RETRACTED: Overlapping signal sequences control nuclear localization and endoplasmic reticulum retention of GRP58

Abstract

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). This article has been retracted: please see Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy). The University of Maryland, Baltimore conducted an internal investigation which found that the article was compromised and a preponderance of evidence supports retraction of the publication in order to correct the scientific record and ensure its integrity. The Editor-in-Chief has decided to retract this article. This article has been found to contain manipulated and enhanced figures, namely figures 1D and 1E, 4A and 4B.

Fig. 1

Nuclear localization (NLS)and ER retention signals. (A). Schematic representation of mouse GRP58 wild type and GRP58 C-terminal truncated mutant (M1 to -M5) proteins. (B). Amino acid sequence alignment of mouse, rat and human GRP58 showing putative NLS and ER retention signals conserved at the C-termini. (C). Subcellular distribution of GRP58 wild type and GRP58 mutants M1–M5 in HCT116 cells. Cells were transfected with plasmids for V5-tagged GRP58-WT or mutants, indicated in Fig. 1A. In a related experiment, HCT116 cells transfected with GRP58 mutant M5 was treated with either DMSO or MG132 (20 μM) for 8 hours. 24h after transfection, cells were harvested, total cell lysates (TCL), cytosolic (C), nuclear (N) and endoplasmic reticulum (ER) fractions were made and analyzed by Western blotting. The immunoblots were stripped and re-probed with anti-lactate dehydrogenase (LDH, cytosolic), anti-lamin B (nuclear) and anti-calnexin (microsomal) antibodies to confirm purity of fractionation. Same experiment with similar results was also done in Hepa-1 cells (data not shown). (D/E). Effect of deletion/mutation of the putative NLS sequence on nuclear localization of GRP58. HCT116 and Hepa-1 cells were transfected with pcDNA-V5 plasmids for GRP58-WT or GRP58-ΔNLS mutant (D) or GRP58-NLS K-A mutant (E) in separate experiments. The cells were harvested 24h post-transfection, cytosolic and nuclear fractions were made and analyzed by Western blotting with anti-V5 antibody. The same blots were also re-probed with anti-LDH and anti-laminB antibodies to show purity of fractionation.

Fig. 2

Immunohistochemical analysis. (A). Immunofluorescence localization of GRP58-WT, GRP58-NLS mutant proteins in Hepa-1 cells. Cells were transfected with pcDNA-V5 plasmids encoding GRP58-WT or GRP58-ΔNLS mutant or GRP58-NLS K-A mutant proteins. After 24h, cells were fixed, permeabilized and indirect immunostaining was performed with anti-V5-FITC antibody to visualize localization of GRP58 wild type and mutant proteins under flourescence microscope and then counterstained with DAPI to show the nucleus. (B). Nuclear localization activity/potential of GRP58-NLS sequence. Hepa-1 cells were transfected with plasmids encoding green fluorescent protein (GFP) alone or GFP fused to GRP58-NLS. GFP fluorescence was visualized in the cells at 24h post-transfection by flourescence microscopy. DAPI staining was also done to visualize the nucleus.

Fig. 3

Effect of individual amino acid mutation in GRP58 NLS sequence on subcellular localization of the protein. GRP58 wild type and alanine substitution mutations of individual amino acids in GRP58 NLS sequence were transfected and total cell lysate (TCL) or cytosolic, nuclear and ER fractions were generated. 50μg of cellular fractions were used for SDS-PAGE, immunoblotted with anti-V5 antibody. The blots were stripped and re-probed with sucellular specific antibodies.

Fig. 4

ER retention of GRP58. (A). Effect of deletion of QEDL sequence on ER localization of GRP58. HCT116 and Hepa-1 cells in separate experiments were transfected with pcDNA-GRP58-WT-V5 or pcDNA-GRP58-ΔER-V5. Cytosolic, nuclear and ER fractions were made and analyzed by Western blotting and probing with anti-V5 antibody. (B). Requirement of both NLS and ER retention (QEDL) sequences for ER retention of GRP58. NLS and putative ER retention (QEDL) sequences of GRP58 were both deleted (GRP58 NLS/ER DD) or alanine substituted (GRP58 NLS/ER DM) and cloned in pcDNA-V5 expression plasmid. HCT116 cells were transfected with GRP58-WT, GRP58 NLS/ER DD, GRP58 NLS/ER DM and GRP58-ΔER plasmids. After 24h, cells harvested and subcellular fractions (cytosol, nuclear and ER) were made and analyzed by immunoblotting with anti-V5, anti-LDH, anti-laminB, anti-calnexin and anti-βactin antibodies. Similar results were observed in Hepa-1 cells (data not shown).