JFC1, a novel tandem C2 domain-containing protein associated with the leukocyte NADPH oxidase.
We have employed a yeast two-hybrid system to screen a B lymphoblast-derived cDNA library, searching for regulatory components of the NADPH oxidase. Using as bait the C-terminal half of p67(phox), which contains both Src homology 3 domains, we have cloned JFC1, a novel human 62-kDa protein. JFC1 possesses two C2 domains in tandem. The C2A domain shows homology with the C2B domain of synaptotagmins. JFC1 mRNA was abundantly expressed in bone marrow and leukocytes. The expression of JFC1 in neutrophils was restricted to the plasma membrane/secretory vesicle fraction. We confirmed JFC1-p67(phox) association by affinity chromatography. JFC1-containing beads pulled down both p67(phox) and p47(phox) subunits from neutrophil cytosol, but when the recombinant proteins were used, only p67(phox) bound to JFC1, indicating that JFC1 binds to the cytosolic complex via p67(phox) without affecting the interaction between p67(phox) and p47(phox). In contrast to synaptotagmins, JFC1 was unable to bind to inositol 1,3,4,5-tetrakisphosphate but did bind to phosphatidylinositol 3,4,5-trisphosphate and to a lesser extent to phosphatidylinositol 3,4-diphosphate. From the data presented here, it is proposed that JFC1 is acting as an adaptor protein between phosphatidylinositol 3-kinase products and the oxidase cytosolic complex.
McAdara Berkowitz
J K
JK
Department of Molecular and Experimental Medicine, Division of Biochemistry, The Scripps Research Institute, La Jolla, California 92037, USA.
Catz
S D
SD
Johnson
J L
JL
Ruedi
J M
JM
Thon
V
V
Babior
B M
BM
D000595:Amino Acid Sequence; D001483:Base Sequence; D015152:Blotting, Northern; D002135:Calcium-Binding Proteins; D002462:Cell Membrane / Q000378:metabolism; D002846:Chromatography, Affinity; D003600:Cytosol / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D015723:Gene Library; D006801:Humans; D007295:Inositol Phosphates / Q000378:metabolism; D007962:Leukocytes / Q000201:enzymology* / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism*; D008969:Molecular Sequence Data; D019255:NADPH Oxidases / Q000737:chemistry* / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000378:metabolism; D009504:Neutrophils / Q000378:metabolism; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D011485:Protein Binding; D014176:Protein Biosynthesis; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D050857:Synaptotagmins; D013755:Tetradecanoylphorbol Acetate / Q000378:metabolism; D014018:Tissue Distribution; D020798:Two-Hybrid System Techniques; D018909:src Homology Domains
D002135:Calcium-Binding Proteins; D018076:DNA, Complementary; D007295:Inositol Phosphates; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D018129:Phosphatidylinositol Phosphates; D010750:Phosphoproteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; C431146:SYTL1 protein, human; C079789:neutrophil cytosol factor 67K; C060974:phosphatidylinositol 3,4,5-triphosphate; C060980:phosphatidylinositol 3,4-diphosphate; C081655:inositol-1,3,4,5-tetrakisphosphate; D050857:Synaptotagmins; D019255:NADPH Oxidases; D013755:Tetradecanoylphorbol Acetate