JFC1, a Novel Tandem C2 Domain-containing Protein Associated with the Leukocyte NADPH Oxidase

Abstract

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 p67phox, 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-p67phox association by affinity chromatography. JFC1-containing beads pulled down both p67phox and p47phox 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 p67phox without affecting the interaction between p67phox and p47phox. 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.