Another biological effect of tosylphenylalanylchloromethane (TPCK): it prevents p47phox phosphorylation and translocation upon neutrophil stimulation.
TPCK (tosylphenylalanylchloromethane), first discovered as a serine protease inhibitor, has been described to affect in diverse systems a number of physiological events probably unrelated to its antiprotease effect, such as proliferation, apoptosis and tumour formation. In the present study, we focus on its inhibition of the neutrophil respiratory burst, an important element of non-specific immunological defence. The superoxide anion-producing enzyme, NADPH oxidase, is quiescent in resting cells. Upon cell stimulation, the redox component, membrane-bound flavocytochrome b558, is activated when the cytosolic factors (p47phox, p67phox and p40phox, as well as the small GTPase Rac) associate with it after translocating to the membrane. This requires the phosphorylation of several p47phox serine residues. The signal transduction events leading to enzyme activation are not completely understood. In the past, the use of diverse protease inhibitors suggested that proteases were involved in NADPH oxidase activation. We suggested previously that TPCK could prevent enzyme activation by the phorbol ester PMA, not due to inhibition of a protease, but possibly to inhibition of the cytosolic factor translocation [Chollet-Przednowed and Lederer (1993) Eur. J. Biochem. 218, 83-93]. In the present work, we show that TPCK, when added to cells before PMA, prevents p47phox phosphorylation and hence its translocation; moreover, when PMA-stimulated cells are incubated with TPCK, p47phox is dephosphorylated and dissociates from the membrane. These results are in line with previous suggestions that the respiratory burst is the result of a series of continuous phosphorylation and dephosphorylation events. They suggest that TPCK leads indirectly to activation of a phosphatase or inactivation of a kinase, and provide the first clue towards understanding the steps leading to its inhibition of NADPH oxidase activation.
Gillibert
Maggaly
M
Laboratoire d'Enzymologie et Biochimie Structurales, CNRS UPR 9063, 91198 Gif-sur-Yvette Cedex, France.
Dehry
Zakia
Z
Terrier
Micheline
M
El Benna
Jamel
J
Lederer
Florence
F
D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D006801:Humans; D019255:NADPH Oxidases / Q000378:metabolism; D018375:Neutrophil Activation / Q000187:drug effects*; D009504:Neutrophils / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects; D016897:Respiratory Burst / Q000187:drug effects; D013481:Superoxides / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014108:Tosylphenylalanyl Chloromethyl Ketone / Q000494:pharmacology*
D010750:Phosphoproteins; D013481:Superoxides; D014108:Tosylphenylalanyl Chloromethyl Ketone; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; D013755:Tetradecanoylphorbol Acetate