The C2A domain of JFC1 binds to 3′-phosphorylated phosphoinositides and directs plasma membrane association in living cells

Abstract

Phosphatidylinositol 3-kinase products play a central role in the regulation of several intracellular pathways via adaptor proteins that share the ability to bind to 3 -phosphoinositides with high affinity and specificity. JFC1 is a C2 domain-containing protein involved in cellular trafficking that has been shown to bind 3 -phosphoinositides in vitro. In this work, we demonstrate that the C2A domain of JFC1 is the module responsible for its binding to the plasma membrane via 3 -phosphoinositides in vivo. We show that the C2A domain of JFC1 is the only domain present in this protein that localizes to the plasma membrane in living cells. Moreover, the C2A domain of JFC1 binds 3 -phosphoinositides in vitro with similar specificity as that described for full-length JFC1, suggesting that the domain mediates the specific membrane localization of the full-length protein. Furthermore, the C2A domain of JFC1 colocalized with the pleckstrin homology domain of Akt in vivo, and both the JFC1 C2A domain and the full-length JFC1 dissociated from the membrane in the presence of PI 3-kinase specific inhibitors. We also show that the association of the C2A domain to the membrane is modulated by calcium. From these results we analyze possible mechanisms for the role of JFC1 in cellular trafficking. P hosphoinositides are specialized phospholipids known to localize to the inner leaflet of the plasma membrane, vesicles, and the nuclear envelope and participate in several signaling pathways (1). One of these phosphoinositides, phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5)P 2 ], plays a central role in the regulation of several intracellular pathways, from actin cytoskeleton modulation and vesicle trafficking (2) to modulation of the transcription factor Tubby protein (3). Furthermore, PtdIns(4,5)P 2 is a precursor for the second messenger phosphatidylinositol (3,4,5) trisphosphate [PtdIns(3,4,5)P 3 ] via the phosphoinositide 3-kinases ref. 4). PI 3-kinases and their products 3Ј-phosphorylated phosphoinositides (3Ј-phosphoinositides) are also involved in many crucial cell functions including vesicle trafficking (1, 2), cytoskeleton modulation, and cell survival (5). It is now clear that most of the functions regulated by phosphoinositides are mediated by adaptor proteins that share the ability to bind to PtdIns(4,5)P 2 and PI 3-kinase products with high affinity and specificity. These effectors are characterized by the presence of specialized phosphoinositide-binding domains. These domains include the pleckstrin homology (PH) domain (6), the FYVE finger (7), and the recently discovered PHOX homology (PX) domain (8). Furthermore, C2 domains have been shown to bind to phospholipids or phosphoinositides in the presence or absence of calcium (9). Proteins that contain C2 domains could generally be categorized as signal transduction mediators or membrane traffic effectors (10). The most extensively studied proteins included in the last group are the synaptotagmins (Syts). Syts are a group of transmembrane proteins that contain tan...

Structural and Membrane Binding Analysis of the Phox Homology Domain of Phosphoinositide 3-Kinase-C2α

“…Our quantitative SPR measurements confirm that the C2 domain of PI3K-C2␣ can indeed bind PtdIns(4,5)P 2 and PtdIns(3,4)P 2 ; however, the C2 domain lacks PI specificity and has about 20-fold lower affinity for PtdIns(4,5)P 2 -containing vesicles than the PX domain. The promiscuity and lower affinity of the C2 domain for PIs has been reported for other C2 domains from synaptotagmin I (73), JFC1 (74), and Rsp5p (75). Also, unlike the PX domain, the C2 domain of PI3K-C2␣ is not able to penetrate the lipid monolayer with the packing density comparable with that of cell membranes with or without PI.…”

Physiological Roles of Rab27 Effectors in Regulated Exocytosis

“…JFC1 is colocalized with a minor population of azurophilic granules and forms an endogenous complex with Rab27a in neutrophils [46]. The C2A domain of JFC1 expressed in cultured cells is exclusively localized to the plasma membrane, whereas the C2B domain is distributed throughout the cytoplasm [47]. These findings suggests that the C2A domain is responsible for the membrane-binding capacity of JFC1, similar to the case of exophilin4 [29,43].…”

“…The C2A domain of JFC1 preferentially binds to 3'-phosphorylated phosphoinositides, and most strongly to phosphatidylinositol (3,4,5) triphosphate (PIP 3 ). Although the Ca 2+ effect on the PIP 3 -binding activity in vitro has not directly been investigated, the C2A domain of JFC1 expressed in NIH3T3 cells is dissociated from the plasma membrane after a calcium influx induced by ionomycin [47]. JFC1 is expressed in LNCap prostate carcinoma cells and preferentially colocalizes with prostatic-specific acid phosphatase but rarely with prostate-specific antigen in prostate granules [48].…”

The C2 domains of the class I Rab11 family of interacting proteins target recycling vesicles to the plasma membrane

“…Recently, this technique has been successfully used to identify the phospholipid binding partners of many proteins (Catz et al, 2002;Chiang et al, 2003;Dowler et al, 1999;Gozani et al, 2003). Using this technique, we determined that the three C2 domains of the class I Rab11-FIPs preferentially bound the phospholipids PtdIns(3,4,5)P3 and PA.…”

“…Catz and co-workers recently demonstrated that the C2A domain of the tandem C2 domain-containing ATPase JFC1 binds preferentially to PtdIns(3,4,5)P 3 (Catz et al, 2002). JFC1 is a Rab27a effector protein and these authors propose that it positions Rab27a-containing vesicles to a specific docking point on the plasma membrane.…”