Possible involvement of optimally phosphorylated L-plastin in activation of superoxide-generating NADPH oxidase.
The involvement of protein phosphatases in the activation of superoxide (O2-)- generating enzyme in human neutrophils was examined using calyculin A, an inhibitor of protein phosphatase type 1 and 2A. Calyculin A inhibited the phorbol myristate acetate (PMA)- and opsonized zymosan (OZ)-activated O2- generation by human neutrophils. This inhibitory effect of calyculin A on PMA-activated O2- generation was reversed by the addition of KT5926, a specific inhibitor of myosin light chain kinase and Ca2+/calmodulin-dependent protein kinase II. These results suggest that the addition of calyculin A may cause hyperphosphorylation of some protein(s) that plays a crucial role in the PMA-dependent activation of O2- generating enzyme, and that this protein hyperphosphorylation may be evoked by a KT5926-sensitive kinase or its downstream kinase. Whereas two-dimensional analysis involving 32P revealed that calyculin A caused the hyperphosphorylation of many proteins, KT5926 mainly reduced the calyculin A-induced hyperphosphorylation of a 67 kDa protein in activated neutrophils, suggesting that the hyperphosphorylation of the 67 kDa protein might inhibit the PMA-dependent activation of NADPH oxidase. The 67 kDa cytosolic protein was moderately phosphorylated on the addition of PMA. On the other hand, in the absence of calyculin A, KT5926 inhibited both PMA-induced O2- generation and phosphorylation of the 67 kDa protein. Amino acid sequence analysis of peptides derived from the 67 kDa protein revealed that the 67 kDa protein was identical to L-plastin, an actin-bundling protein. We conclude that optimally phosphorylated L-plastin may play some crucial role in the activation of NADPH oxidase.
Oshizawa
Tadashi
T
Division of Cellular and Gene Therapy Products, and Division of Biosignaling, National Institute of Health Sciences, Kamiyoga 1-18-1, Setagaya-ku, Tokyo 158-8501.
Yamaguchi
Teruhide
T
Suzuki
Kazuhiro
K
Yamamoto
Yukio
Y
Hayakawa
Takao
T
D000595:Amino Acid Sequence; D002227:Carbazoles / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D008387:Marine Toxins; D008562:Membrane Glycoproteins; D008840:Microfilament Proteins; D008969:Molecular Sequence Data; D009219:Myosin-Light-Chain Kinase / Q000037:antagonists & inhibitors; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism; D010080:Oxazoles / Q000037:antagonists & inhibitors / Q000494:pharmacology; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D010766:Phosphorylation / Q000187:drug effects; D013481:Superoxides / Q000037:antagonists & inhibitors / Q000378:metabolism*
D002227:Carbazoles; D004791:Enzyme Inhibitors; D007211:Indoles; D008387:Marine Toxins; D008562:Membrane Glycoproteins; D008840:Microfilament Proteins; D010080:Oxazoles; D010750:Phosphoproteins; C026742:plastin; D013481:Superoxides; C063769:KT 5926; C059041:calyculin A; D019255:NADPH Oxidases; D009219:Myosin-Light-Chain Kinase