Cullin-associated NEDD8-dissociated protein 1, a novel interactor of rabphilin-3A, deubiquitylates rabphilin-3A and regulates arginine vasopressin secretion in PC12 cells

Abstract

Abstract. The molecular mechanism involved in the exocytosis of arginine vasopressin (AVP) is not fully known. Rabphilin-3A has been suggested as a novel autoantigen in infundibulo-neurohypophysitis (LINH), which leads to central diabetes insipidus through insufficient secretion of AVP. However, the role of rabphilin-3A in the pathogenesis of LINH remains unclear. Thus, the aim of the present study was to identify proteins binding rabphilin-3A in the posterior pituitary. Using glutathione S-transferase (GST)-pulldown assays and proteomic analyses, cullin-associated NEDD8-dissociated protein 1 (CAND1) was identified as a rabphilin-3A-binding protein in the posterior pituitary. Co-immunoprecipitation assays indicated that CAND1 interacted endogenously with rabphilin-3A. In addition, immunohistochemistry experiments showed that CAND1 immunoreactivity was detected mainly in the posterior pituitary, intermediate lobe, and the supraoptic nucleus in the hypothalamus, and less in the anterior lobe, partially co-localizing with rabphilin-3A. Overexpression of CAND1 resulted in deubiquitylation of rabphilin-3A in PC12 cells. Moreover, overexpression of CAND1 in PC12 cells co-transfected with AVP enhanced both basal and KCl-stimulated AVP secretion. The findings indicate that CAND1 inhibits the ubiquitylation of rabphilin-3A and positively regulates AVP secretion. These data shed light on a novel potential mechanism involving rabphilin-3A in AVP secretion, and suggest a new role of CAND1 as a regulator of hormone or neurotransmitter secretion.Key words: Vasopressin, Rabphilin-3A, Cullin-associated NEDD8-dissociated protein 1, Deubiquitylation, Secretion doi:10.1507/endocrj.EJ17-0399 ARGININE VASOPRESSIN (AVP) is a hormone secreted from the posterior pituitary through exocytosis, a process involving numerous steps [1][2][3]. Rabphilin-3A has been suggested as a major autoantigen in infundibuloneurohypophysitis (LINH) and autoantibodies against rabphilin-3A may be useful as diagnostic markers for LINH [4]. Rabphilin-3A is a specific GTP-Rab3A-binding protein on secretory granules [5,6], regulating neurotransmitter release, and acting in conjunction with synaptosomal-associated protein 25 (SNAP25), Rab27A, and syntaxin 1 [7][8][9][10][11]. Although the involvement of Submitted Sep. 11, 2017; Accepted Nov. 30, 2017 as EJ17-0399 Released online in J-STAGE as advance publication Jan. 23, 2018Correspondence to: Yoshihisa Sugimura, Division of Endocrinology and Metabolism, Department of Internal Medicine, Fujita Health University, 1-98 Dengakugakubo, Toyoake, Aichi 470-1192, Japan. E-mail: sugiyosi@med.nagoya-u.ac.jp rabphilin-3A in AVP secretion has not been previously characterized, preliminary data showed that rabphilin-3A may be involved in the docking and fusion of AVP vesicles, thereby regulating AVP release and water homeostasis in the body. However, the detailed mechanism of AVP release mediated by rabphilin-3A in the posterior pituitary is largely unknown.The aim of this study was to identify new interactors of rabphili...

Studies on anti-rabphilin-3A antibodies in 15 consecutive patients presenting with central diabetes insipidus at a single referral center

“…Rabphilin-3A is a specific GTP-Rab3A-binding protein localized in secretory granules, where it regulates neurotransmitter release 47 – 50 . Regarding the involvement of rabphilin-3A in AVP secretion, AVP secretion was reported to be mediated by the interaction between rabphilin-3A and cullin-associated and neddylation-dissociated protein 1 51 . Rabphilin-3A may be a pathogenic antigen, and T cells specific for rabphilin-3A may be involved in the pathogenesis of neurohypophysitis in mice 52 .…”

Transcriptome-Wide Detection of Intron/Exon Definition in the Endogenous Pre-mRNA Transcripts of Mammalian Cells and Its Regulation by Depolarization

“…Instead, it gave rise to clusters for more specific pituitary functions: genes with alternative splicing, prenylation, nuclear functions, and protein interactions ( Figure 7 B). For example, the “alternative splicing” cluster includes the pituitary-specific transcription factor Pou1f1 ( Pit1, see also Figure 6 A), essential for pituitary cell differentiation and transcription of prolactin and growth hormone [ 40 , 41 ], and Sequestosome 1 ( p62 ), essential for luteinizing hormone production [ 42 ], as well as the cullin-associated and neddylation-dissociated 2 ( Cand2 ) with a homolog Cand1 regulating vasopressin secretion [ 43 ]. Thus, the detected RISE-associated genes cluster for either basic or tissue-specific cellular functions depending on their regulation status by depolarization.…”

A novel crosstalk between Nrf2 and Smad2/3 bridged by two nuanced Keap1 isoforms

“…In addition to its interactors Smad2/3, Keap1-putdown interactome analysis also revealed that it may act as a key player in the process to sequentially add and remove ubiquitin by dynamic formation of distinctive functional complexes with ubiquitin conjugating enzymes (e.g., Cullin-1, 2, 3 , 4B, 5, ATG3, UBE3A, and UBE3C) and deubiquitinating enzymes (e.g., USP5, 7, 10, 12, 14, 15, 17L, 22, 24, 47 and UCHL5), as well as with cullin-associated NEDD8-dissociated protein 1 (CAND1, as an inhibitor promoting dissociation of substrate receptor components from the Cullin RING ligases 50 ). For this, it inferable that the selective dynamic formation of such opposite functional complexes is much likely to depend on distinctive tempo-spatial contexts of between Smad2/3 and Nrf2.…”