JAB1/CSN5 inhibits the activity of Luman/CREB3 by promoting its degradation - PubMed (original) (raw)

JAB1/CSN5 inhibits the activity of Luman/CREB3 by promoting its degradation

Lisa M DenBoer et al. Biochim Biophys Acta. 2013 Sep.

Abstract

Luman/CREB3 (also called LZIP) is an endoplasmic reticulum (ER)-bound transcription factor that has been implicated in the ER stress response. In this study, we used the region of Luman containing the basic DNA-binding domain as bait in a yeast two-hybrid screen and identified the Jun activation domain-binding protein 1 (JAB1) or the COP9 signalosome complex unit 5 (CSN5) as an interacting protein. We confirmed their direct binding by glutathione S-transferase pull-down assays, and verified the existence of such interaction in the cellular environment by mammalian two-hybrid and co-immunoprecipitation assays. Deletion mapping studies revealed that the MPN domain in JAB1 was essential and sufficient for the binding. JAB1 also colocalized with Luman in transfected cells. More interestingly, the nuclear form of Luman was shown to promote the translocation of JAB1 into the nucleus. We found that overexpression of JAB1 shortened the half-life of Luman by 67%, and repressed its transactivation function on GAL4 and unfolded protein response element (UPRE)-containing promoters. We therefore propose that JAB1 is a novel binding partner of Luman, which negatively regulates the activity of Luman by promoting its degradation.

Keywords: ER; ER-associated degradation; ERAD; HSV; JAB1/CSN5; Luman/CREB3/LZIP; Protein degradation; Transcription factor; UPR; UPRE; Unfolded protein response; endoplasmic reticulum; herpes simplex virus; unfolded protein response.

Crown Copyright © 2013. Published by Elsevier B.V. All rights reserved.

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Figures

Fig. 1

Fig. 1

Luman interacts with JAB1 both in vitro and in vivo. (A) Direct binding of Luman to JAB1 in GST pull-down assays. GST, GST-Luman(1–215) and GST-JAB1 proteins were coupled to glutathione-sepharose beads and incubated with [35S]-labeled GAL4 AD-GFP, FLAG-Luman and HA-JAB1. After extensive washing, proteins were eluted from the beads, separated on a 10% SDS-PAGE gel and visualized by autoradiography. The input lanes have 10% of the radio-labeled protein used in each pull-down assay. (B) Luman interacts with JAB1 in the cell as demonstrated by the mammalian two-hybrid assay. HEK-293 cells were transiently transfected with a combination of pM-JAB1 and a Luman construct, along with the reporter plasmids p5×GAL4 luciferase and pRL-SV40. Dual luciferase activities were measured 24 h post-transfection. Values are normalized to Renilla luciferase before being referenced to the pM-JAB1 background control. Data are based on 6 independent assays and shown with standard errors. (C) Luman interacts with JAB1 in mammalian cells as demonstrated by co-immunoprecipitation. HEK-293 cells were transiently transfected with pcDNA-Luman, pFLAG-JAB1 or both. Cells were treated with proteasome inhibitor MG132 for the last 6 h, prior to harvest of cell lysates at 24 h post-transfection. Cleared lysates were incubated with either a Luman- or FLAG-specific antibody, followed by precipitation with Protein G beads. Precipitated samples were subjected to SDS-PAGE and probed in Western blotting with antibodies against FLAG (top panel), Luman (middle panel) or HCF-1 (bottom panel). HCF-1 was included as a positive control for Luman since it is a known interacting protein. The input lanes have 2% of cleared cell lysates used in the immunoprecipitation assays. Abbreviations: IP, immunoprecipitation antibody; WB, Western blot antibody.

Fig. 2

Fig. 2

Co-localization of JAB1 with Luman demonstrated by confocal fluorescence microscopy. Vero cells were transiently transfected with plasmids expressing JAB1 or GFP-Luman protein. JAB1 was visualized indirectly with a JAB1-specific antibody and a fluorophore Alexa488- or Alexa594-conjugated secondary antibody. DAPI was used to counter-stain the nuclei of the cells.

Fig. 3

Fig. 3

The MPN domain of JAB1 is necessary and sufficient for interaction with Luman. (A) Schematic diagram showing the JAB1 deletion mutants used in the study. (B) GST and GST-Luman(1–215) proteins were coupled to glutathione-sepharose beads and incubated with 35S-labeled HA-JAB1 proteins, as well as with the controls FLAG-Luman and GAL4 AD-GFP. After extensive washing, proteins were eluted from the beads, separated on a 10% SDS-PAGE gel and visualized by autoradiography. The input lanes have 10% of the radio-labeled protein used in each assay. (C) To confirm the pulldown results of JAB1(190–334), the experiment was repeated with the full-length JAB1 protein and Luman as positive controls.

Fig. 4

Fig. 4

JAB1 represses transactivational activity of Luman and promotes degradation of the Luman protein. (A) Dual luciferase reporter assays. HEK-293 cells were transiently transfected with pHA-JAB1 and FLAG-Luman (top panel) or Gal DBD-Luman (bottom panel) constructs, along with the firefly luciferase reporter plasmids 5×UPRE or 5×GAL4 respectively. The control Renilla luciferase reporter plasmid pRL-SV40 was included in all samples. The firefly luciferase values are normalized to Renilla luciferase before being referenced to the pcDNA control. Data are based on 3 independent assays and shown with standard errors. (B) Pulse-chase assays. HEK-293 cells were transiently transfected with pcFLAG-Luman alone, or co-transfected with HA-JAB1. The cells were methionine/cysteine starved for 1 h, pulse-labeled with [35S]-methionine/cysteine for 1.5 h and chased for the time as indicated. The Luman protein was then immunoprecipitated with an anti-FLAG antibody, resolved by SDS-PAGE, and visualized by autoradiography. The negative control represents a pooled sample immunoprecipitated with a non-specific antibody. The half-life of the protein was determined by densitometric analysis using data from two independent trials collected on a Typhoon 9400 Phosphorimager. Values are plotted with standard errors.

Fig. 5

Fig. 5

JAB1 siRNA knockdown increases the levels of Luman and the downstream target Herp. (A) HEK293 cells treated with either scrambled siRNA or JAB1 siRNA, were immunoprecipitated using an anti-Luman antibody. The flowthrough (FT) and eluted (E) samples were separated on a 10% SDS-PAGE, and analyzed by Western blotting using both Luman and ubiquitin antibodies. The left panel depicts the levels of Luman, JAB1 and β-actin in the input lysates prior to immunoprecipitation. The right panel shows the levels of total Luman and ubiquitinated Luman in the precipitates. (B) RT-PCR analysis of Herp and GAPDH in HEK293 cells which were transfected with either scrambled siRNA or JAB1 siRNA or Luman, with or without tunicamycin (Tm) treatment. Densitometric analysis of the HERP bands normalized against GAPDH was performed. The experiment was repeated three times, and fold changes over the control siRNA sample were shown, along with standard errors.

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References

    1. Lu R, Yang P, O’Hare P, Misra V. Luman, a new member of the CREB/ATF family, binds to herpes simplex virus VP16-associated host cellular factor. Mol Cell Biol. 1997;17:5117–5126. - PMC - PubMed
    1. Freiman RN, Herr W. Viral mimicry: common mode of association with HCF by VP16 and the cellular protein LZIP. Genes Dev. 1997;11:3122–3127. - PMC - PubMed
    1. Kristie TM, Sharp PA. Purification of the cellular C1 factor required for the stable recognition of the Oct-1 homeodomain by the herpes simplex virus alpha-trans-induction factor (VP16) J Biol Chem. 1993;268:6525–6534. - PubMed
    1. Wilson AC, LaMarco K, Peterson MG, Herr W. The VP16 accessory protein HCF is a family of polypeptides processed from a large precursor protein. Cell. 1993;74:115–125. - PubMed
    1. Lu R, Yang P, Padmakumar S, Misra V. The herpesvirus transactivator VP16 mimics a human basic domain leucine zipper protein, Luman, in its interaction with HCF. J Virol. 1998;72:6291–6297. - PMC - PubMed

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