title: Novel phosphorylation of aquaporin-5 at its threonine 259 through cAMP signaling in salivary gland cells.
abstract: Aquaporin-5 (AQP5), a water channel, plays key roles in salivary secretion. The novel phosphorylation of AQP5 was investigated by using human salivary gland (HSG) cells and mouse salivary glands. In the HSG cells stably transfected with a wild-type mouse AQP5 construct, a protein band immunoreactive with antibody against phosphorylated PKA substrate was detected in the AQP5 immunoprecipitated sample, and its intensity was enhanced by short-term treatment of the cells with 8-bromo-cAMP, forskolin, or phorbol 12-myristate 13-acetate, but not by that with A23187 calcium ionophore. Such enhancement was inhibited in the presence of H-89, a PKA inhibitor. An AQP5 mutant (AQP5-T259A) expressed by transfection of HSG cells was not recognized by anti-phosphorylated PKA substrate antibody, even when the cells were stimulated with the protein kinase activators. Immunoblotting and immunofluorescence studies using a specific antibody detecting AQP5 phosphorylated at its Thr259 demonstrated that AQP5 was rapidly and transiently phosphorylated at the apical membrane of acinar cells in the submandibular and parotid glands after administration of isoproterenol, but not pilocarpine. Furthermore, both AQP5 and AQP5-T259A were constitutively localized at the plasma membrane in HSG cells under the resting and forskolin-stimulated conditions. These results suggest that AQP5 is phosphorylated at its Thr259 by PKA through cAMP, but not Ca(2+), signaling pathways, and that this phosphorylation does not contribute to AQP5 trafficking in the salivary gland cells.
authors.affiliation: Dept. of Molecular Oral Physiology, Institute of Health Biosciences, The Univ. of Tokushima Graduate School, Japan.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Hasegawa
authors.forename: Ahmad
authors.initials: A
authors.lastname: Azlina
authors.forename: Purevjav
authors.initials: P
authors.lastname: Javkhlan
authors.forename: Chenjuan
authors.initials: C
authors.lastname: Yao
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Akamatsu
authors.forename: Kazuo
authors.initials: K
authors.lastname: Hosoi
mesh_terms: D019943:Amino Acid Substitution / Q000502:physiology; D000818:Animals; D000906:Antibodies / Q000276:immunology; D000918:Antibody Specificity / Q000276:immunology; D051402:Aquaporin 5 / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D000001:Calcimycin / Q000494:pharmacology; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D003902:Detergents / Q000737:chemistry; D006801:Humans; D007545:Isoproterenol / Q000494:pharmacology; D007546:Isoquinolines / Q000494:pharmacology; D008297:Male; D051379:Mice; D008813:Mice, Inbred ICR; D010306:Parotid Gland / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects / Q000502:physiology; D010862:Pilocarpine / Q000494:pharmacology; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology; D012469:Salivary Glands / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D013363:Submandibular Gland / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D013449:Sulfonamides / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013912:Threonine / Q000378:metabolism*; D014162:Transfection
chemical_list: D000906:Antibodies; D051402:Aquaporin 5; D003902:Detergents; D007546:Isoquinolines; D047428:Protein Kinase Inhibitors; D013449:Sulfonamides; D010862:Pilocarpine; D005576:Colforsin; D013912:Threonine; D000001:Calcimycin; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D007545:Isoproterenol; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide; D013755:Tetradecanoylphorbol Acetate