The phosphatase inhibitor okadaic acid induces AQP2 translocation independently from AQP2 phosphorylation in renal collecting duct cells.
Phosphorylation by kinases and dephosphorylation by phosphatase markedly affect the biological activity of proteins involved in intracellular signaling. In this study we investigated the effect of the serine/threonine phosphatase inhibitor okadaic acid on water permeability properties and on aquaporin2 (AQP2) translocation in AQP2-transfected renal CD8 cells. In CD8 cells both forskolin alone and okadaic acid alone increased the osmotic water permeability coefficient P(f) by about 4- to 5-fold. In intact cells, in vivo phosphorylation studies revealed that forskolin stimulation resulted in a threefold increase in AQP2 phosphorylation. In contrast, okadaic acid treatment promoted only a 60% increase in AQP2 phosphorylation which was abolished when this treatment was performed in the presence of 1 microM H89, a specific protein kinase A (PKA) inhibitor. Nevertheless, in this latter condition, confocal microscopy analysis revealed that AQP2 translocated and fused to the apical membrane. Okadaic acid-induced AQP2 translocation was dose dependent having its maximal effect at a concentration of 1 microM. In conclusion, our results clearly indicate that okadaic acid exerts a full forskolin-like effect independent from AQP2 phosphorylation. Thus AQP2 phosphorylation is not essential for water channel translocation in renal cells, indicating that different pathways might exist leading to AQP2 apical insertion and increase in P(f).
Valenti
G
G
Dipartimento di Fisiologia Generale ed Ambientale, Universita' degli Studi, Via Amendola 165/A, Italy. g.valenti@biologia.uniba.it.
Procino
G
G
Carmosino
M
M
Frigeri
A
A
Mannucci
R
R
Nicoletti
I
I
Svelto
M
M
D000199:Actins / Q000378:metabolism; D000818:Animals; D051399:Aquaporin 2; D051403:Aquaporin 6; D020346:Aquaporins / Q000378:metabolism*; D001692:Biological Transport / Q000187:drug effects / Q000502:physiology; D002460:Cell Line; D005576:Colforsin / Q000494:pharmacology; D003599:Cytoskeleton / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D007685:Kidney Tubules, Collecting / Q000166:cytology* / Q000201:enzymology; D019319:Okadaic Acid / Q000494:pharmacology*; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors*; D010766:Phosphorylation; D011817:Rabbits
D000199:Actins; D051399:Aquaporin 2; D051403:Aquaporin 6; D020346:Aquaporins; D004791:Enzyme Inhibitors; D005576:Colforsin; D019319:Okadaic Acid; D010744:Phosphoric Monoester Hydrolases