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

• Published in: ENDOCRINE JOURNAL 2018, Vol.65(3), pp.325-334
• Main Authors: Nakashima, Kohtaro, Takeuchi, Seiji, Iwama, Shintaro, Kiyota, Atsushi, Yasuda, Yoshinori, Iwata, Naoko, Enomoto, Atsushi, Arima, Hiroshi, Sugimura, Yoshihisa
• Format: Article
• Language: English
• Subjects: Adaptor Proteins, Signal Transducing - metabolism; Animals; Arginine Vasopressin - metabolism; Argipressin; Chlorides; Cullin; Cullin-associated NEDD8-dissociated protein 1; Deubiquitinating Enzymes - genetics; Deubiquitinating Enzymes - metabolism; Deubiquitylation; Diabetes insipidus; Exocytosis; Glutathione transferase; Hypothalamus; Immunohistochemistry; Immunoprecipitation; Nerve Tissue Proteins - metabolism; PC12 Cells; Pheochromocytoma cells; Pituitary (posterior); Pituitary Gland, Posterior - metabolism; Potassium chloride; Proteins; Rabphilin-3A; Rats; Rats, Sprague-Dawley; Secretion; Supraoptic nucleus; Transcription Factors - genetics; Transcription Factors - metabolism; Ubiquitin; Vasopressin; Vesicular Transport Proteins - metabolism
• ISSN: 0918-8959; 1348-4540
• DOI: 10.1507/endocrj.EJ17-0399

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.