Activation of dual oxidases Duox1 and Duox2: differential regulation mediated by camp-dependent protein kinase and protein kinase C-dependent phosphorylation.
Dual oxidases were initially identified as NADPH oxidases producing H(2)O(2) necessary for thyroid hormone biosynthesis. The crucial role of Duox2 has been demonstrated in patients suffering from partial iodide organification defect caused by bi-allelic mutations in the DUOX2 gene. However, the Duox1 function in thyroid remains elusive. We optimized a functional assay by co-expressing Duox1 or Duox2 with their respective maturation factors, DuoxA1 and DuoxA2, to compare their intrinsic enzymatic activities under stimulation of the major signaling pathways active in the thyroid in relation to their membrane expression. We showed that basal activity of both Duox isoenzymes depends on calcium and functional EF-hand motifs. However, the two oxidases are differentially regulated by activation of intracellular signaling cascades. Duox1 but not Duox2 activity is stimulated by forskolin (EC(50) = 0.1 microm) via protein kinase A-mediated Duox1 phosphorylation on serine 955. In contrast, phorbol esters induce Duox2 phosphorylation via protein kinase C activation associated with high H(2)O(2) generation (phorbol 12-myristate 13-acetate EC(50) = 0.8 nm). These results were confirmed in human thyroid cells, suggesting that Duox1 is also involved in thyroid hormonogenesis. Our data provide, for the first time, detailed insights into the mechanisms controlling the activation of Duox1-2 proteins and reveal additional phosphorylation-mediated regulation.
Rigutto
Sabrina
S
Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire, Université Libre Bruxelles, Brussels, Belgium. sabrina.rigutto@ulb.ac.be
Hoste
Candice
C
Grasberger
Helmut
H
Milenkovic
Milutin
M
Communi
David
D
Dumont
Jacques E
JE
Corvilain
Bernard
B
Miot
Françoise
F
De Deken
Xavier
X
D000818:Animals; D019556:COS Cells; D002273:Carcinogens / Q000494:pharmacology; D002462:Cell Membrane / Q000201:enzymology* / Q000235:genetics; D002522:Chlorocebus aethiops; D005576:Colforsin / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism*; D000074623:Dual Oxidases; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D007454:Iodides / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects / Q000502:physiology; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013959:Thyroid Diseases / Q000201:enzymology / Q000235:genetics; D013961:Thyroid Gland / Q000201:enzymology*; D013963:Thyroid Hormones / Q000096:biosynthesis
D002273:Carcinogens; C513411:DUOXA2 protein, human; D007454:Iodides; D008565:Membrane Proteins; D013963:Thyroid Hormones; D005576:Colforsin; D006861:Hydrogen Peroxide; D000074623:Dual Oxidases; D019255:NADPH Oxidases; C431513:DUOX1 protein, human; C431515:DUOX2 protein, human; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate