The palmitoylation of the N-terminal extracellular Cys37 mediates the nuclear translocation of VPAC1 contributing to its anti-apoptotic activity

Rongjie Yu; Hongyu Liu; Xinhe Peng; Yue Cui; Suqin Song; Like Wang; Huahua Zhang; An Hong; Tianhong Zhou

doi:10.18632/oncotarget.17449

The palmitoylation of the N-terminal extracellular Cys37 mediates the nuclear translocation of VPAC1 contributing to its anti-apoptotic activity

Oncotarget. 2017 Jun 27;8(26):42728-42741. doi: 10.18632/oncotarget.17449.

Authors

Rongjie Yu^{1

2}, Hongyu Liu^{1

2}, Xinhe Peng^{1

2}, Yue Cui^{1

2}, Suqin Song^{1

2}, Like Wang^{1

2}, Huahua Zhang³, An Hong^{1

2}, Tianhong Zhou⁴

Affiliations

¹ Institute of Biomedicine, School of Life Science and Technology, Jinan University, Guangzhou, Guangdong, China.
² National Engineering Research Center of Genetic Medicine, Jinan University, Guangzhou, Guangdong, China.
³ Department of Medical Genetics, Guangdong Medical University, Dongguan, Guangdong, China.
⁴ Department of Bioengineering, School of Life Science and Technology, Jinan University, Guangzhou, Guangdong, China.

Abstract

VPAC1 is class B G protein-coupled receptors (GPCR) shared by pituitary adenylate cyclase activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP). The first cysteine (Cys37) in the N-terminal extracellular domain of mature VPAC1 is a free Cys not involved in the formation of conserved intramolecular disulfide bonds. In order to investigate the biological role of this Cys37 in VPAC1, the wild-type VPAC1 and Cys37/Ala mutant (VPAC1-C37/A) were expressed stably as fusion proteins with enhanced yellow fluorescent protein (EYFP) respectively in Chinese hamster ovary (CHO) cells. Both VPAC1-EYFP and VPAC1-C37/A-EYFP trafficked to the plasma membrane normally, and CHO cells expressing VPAC1-EYFP displayed higher anti-apoptotic activity against camptothecin (CPT) induced apoptosis than the cells expressing VPAC1-C37/A-EYFP, while VPAC1-C37/A-CHO cells showed higher proliferative activity than VPAC1-CHO cells. Confocal microscopic analysis, western blotting and fluorescence quantification assay showed VPAC1-EYFP displayed significant nuclear translocation while VPAC1-C37/A-EYFP did not transfer into nucleus under the stimulation of VIP (0.1 nM). Acyl-biotin exchange assay and click chemistry-based palmitoylation assay confirmed for the first time the palmitoylation of Cys37, which has been predicted by bioinformatics analysis. And the palmitoylation inhibitor 2-bromopalmitate significantly inhibited the nuclear translocation of VPAC1-EYFP and its anti-apoptotic activity synchronously. These results indicated the palmitoylation of the Cys37 in the N-terminal extracellular domain of VPAC1 mediates the nuclear translocation of VPAC1 contributing to its anti-apoptotic activity. These findings reveal for the first time the lipidation-mediating nuclear translocation of VPAC1 produces a novel anti-apoptotic signal pathway, which may help to promote new drug development strategy targeting VPAC1.

Keywords: anti-apoptotic activity; cysteine (Cys); nuclear translocation; palmitoylation; vasoactive intestinal peptide receptor 1 (VPAC1).

MeSH terms

Animals
Apoptosis / physiology
CHO Cells
Cells, Cultured
Cricetulus
Cysteine / metabolism*
Lipoylation
Transfection
Vasoactive Intestinal Peptide / metabolism*

Substances

Vasoactive Intestinal Peptide
Cysteine