Identification of an actin-binding site in p47phox an organizer protein of NADPH oxidase.
Actin has been reported to enhance the superoxide-generating activity of neutrophil NADPH oxidase in a cell-free system and to interact with p47phox, a regulatory subunit of the oxidase. In the present study, we searched for an actin-binding site in p47phox by far-western blotting and blot-binding assays using truncated forms of p47phox. The amino-acid sequence 319-337 was identified as an actin-binding site, and a synthetic peptide of this sequence bound to actin. The sequence shows no homology to other known actin-binding motifs. It is located in the autoinhibitory region of p47phox and includes Ser-328, a phosphorylation site essential for unmasking. Although a phosphorylation-mimetic p47phox mutant bound to actin with a lower affinity than the wild type, the same mutant interacted with filamentous actin more efficiently than the wild type. A mutant peptide p47phox (319-337, Ser328Glu) bound to filamentous actin more tightly than to monomer actin. These results suggest that p47phox moves to cortical actin when it becomes unmasked in the cells.
Tamura
Minoru
M
Department of Applied Chemistry, Faculty of Engineering, Ehime University, Matsuyama, Ehime 790-8577, Japan. tamura@eng.ehime-u.ac.jp
Itoh
Katsunori
K
Akita
Hiroshi
H
Takano
Keiko
K
Oku
Satoshi
S
D008841:Actin Cytoskeleton / Q000235:genetics / Q000378:metabolism*; D000199:Actins / Q000235:genetics / Q000378:metabolism*; D020816:Amino Acid Motifs / Q000235:genetics; D000595:Amino Acid Sequence / Q000235:genetics; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D002474:Cell-Free System; D006801:Humans; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism; D010455:Peptides / Q000235:genetics / Q000378:metabolism*; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D017354:Point Mutation; D011485:Protein Binding / Q000235:genetics
D000199:Actins; D010455:Peptides; D010750:Phosphoproteins; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1