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 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.

Characterization of the Rab8-specific membrane traffic route linked to protrusion formation

“…It interacts with Akt, Rab27a and the NADPH oxidase (Johnson et al, 2005a;Johnson et al, 2005b;MacAdara et al, 2001). JFC1 is known to regulate androgendependent secretion of prostate-specific antigen and prostaticspecific acid phosphatase (Johnson et al, 2005b).…”

“…We found several identical clones that corresponded to the open reading frame encoding the human JFC1 protein, the human homolog of the mouse synaptotagmin-like protein 1 (Slp1) (Fukuda and Mikoshiba, 2001;MacAdara-Berkowitz et al, 2001). JFC1 specifically interacted with wild-type Rab8 and the active GTP-bound mutant Rab8-67L, but not with the dominant-negative GDPbound mutant Rab8-22N (Fig.…”

Regulation of vesicular trafficking and leukocyte function by Rab27 GTPases and their effectors

“…The Slp family of Rab27 effectors was the first identified [43]. It is composed of five members: Slp1/ JFC1/exophilin7 [35], Slp2a/exophilin4, Slp3/exophilin6, Slp4-a/granuphilin-a/exophilin2 [44], and Slp5/exophilin9 [45], encoded by genes SYTL1-5, located in loci 1p36.11, 11q14.1, 6q25.3, Xq21.33, and Xp21.1, respectively. Slp1, also known as JFC1, was the first Rab27 effector identified in hematopoietic cells, and expression of Slp1/JFC1 in human neutro-phils was demonstrated [35].…”

“…It is composed of five members: Slp1/ JFC1/exophilin7 [35], Slp2a/exophilin4, Slp3/exophilin6, Slp4-a/granuphilin-a/exophilin2 [44], and Slp5/exophilin9 [45], encoded by genes SYTL1-5, located in loci 1p36.11, 11q14.1, 6q25.3, Xq21.33, and Xp21.1, respectively. Slp1, also known as JFC1, was the first Rab27 effector identified in hematopoietic cells, and expression of Slp1/JFC1 in human neutro-phils was demonstrated [35]. Proteomics analysis of neutrophil granules has confirmed the expression of Slp1/JFC1 in human neutrophils and its localization at azurophilic granules [46].…”

Upregulation of the Rab27a-Dependent Trafficking and Secretory Mechanisms Improves Lysosomal Transport, Alleviates Endoplasmic Reticulum Stress, and Reduces Lysosome Overload in Cystinosis

“…Rab27a and its specific effectors are master regulators of vesicular transport and exocytosis (19,20). They are expressed in multiple cells and tissues with secretory functions, including the kidney (23)(24)(25)(26) and eye (21,22). Rab27b, which has 72% homology with Rab27a at the amino acid level, is also expressed in the kidney's transitional epithelium and in corneal epithelial cells (23).…”