A novel ER–microtubule-binding protein, ERLIN2, stabilizes Cyclin B1 and regulates cell cycle progression

The gene encoding endoplasmic reticulum (ER) lipid raft-associated protein 2 (ERLIN2) is amplified in human breast cancers. ERLIN2 gene mutations were also found to be associated with human childhood progressive motor neuron diseases. Yet, an understanding of the physiological function and mechanism for ERLIN2 remains elusive. In this study, we reveal that ERLIN2 is a spatially and temporally regulated ER–microtubule-binding protein that has an important role in cell cycle progression by interacting with and stabilizing the mitosis-promoting factors. Whereas ERLIN2 is highly expressed in aggressive human breast cancers, during normal development ERLIN2 is expressed at the postnatal stage and becomes undetectable in adulthood. ERLIN2 interacts with the microtubule component α-tubulin, and this interaction is maximal during the cell cycle G2/M phase where ERLIN2 simultaneously interacts with the mitosis-promoting complex Cyclin B1/Cdk1. ERLIN2 facilitates K63-linked ubiquitination and stabilization of Cyclin B1 protein in G2/M phase. Downregulation of ERLIN2 results in cell cycle arrest, represses breast cancer proliferation and malignancy and increases sensitivity of breast cancer cells to anticancer drugs. In summary, our study revealed a novel ER–microtubule-binding protein, ERLIN2, which interacts with and stabilizes mitosis-promoting factors to regulate cell cycle progression associated with human breast cancer malignancy.

All cell lines were maintained at 37°C, in a 5% CO2 environment. The lentiviral expression construct expressing the human ERLIN2 tagged with or without V5 (pLenti-ERLIN2) was established as previously described (Wang et al., 2012). The pLenti-ERLIN2 virus was used to infect CHO-k1 or Huh-7 cells to produce CHO or Huh-7 cell lines that stably express different levels of V5 tagged-ERLIN2. The pLenti-LacZ virus was included as an experimental control.
Selection with 10 μg/mL blasticidin was started from 48 hours after infection.

Western Blot and Immunoprecipitation (IP)-Western blot analyses -To determine expression
levels of ERLIN2, α-tubulin, Cyclin B1, or GAPDH, total cell lysates were prepared from cultured cells or various tissues from wild-type mice using NP-40 lysis buffer. Denatured proteins were separated by SDS-PAGE on 10% Tris-glycine polyacrylamide gels and transferred onto a 0.45mm PVDF membrane (Fisher Scientific). Membrane-bound antibodies were detected by an enhanced chemiluminescence detection reagent (Fisher Scientific). For IP-Western blot analysis, total protein lysates from in vitro cultured cells were immunoprecipitated with anti-α-tubulin, antiβ-tubulin, anti-Cyclin B1, anti-CDK1 or anti-ERLIN2 antibodies, followed by Western blot analysis using the anti-ERLIN2 or anti-α-tubulin antibody to detect protein interactions. Data shown was a representative of at least 3 independent experiments.
Immunofluorescence -Indirect immunofluorescence was performed as described previously (Zhang et al., 2008). In brief, cells were grown on glass coverslips, fixed with 4% paraformaldehyde in PBS for 10min followed by blocking with 5% normal goat serum in 0.1 Triton X-100/PBS before incubation with primary antibodies. Secondary antibodies used were Alexa Fluor 594-or Fluor 488-conjugated chicken anti-mouse or anti-rabbit IgG (Invitrogen).
Slides were mounted with SlowFade Gold anti-fade reagent containing DAPI. Fluorescent signals were observed and photographed using a fluorescence microscope, and the images were acquired with AxioVisionrel 4.6.
Tissue array and immunohistochemistry (IHC) staining -Human breast cancer tissue array was obtained from Nuclea Biotechnologies (US Biolab, Washington). Immunohistochemistry was performed on tumor tissue sections using the standard laboratory protocols. Briefly, after deparaffinizing and hydrating with phosphate-buffered saline (PBS) buffer (pH 7.4), the sections were pretreated with hydrogen peroxide (3%) for 10 min to remove endogenous peroxidase, followed by antigen retrieval via steam bath for 20 min in EDTA. The rabbit anti-ERLIN2 primary antibody was applied, followed by washing and incubation with the biotinylated secondary antibody for 30 min at room temperature. Detection was performed with diaminobenzidine (DAB) and counterstaining with Mayer hematoxylin followed by dehydration and mounting. Cell proliferation assay -Cell proliferation rates were determined using CellTiter 96 nonradioactive cell proliferation MTT assay kits (Promega). Briefly, approximately 5000 cells per well were seeded in triplicate in 96-well culture plates. After cell culture for 24, 48, and 72 hrs, 20 μl of MTT (5 mg/ml) solution was added to 200 μl of medium in each well. Cells were cultured for an additional 4 hrs to allow MTT to be well metabolized. After that, the medium was aspirated, and 200 μl of DMSO was added into the well to dissolve the purple formazan crystals. The absorbance of the plate was measured at 570 nm using a plate reader.  Fig 3: Predicted topology of the human ERLIN2 based on the Mobyle topology prediction program for membrane proteins (TopPred 1.10; http://mobyle.pasteur.fr). In two speculative models, N-terminal (NH3) is located in the cytosol and the ER lumen, respectively. There are two candidate membrane-spanning (transmembrane) segments: (1) amino acids 4 to 24; and (2) amino acids 38 to 58. In both models, partial of the SPFH domain is exposed to the cytosol.