PHOSPHORYLATION DOWN-REGULATES THE STORE-OPERATED CA2+ ENTRY PATHWAY OF HUMAN NEUTROPHILS

We have reported previously that the chemotactic peptide N-formyl-meth ionyl-leucyl-phenylalanine (fMLP) inhibits transiently Ca2+ entry thro ugh the plasma membrane Ca2+ pathway activated by emptying the intrace llular Ca2+ stores (Montero, M., Garcia-Sancho, J., and Alvarez, J. (1 993) J. Biol. Chem. 268, 13055-13061). We show here that calyculin A a nd 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 incubatio n 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 modif ied by phosphatase inhibitors. These results provide additional suppor t for the view that fMLP inhibits SOCP through phosphorylation of eith er the SOCP protein or a regulatory protein and indicate that dephosph orylation mediated by protein phosphatases 1 and/or 2A restores the ac tivity of SOCP after inhibition by fMLP. The time course of the inhibi tion of SOCP by fMLP was similar to the one reported previously for th e transient fMLP-induced phosphorylation of a 47-kDa protein involved in the generation of respiratory burst, which was similarly affected b y the phosphatase inhibitors.