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Involvement of tyrosine phosphorylation in inhibition of fMLP‐induced PLD activation by N ‐acetyl‐L‐cysteine in differentiated HL60 cells
Author(s) -
Nakamura Mitsuhiro,
Nakashima Shigeru,
Katagiri Yoshihiro,
Nozawa Yoshinori
Publication year - 1998
Publication title -
journal of leukocyte biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.819
H-Index - 191
eISSN - 1938-3673
pISSN - 0741-5400
DOI - 10.1002/jlb.63.6.781
Subject(s) - tyrosine phosphorylation , phosphorylation , phospholipase d , tyrosine , wortmannin , biochemistry , biology , dithiothreitol , microbiology and biotechnology , phospholipase c , phosphatidylinositol , signal transduction , enzyme
N ‐acetyl‐L‐cysteine (NAC), which is known as a multipotential agent; an antioxidant, a thiol reagent, or a tyrosine kinase inhibitor, inhibited N ‐formyl‐methionyl‐leucyl‐phenylalanine (fMLP)‐induced phospholipase D (PLD) activation in HL60 cells in a concentration‐dependent manner (IC 50 = 2 mM). Its inhibitory mechanism was examined in this study to gain insight into the regulation of PLD activity. NAC had no direct effect on membrane PLD activity in an in vitro assay system. fMLP‐induced formation of inositol phosphates via phospholipase C (PLC) was not affected by the drug, suggesting that the receptor‐G protein coupling was not inhibited. H 2 O 2 , which is known to induce PLD activation in several types of cells, failed to activate PLD in HL60 cells. Pretreatment of 3‐amino‐1,2,4‐triazole (ATZ), a catalase inhibitor, did not enhance fMLP‐induced PLD activation. NAC inhibited fMLP‐induced tyrosine phosphorylation of several protein bands (42, 44, 64, and 138 kDa) in a concentration‐dependent manner. The temporal and concentration‐dependent inhibitory profiles for tyrosine phosphorylation of 64‐ and 138‐kDa proteins were well correlated with PLD activation. However, thiol reagents, 1 mM 2,3‐dimercapto‐1‐propanol (2,3‐DMP), 1 mM dithiothreitol (DTT), and 2 mM cysteine also did not suppress protein tyrosine phosphorylation or PBut formation by fMLP. Wortmannin, a selective phosphatidylinositol 3‐kinase (PI 3‐kinase) inhibitor, inhibited these two tyrosine phosphorylation bands. These results suggest that NAC inhibits fMLP‐induced PLD activation through blockage of protein tyrosine phosphorylation, which is located at the downstream of PI‐3 kinase. J. Leukoc. Biol . 63: 781–789; 1998.