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. 2021 May 7;28(1):35.
doi: 10.1186/s12929-021-00731-9.

NEDD4 regulates ubiquitination and stability of the cell adhesion molecule IGPR-1 via lysosomal pathway

Linzi Sun  1 , Razie Amraei  1 , Nader Rahimi  2
Affiliations

NEDD4 regulates ubiquitination and stability of the cell adhesion molecule IGPR-1 via lysosomal pathway

Linzi Sun et al. J Biomed Sci. .

Abstract

Background: The cell adhesion molecule IGPR-1 regulates various critical cellular processes including, cell-cell adhesion, mechanosensing and autophagy and plays important roles in angiogenesis and tumor growth; however, the molecular mechanism governing the cell surface levels of IGPR-1 remains unknown.

Results: In the present study, we used an in vitro ubiquitination assay and identified ubiquitin E3 ligase NEDD4 and the ubiquitin conjugating enzyme UbcH6 involved in the ubiquitination of IGPR-1. In vitro GST-pulldown and in vivo co-immunoprecipitation assays demonstrated that NEDD4 binds to IGPR-1. Over-expression of wild-type NEDD4 downregulated IGPR-1 and deletion of WW domains (1-4) of NEDD4 revoked its effects on IGPR-1. Knockdown of NEDD4 increased IGPR-1 levels in A375 melanoma cells. Deletion of 57 amino acids encompassing the polyproline rich (PPR) motifs on the C-terminus of IGPR-1 nullified its binding with NEDD4. Furthermore, we demonstrate that NEDD4 promotes K48- and K63-dependent polyubiquitination of IGPR-1. The NEDD4-mediated polyubiquitination of IGPR-1 stimulates lysosomal-dependent degradation of IGPR-1 as the treatment of cells with the lysosomal inhibitors, bafilomycine or ammonium chloride increased IGPR-1 levels ectopically expressed in HEK-293 cells and in multiple endogenously IGPR-1 expressing human skin melanoma cell lines.

Conclusions: NEDD4 ubiquitin E3 ligase binds to and mediates polyubiquitination of IGPR-1 leading to its lysosomal-dependent degradation. NEDD4 is a key regulator of IGPR-1 expression with implication in the therapeutic targeting of IGPR-1 in human cancers.

Keywords: CD28H; IGPR-1; K48-linked ubiquitination; K63-linked ubiquitination; Lysosomal degradation; Melanoma; NEDD4; TMIGD2; UbcH6; Ubiquitination.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
NEDD4 through its WW domain 4 binds to IGPR-1. a A schematic of IGPR-1 and the locations of the poly-proline rich motifs at the C-terminus. b Schematic of NEED4 domains and GST constructs corresponding to WW domains (#1,3,4). c Coomassie blue staining of purified GST-NEDD4 fusion WW domains. d HEK-293 cells expressing empty vector (EV) or IGPR-1-Myc were subjected to an in vitro GST-pull-down assay corresponding to WW domains (1, 3 and 4) of NEDD4, followed by Western blot analysis using an anti-IGPR-1 antibody. The same membrane was blotted with an anti-GST antibody for GST-NEDD4 fusion proteins. e Western blot analysis of whole cell lysate of HEK-293 cells expressing EV or IGPR-1-Myc immunoblotted for IGPR-1 or GAPDH. f HEK-293 cells expressing EV or IGPR-1-Myc were transfected with NEDD4-HA. After 48 h post-transfection, cells were lysed and subjected to co-immunoprecipitation assay via anti-IGPR-1 antibody followed by Western blot analysis using HA antibody. The membrane was re-blotted with anti-IGPR-1 antibody. Whole cell lysate (WCL)
Fig. 2
Fig. 2
Deletion of proline-rich motifs on IGPR-1 eliminates its binding with NEDD4: a Schematic of IGPR-1 and Δ57/IGPR-1. PPR motif, poly-proline-rich motif, TM, transmembrane domain. b Whole cell lysates from HEK-293 cells expressing empty vector (EV), IGPR-1-Myc or Δ57/IGPR-1-Myc were subjected to in vitro GST-pull down assay using GST-NEDD4-WW#4 followed by Western blot analysis using anti-Myc antibody. The same membrane was blotted with anti-GST antibody. c Whole cell lysates corresponding to IGPR-1-Myc or Δ57/IGPR-1-Myc expressed in HEK-293 cells and loading control, GAPDH. d Whole cell lysates from HEK-293 cells expressing IGPR-1-Myc or Δ57/IGPR-1-Myc were subjected to co-immunoprecipitation assay using anti-Myc antibody followed by Western blot analysis using anti-HA antibody. e Whole cell lysates (WCL) corresponding to IGPR-1-Myc, Δ57/IGPR-1-Myc, HA-NEDD4 expressed in HEK-293 cells and loading control, GAPDH
Fig. 3
Fig. 3
NEDD4 mediates mono- and polyubiquitination of IGPR-1. a A schematic of in vitro ubiquitination assay is shown. b In vitro ubiquitination assay was carried out as described in the material and method section using biotinylated ubiquitin (Ub), E2 conjugating enzymes, purified GST-NEDD4 and GST-cytoplasmic domain of IGPR-1. The samples were resolved on SDS-PAGE and ubiquitinated GST-IGPR-1 was detected by Western blotting via Streptavidin-HRP antibody. c–e Coomassie blue staining of GST-IGPR-1, GST-NEDD4 and E2 conjugating enzymes. f HEK-293 cells expressing IGPR-1-Myc were transfected with an empty vector or wild-type NEDD4-HA, cells were lysed and whole cell lysates were immunoprecipitated with an anti-Myc antibody followed by Western blot analysis using anti-ubiquitin (FK2) antibody, K63-poly-ubiqutin antibody or K48-poly-ubiqutin antibody. Whole cell lysates (WCL) were blotted for IGPR-1-Myc and NEDD4-HA
Fig. 4
Fig. 4
NEED4 mediates K48- and K63-dependent polyubiquitination and downregulation of IGPR-1. a A schematic of ubiquitin (Ub) constructs are shown. b HEK-293 cells expressing IGPR-1 were transfected with wild-type Ub, KO-Ub, K48-Ub or K63-Ub constructs. After 48 h, cells were lysed and whole cell lysates (WCL) were immunoprecipitated with anti-Myc and blotted with anti-HA antibody for ubiquitin. The same membrane was re-blotted for IGPR-1. WCL was blotted for total IGPR-1, and loading control GAPDH. c IGPR-1/HEK-293 and IGPR-1/HEK-293 cells transfected with NEDD4 were subjected to cycloheximide pulse-chase assay. Cells were lysed at the indicated time points and whole cell lysates was subjected to Western blot analysis using anti-Myc antibody. The graph is representative of three independent experiments. Expression of NEDD4 and loading control GAPDH are shown. d HEK-293 cells expressing empty vector (EV), IGPR-1-Myc alone or co-expressing with NEDD4-HA were stained with anti-Myc antibody and subjected to immunofluorescence microscopy. Scale bar, 10 μm
Fig. 5
Fig. 5
NEDD4 promotes lysosomal degradation of IGPR-1. a HEK-293 cells expressing IGPR-1 were transfected with an empty vector or wild-type NEDD4-HA or WW domain truncated NEDD4, where all the four WW domains was deleted (ΔWW-NEDD4). Cells were lysed and whole cell lysates were immunoblotted with an anti-IGPR-1 antibody, anti-HA antibody or Anti-Myc antibody for ΔWW-NEDD4. The membrane was also blotted for GAPDH for protein loading control. b HEK-293 cells expressing IGPR-1 were treated with MG132 or bortezomib for two hours, cells were lysed and whole cell lysates were blotted for IGPR-1 or GAPDH. c HEK-293 cells expressing IGPR-1 were treated with control vehicle, bafilomycine or NH4Cl for 18 h. Cells were lysed and whole cell lysates were blotted for IGPR-1 or GAPDH. d HEK-293 cells expressing IGPR-1 were transfected with an empty vector or wild-type NEDD4-HA. After 46 h of transfection, cells were with control vehicle or bafilomycine and incubated for additional 18 h. Cells were lysed and whole cell lysates were blotted for IGPR-1 or GAPDH. e Melanoma cell lines (MEL-JoSo, SK-Mel-28 and A375) were treated with control vehicle or bafilomycine for 18 h. Cells were lysed and whole cell lysates were blotted for IGPR-1 or GAPDH. The graphs a–e are representative of three independent experiments. P < 0.05
Fig. 6
Fig. 6
IGPR-1 is expressed in human melanoma and knockdown of NEDD4 increases IGPR-1 expression. a, b Expression of IGPR-1 in human melanoma. Data extracted from TCGA dataset via cBioportal (http://www.cbioportal.org/). c The volcano plot of IGPR-1 is shown. d Expression of IGPR-1 and NEDD4 in human melanoma cell lines. e A375 melanoma cells were transfected with human NEDD4 shRNA and after selection with puromycin cells were lysed and subjected Western blot analysis using anti-NEDD4, anti-IGPR-1 antibodies. Also shown is the Western blot analysis of A375 cells expressing NEDD4-shRNA with NEDD4-HA. The graph is representative of three independent experiments. P < 0.05
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