Regulation of thromboxane receptor (TP) phosphorylation by protein phosphatase 1 (PP1) and PP2A.
To investigate the protein phosphatases that dephosphorylate TP, human embryonic kidney cells (HEK293 cells) stably transfected with 12CA5-tagged TP were treated with TP agonist, washed, and then allowed to recover in the presence or absence of the cell-permeable PP1 and PP2A inhibitors calyculin or okadaic acid (OKA). After recovery, cells were rechallenged with TP agonist and TP responsiveness was assessed by measuring inositol trisphosphate generation. TP responsiveness recovered over a 20-min time period. Recovery of TP responsiveness was inhibited by calyculin and OKA and was associated with dephosphorylation of receptor proteins. To further identify the TP phosphatase, TP phosphorylated in the intact cell were isolated by immunoprecipitation and were used as substrate for protein phosphatases prepared from HEK293 cells. TP were dephosphorylated by whole-cell homogenates. Dephosphorylation of TP was completely inhibited by the PP1 and PP2A inhibitors calyculin and microcystin-LR, suggesting that the decrease in TP phosphorylation was not due to receptor degradation. TP phosphatase activity was partially blocked by 1) inhibitor 2, a specific protein inhibitor of PP1; and 2) OKA at concentrations (1 nM) that specifically inhibit PP2A. TP phosphatase activity did not have an absolute requirement for divalent cations and was found primarily in cytosolic fractions of the cell. These data suggest that PP1- and PP2A-like protein phosphatases dephosphorylate TP. By regulating the phosphorylation state of TP, protein phosphatases may modulate tissue responsiveness to thromboxane.
Spurney
R F
RF
Division of Nephrology, Department of Medicine, Duke University Medical Centers, Durham, North Carolina 27710, USA. spurn002@mc.duke.edu
D002412:Cations / Q000494:pharmacology; D002460:Cell Line; D003600:Cytosol / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007295:Inositol Phosphates / Q000378:metabolism; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D019319:Okadaic Acid / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D054645:Protein Phosphatase 1; D017482:Receptors, Thromboxane / Q000235:genetics / Q000378:metabolism*; D013347:Subcellular Fractions / Q000187:drug effects / Q000201:enzymology; D014162:Transfection
D002412:Cations; D004791:Enzyme Inhibitors; D007295:Inositol Phosphates; D017482:Receptors, Thromboxane; D019319:Okadaic Acid; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1