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Phosphorylation down-regulates the store-operated Ca2+ entry pathway of human neutrophils
M Montero1, J García-Sancho, J Alvarez
1Departamento de Bioquímica y Biología Molecular y Fisiología, Facultad de Medicina, Universidad de Valladolid, Spain.
Abstract:
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.
Insights
Protein phosphatase inhibitors prevent the recovery of calcium (Ca2+) entry after N-formyl-methionyl-leucyl-phenylalanine (fMLP) inhibition. Dephosphorylation by phosphatases 1 and/or 2A is crucial for restoring store-operated calcium entry pathway (SOCP) activity.
Area of Science:
- Cellular Biology
- Immunology
- Biochemistry
Background:
- The chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP) was previously shown to transiently inhibit calcium (Ca2+) entry via the plasma membrane Ca2+ channel, a process activated by the depletion of intracellular Ca2+ stores.
- This inhibition affects the store-operated calcium entry pathway (SOCP).
Purpose of the Study:
- To investigate the role of protein phosphatases in the regulation of fMLP-induced inhibition of Ca2+ entry.
- To determine if dephosphorylation is necessary for the restoration of SOCP activity.
Main Methods:
- Treatment of cells with calyculin A and okadaic acid (inhibitors of protein phosphatases 1 and 2A).
- Assessment of Ca2+ and Mn2+ entry.
- Comparison with fMLP-induced inhibition and phorbol dibutyrate inhibition.
Main Results:
- Calyculin A and okadaic acid prevented the spontaneous reversion of fMLP-induced inhibition, leading to permanent inhibition of Ca2+ and Mn2+ entry.
- At high concentrations or prolonged incubation, these phosphatase inhibitors also inhibited SOCP independently of fMLP.
- Inhibition of SOCP by phorbol dibutyrate was unaffected by phosphatase inhibitors.
- The time course of fMLP-induced SOCP inhibition mirrored that of fMLP-induced phosphorylation of a 47-kDa protein involved in respiratory burst.
Conclusions:
- fMLP inhibits SOCP likely through phosphorylation of the SOCP protein or a regulatory protein.
- Dephosphorylation mediated by protein phosphatases 1 and/or 2A is essential for restoring SOCP activity after fMLP-induced inhibition.
- These findings highlight the critical role of phosphatase activity in regulating calcium signaling pathways.