of Zranb2 inhibited BMP activities in C2C12 myoblasts in vitro, and the injection of Zranb2 mRNA into zebrafish embryos induced weak dorsalization. Deletion analyses of Zranb2 indicated that the serine/arginine‐rich (SR) domain and the glutamine‐rich domain were required for the inhibition of BMP activity and the interaction with Smad1, respectively. Zranb2 was found to be localized in the nucleus; however, the SR domain‐deleted mutant localized to the cytoplasm. The knockdown of endogenous Zranb2 in C2C12 cells enhanced BMP activity. Zranb2 suppressed Smad transcriptional activity without affecting Smad phosphorylation, nuclear localization, or DNA binding. Taken together, these findings suggested that Zranb2 is a novel BMP suppressor that forms a complex with Smads in the nucleus. J. Cell. Biochem. 113: 808–814, 2012.

TBL2 Is a Novel PERK-Binding Protein that Modulates Stress-Signaling and Cell Survival during Endoplasmic Reticulum Stress

Under ER stress, PKR-like ER-resident kinase (PERK) phosphorylates translation initiation factor eIF2α, resulting in repression of global protein synthesis and concomitant upregulation of the translation of specific mRNAs such as activating transcription factor 4 (ATF4). This PERK function is important for cell survival under ER stress and poor nutrient conditions. However, mechanisms of the PERK signaling pathway are not thoroughly understood. Here we identify transducin (beta)-like 2 (TBL2) as a novel PERK-binding protein. We found that TBL2 is an ER-localized type-I transmembrane protein and preferentially binds to the phosphorylated form of PERK, but not another eIF2α kinase GCN2 or ER-resident kinase IRE1, under ER stress. Immunoprecipitation analysis using various deletion mutants revealed that TBL2 interacts with PERK via the N-terminus proximal region and also associates with eIF2α via the WD40 domain. In addition, TBL2 knockdown can lead to impaired ATF4 induction under ER stress or poor nutrient conditions such as glucose and oxygen deprivation. Consistently, TBL2 knockdown rendered cells vulnerable to stresses similarly to PERK knockdown. Thus, TBL2 serves as a potential regulator of the PERK pathway.

The Role of Acetylation in the Subcellular Localization of an Oncogenic Isoform of Translation Factor eIF5A

Mammalian cells express two isoforms of eIF5A, eIF5A1 and eIF5A2, but little is known about the function of eIF5A2. Here we report that eIF5A2 is reversibly acetylated at lysine-47. HDAC6 and SIRT2 were identified as the enzymes responsible for deacetylating eIF5A2. Analysis using acetylation-deficient mutants indicated that acetylation regulates the subcellular localization of eIF5A2.

Ubiquitylation of Ku80 by RNF126 Promotes Completion of Nonhomologous End Joining-Mediated DNA Repair

ABSTRACT Repair of damaged DNA is critical for maintenance of genetic information. In eukaryotes, DNA double-strand breaks (DSBs) are recognized by the Ku70-Ku80 heterodimer, which then recruits proteins that mediate repair by nonhomologous end joining (NHEJ). Prolonged retention of Ku70/80 at DSBs prevents completion of repair, however, with ubiquitylation of Ku80 having been implicated in Ku70/80 dissociation from DNA. Here, we identify RNF126 as a ubiquitin ligase that is recruited to DSBs and ubiquitylates Ku80, with UBE2D3 serving as an E2 enzyme. Knockdown of RNF126 prevented Ku70/80 dissociation from DSBs and inhibited break repair. Attenuation of Ku80 ubiquitylation by replacement of ubiquitylation site lysines with arginine residues delayed Ku70/80 release from chromatin after DSB induction by genotoxic insults. Together, our data indicate that RNF126 is a novel regulator of NHEJ that promotes completion of DNA repair by ubiquitylating Ku80 and releasing Ku70/80 from damaged DNA.

CACUL1/CAC1 Regulates the Antioxidant Response by Stabilizing Nrf2.

Nrf2 is the pre-dominant transcription activator responsible for coordinated up-regulation of ARE-driven antioxidant and detoxification genes. The activity of Nrf2 is tightly regulated at basal levels through its ubiquitination by Cul3-Keap1 and consequential degradation. Upon exposure to stress, the Cul3-Keap1 ligase is inhibited, leading to Nrf2 stabilization and activation. Here we describe CACUL1/CAC1 as a positive regulator of the Nrf2 pathway. We found that CACUL1 is up-regulated by Nrf2-activating oxidative stresses in cells and in mice. The association of CACUL1 with the Cul3-Keap1 complex led to a decrease in Nrf2 ubiquitination levels at non-stressed as well as stressed conditions, and sensitized cells for higher Nrf2 activation. Furthermore, CACUL1 knock-down led to a decrease in Nrf2 activity and cell viability under stress. Our results show that CACUL1 is a regulator of Nrf2 ubiquitination, adding another regulatory layer to the Nrf2 antioxidant stress response.

Corrigendum to ''Acetylation regulates subcellular localization of eukaryotic translation initiation factor 5A (eIF5A)'' (FEBS Lett. 586 (2012) 3236-3241)

http://dx.doi.org/10.1016/j.febslet.2014.06.002 0014-5793/ 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. DOI of original article: http://dx.doi.org/10.1016/j.febslet.2012.06.042 ⇑ Corresponding author at: Chemical Genetic Laboratory, RIKEN Advanced Science Institute, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan. Fax: +81 48 462 46 E-mail address: akihiro-i@riken.jp (A. Ito). Muhammad Ishfaq , Kazuhiro Maeta , Satoko Maeda , Toru Natsume , Akihiro Ito a,b,f,⇑, Minoru Yoshida a,b,c,f