Phosphorylation down-regulates the store-operated Ca2+ entry pathway of human neutrophils.
We have reported previously that the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP) inhibits transiently Ca2+ entry through the plasma membrane Ca2+ pathway activated by emptying the intracellular Ca2+ stores (Montero, M., García-Sancho, J., and Alvarez, J. (1993) J. Biol. Chem. 268, 13055-13061). We show here that calyculin A and okadaic acid, inhibitors of protein phosphatases 1 and 2A, prevent the spontaneous reversion of the fMLP-induced inhibition of the entry of Ca2+ and Mn2+ (used as a Ca2+ surrogate), leading to a permanently inhibited Ca2+ entry pathway. At high concentrations or long incubation times the phosphatase inhibitors were even able to inhibit the store-operated Ca2+ entry pathway (SOCP) in the absence of fMLP. Inhibition of SOCP by phorbol dibutyrate, which is not reversible, was not modified by phosphatase inhibitors. These results provide additional support for the view that fMLP inhibits SOCP through phosphorylation of either the SOCP protein or a regulatory protein and indicate that dephosphorylation mediated by protein phosphatases 1 and/or 2A restores the activity of SOCP after inhibition by fMLP. The time course of the inhibition of SOCP by fMLP was similar to the one reported previously for the transient fMLP-induced phosphorylation of a 47-kDa protein involved in the generation of respiratory burst, which was similarly affected by the phosphatase inhibitors.
Montero
M
M
Departamento de Bioquímica y Biología Molecular y Fisiología, Facultad de Medicina, Universidad de Valladolid, Spain.
García-Sancho
J
J
Alvarez
J
J
D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors; D004988:Ethers, Cyclic / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D008345:Manganese / Q000378:metabolism; D008387:Marine Toxins; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000378:metabolism*; D019319:Okadaic Acid; D010080:Oxazoles / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors*; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D013729:Terpenes / Q000494:pharmacology; D019284:Thapsigargin
D004988:Ethers, Cyclic; D008387:Marine Toxins; D010080:Oxazoles; D010750:Phosphoproteins; D013729:Terpenes; D019319:Okadaic Acid; D008345:Manganese; D009240:N-Formylmethionine Leucyl-Phenylalanine; D019284:Thapsigargin; C059041:calyculin A; D010749:Phosphoprotein Phosphatases; D000252:Calcium-Transporting ATPases; D002118:Calcium