Sec16A, a key protein in COPII vesicle formation, regulates the stability and localization of the novel ubiquitin ligase RNF183

<div><p>We identified 37 ubiquitin ligases containing RING-finger and transmembrane domains. Of these, we found that RNF183 is abundantly expressed in the kidney. RNF183 predominantly localizes to the endoplasmic reticulum (ER), Golgi, and lysosome. We identified Sec16A, which is involved in coat protein complex II vesicle formation, as an RNF183-interacting protein. RNF183 colocalized with Sec16A and interacted through the central conserved domain (CCD) of Sec16A. Although Sec16A is not a substrate for RNF183, RNF183 was more rapidly degraded by the ER-associated degradation (ERAD) in the absence of Sec16A. Sec16A also stabilized the interacting ubiquitin ligase RNF152, which localizes to the lysosome and has structural similarity with RNF183. These results suggest that Sec16A appears to regulate the protein stability and localization of lysosomal ubiquitin ligases.</p></div

Effects of Sec16 on RNF183 subcellular localization.

<p>(A) Effect of Sec16A downregulation on RNF183 subcellular localization. HeLa cells stably expressing RNF183-V5 were transfected with NC (1st, 3rd, 5th panels) or Sec16A (2nd, 4th, 6th panels) siRNAs. At 48 h after transfection, cells were subjected to immunofluorescence staining with anti-V5 (<i>green</i>) and anti-calnexin, GM130, or LAMP1 (<i>red</i>) antibodies, and DAPI (<i>blue</i>). (B) Effect of proteasome inhibition on RNF183 subcellular localization. HeLa cells stably expressing RNF183-V5 were transfected with NC (<i>top</i> panels) or Sec16A (<i>middle</i> and <i>bottom</i> panels) siRNAs. At 36 h after transfection, cells were incubated with (<i>bottom</i> panels) or without (<i>top</i> and <i>middle</i> panels) 10 μM MG132 for 12 h.</p

Proteomics analysis of the RNF183 interacting protein.

<p>Proteomics analysis of the RNF183 interacting protein.</p

Interaction and colocalization of RNF183 and Sec16A.

<p>(A) Interaction of RNF183 and Sec16A. Cell lysates from HEK293 cells stably expressing V5-tagged RNF183 were immunoprecipitated using anti-V5 antibody, and the immune complexes were analyzed by Western blotting with anti-Sec16A (<i>upper</i> panel) or anti-V5 (<i>lower</i> panel) antibodies. (B) Colocalization of RNF183 with Sec16A. HeLa cells stably transfected with RNF183-V5 were subjected to immunofluorescence staining with anti-Sec16A (<i>green</i>) and DAPI (<i>blue</i>). (C) Effect of RNF183 knockdown on Sec16A protein. RNF183 expression in HEK293 cells stably transfected with RNF183-V5 was suppressed using siRNA. Endogenous Sec16A protein levels were detected using Western blotting with anti-Sec16A antibody. NC, negative control. (D) Schematic diagram of the predicted domains of Sec16A. (E) Interaction domain of Sec16A with RNF183. Lysates from HEK293 cells stably expressing RNF183 transiently transfected with GFP-tagged full-length Sec16A (Full) or its deletion mutant constructs were subjected to immunoprecipitation with anti-V5 antibody, followed by immunoblotting with anti-GFP antibody.</p

Effects of Sec16 on RNF183 protein stability.

<p>(A) Effect of Sec16A downregulation on RNF183 protein stability. HeLa cells stably expressing RNF183-V5 were transfected with NC (1st, 3rd, and 5th panels) or Sec16A (2nd, 4th and 6th panels) siRNA. At 44 h after transfection, cells were treated with 30 μg/ml cycloheximide (CHX) and 10 μM MG132 for the indicated periods. Total cell lysates were analyzed by Western blotting with an anti-V5 (1st and 2nd panels), Sec16A (3rd and 4th panels), and β-actin (5th and 6th panels) antibodies. (B) Quantitative curves of data from (A). RNF183 levels at each time point were plotted relative to the level at time 0 (n = 3). Asterisks represent significant differences (Student’s t test with Bonferroni correction, *p < 0.05; NC vs. Sec16A siRNA; #p < 0.05, ##p < 0.01; ##p < 0.001; Sec16A siRNA vs. Sec16A siRNA + MG132).</p

Effects of Sec16 on other ubiquitin ligases.

<p>(A) Interactions of RNF152 and HRD1 with Sec16A. Coimmunoprecipitation was performed in HEK293 cells engineered to stably express V5-tagged RNF183, V5-tagged RNF152, or myc-tagged HRD1. Cell lysates were immunoprecipitated with anti-V5 or anti-myc antibodies or normal mouse immunoglobulin G (IgG; negative control). Immune complexes were analyzed by Western blotting with an anti-Sec16A antibody (<i>top</i> panel) and anti-V5 (<i>second</i> panel) or anti-myc antibodies (<i>third</i> panel). (B, D) Effect of Sec16A downregulation on RNF152 and HRD1 protein stability. Stable RNF152-V5- or HRD1-myc-expressing HEK293 cells were transfected with NC or Sec16A siRNA. At 48 h after transfection, cells were subjected to a CHX assay. (C, E) Asterisks represent significant differences (n = 3; Student’s t test with Bonferroni correction, *p < 0.05, ***p < 0.001; NC vs. Sec16A siRNA).</p

The structure of BBF2H7 and its expression in tumors or cancer cell lines.

<p>(A) Increased expression of <i>Bbf2h7</i> in human cancers. Microarray datasets of tumors were accessed in the ONCOMINE Cancer Profiling Database (version 4.4.4.4, <a href="http://www.oncomine.org/" target="_blank">www.oncomine.org</a>). Box plots showing increased expression of <i>Bbf2h7</i> during tumorigenesis of various cancers were constructed from ONCOMINE. The y-axis represents the log₂ median-centered intensity (normalized expression). The line within the box represents the median expression value for each group, and the upper and lower edges of the box indicate the 75^th and 25^th percentiles of the distribution, respectively. The lines (whiskers) from each box extend to the 90^th and 10^th percentiles of the distribution. The black dots outside the ends of the whiskers represent the largest and smallest data points. Box plots depicting the distribution of <i>Bbf2h7</i> expression within each sample and a Student’s <i>t</i>-test giving a <i>P</i> value for the comparison of <i>Bbf2h7</i> expression between normal and malignant tissue samples were obtained directly through ONCOMINE. Normal colon tissue samples included ascending colon, descending colon and rectum. (B) Predicted peptide features of human BBF2H7. Transcription activation, basic leucine zipper (bZIP) and transmembrane domains, as well as a site-1 protease (S1P) site are indicated. (C) Under ER stress conditions, BBF2H7 is transported from the ER to the Golgi apparatus and cleaved at the S1P site [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125982#pone.0125982.ref009" target="_blank">9</a>]. The cleaved BBF2H7 N-terminus acts as a transcription factor via binding to the cyclic AMP response element (CRE) of target genes [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125982#pone.0125982.ref015" target="_blank">15</a>]. In contrast, the cleaved BBF2H7 C-terminus is extracellularly secreted [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125982#pone.0125982.ref016" target="_blank">16</a>]. (D) Western blotting of the endogenous full-length BBF2H7 and the N- or C-terminus of BBF2H7 using cell lysates (left panel) or culture media (right panel) of several human cancer cell lines. BBF2H7 N-terminus, BBF2H7 C-terminus or IL-6 in the culture media was immunoprecipitated using anti-BBF2H7 N-terminus (top panel), anti-BBF2H7 C-terminus (middle panel), or anti-IL-6 antibodies (bottom panel), respectively. β-actin or IL-6 was used as loading controls. (E) RT-PCR analysis of <i>Xbp1</i> mRNA expression in several human cancer cell lines. <i>Xbp1-s</i> and <i>Xbp1-u</i> indicate spliced and unspliced forms of <i>Xbp1</i>, respectively. Note that <i>Xbp1-s</i> was detected in all cancer cell lines, indicating that these cells are undergoing ER stress. (F) RT-PCR analysis of <i>Xbp1</i> mRNA expression in U251MG cells treated with 2 mM 4-PBA, a chemical chaperon, for 3 h. (G) Quantification of <i>Xbp1-s</i> expression in F. Error bars represent the mean ± SD of five independent experiments. <b>**</b><i>P</i> < 0.01. (H) Western blotting of BBF2H7 in U251MG cells treated with 1 μM thapsigargin (TG), an inhibitor of ER Ca²⁺-ATPase, for 6 h. (I) Immunohistochemistry of brain sections from glioblastoma patients using an anti-BBF2H7 C-terminus-specific antibody. Strong signals were detected both in the cytosol of the cancer cells (indicated with an arrowhead) and in their surrounding extracellular space (indicated with arrows). Left panel shows a low magnification, and right panel shows a higher magnification of the left panel. Scale bars indicate 50 μm (left panel) and 10 μm (right panel).</p

Secreted BBF2H7 C-terminus is involved in the proliferation of cancer cells.

<p>U251MG cells were knocked down by siRNAs, followed by treatment with conditioned medium collected from HEK293T cells transfected with an empty vector (Mock) or BBF2H7 C-terminus (C-Sup.), or by treatment with C-Sup. depleted of BBF2H7 C-terminus by immunoprecipitation using an anti-BBF2H7 C-terminus antibody (C-Sup. + Ab.), and then cell proliferation was monitored by cell counting and WST-8 assays. Scramble and siBBF2H7-1 indicate non-targeting siRNA and <i>Bbf2h7</i>-targeting siRNA, respectively. (A, B) U251MG cells were cultured for 5 days after conditioned medium treatment, and then analyzed by cell counting at day 5 (A) and WST-8 assays at the indicated days (B). Error bars represent the mean ± SD of five independent experiments. <b>*</b><i>P</i> < 0.05, <b>**</b><i>P</i> < 0.01, <b>***</b><i>P</i> < 0.001. (C) RT-PCR analysis of Hh target genes in U251MG cells at 5 days after conditioned medium treatment. (D) Quantitative real-time PCR analyses of Hh target genes expression in C. Error bars represent the mean ± SD of five independent experiments. <b>*</b><i>P</i> < 0.05.</p

Cancer cell proliferation in an Shh-dependent manner.

<p>(A) RT-PCR analysis of Hh target genes in several cancer cell lines treated with 500 ng/ml recombinant Shh for 48 h. (B) Quantitative real-time PCR analyses of Hh target genes expression in A. Error bars represent the mean ± SD of five independent experiments. <b>*</b><i>P</i> < 0.05, <b>**</b><i>P</i> < 0.01. (C) The cancer cell lines were cultured for 5 days after treatment with 500 ng/ml recombinant Shh and analyzed by WST-8 assays at day 5. Error bars represent the mean ± SD of five independent experiments. <b>*</b><i>P</i> < 0.05.</p

Activation of Hh signaling by BBF2H7 C-terminus.

<p>(A, B) <i>Bbf2h7</i>^-/- MEFs were cultured for 5 days after transfection with BBF2H7 full-length (Full), N-terminus (N), C-terminus (C) or an empty vector (Mock), and analyzed by cell counting at day 5 (A) and WST-8 assay at the indicated time points (B). WT and KO indicate wild type MEFs and <i>Bbf2h7</i>^-/- MEFs, respectively. Error bars represent the mean ± SD of five independent experiments. <b>*</b><i>P</i> < 0.05, <b>**</b><i>P</i> < 0.01. (C) RT-PCR analysis of Hh target genes in several cancer cell lines exposed to conditioned medium collected from HEK293T cells transfected with BBF2H7 C-terminus (C-Sup. +) or an empty vector (C-Sup.–) for 48 h. (D) Quantitative real-time PCR analyses of Hh target genes expression in C. Error bars represent the mean ± SD of five independent experiments. <b>*</b><i>P</i> < 0.05, <b>**</b><i>P</i> < 0.01. (E) RT-PCR analysis of Hh target genes in U251MG cells treated with C-Sup., C-Sup. depleted of BBF2H7 C-terminus by immunoprecipitation using an anti-BBF2H7 C-terminus antibody (C-Sup. + Ab.), or C-Sup. in the presence of 5 μM cyclopamine (C-Sup. + CPN). Mock indicates conditioned medium collected from HEK293T cells transfected with an empty vector. (F) Quantitative real-time PCR analyses of Hh target genes expression in E. Error bars represent the mean ± SD of five independent experiments. <b>*</b><i>P</i> < 0.05, <b>**</b><i>P</i> < 0.01. (G, H) U251MG cells were cultured for 5 days after conditioned medium treatment and analyzed by cell counting at day 5 (G) and WST-8 assays at the indicated days (H). Error bars represent the mean ± SD of five independent experiments. <b>*</b><i>P</i> < 0.05, <b>**</b><i>P</i> < 0.01.</p