Interaction Protein for Cytohesin Exchange Factors 1 (IPCEF1) Binds Cytohesin 2 and Modifies Its Activity

Kanamarlapudi Venkateswarlu

doi:10.1074/jbc.m304078200

Abstract

The ADP-ribosylation factor 6 (ARF6) small GTPase functions as a GDP/GTP-regulated switch in the pathways that stimulate actin reorganization and membrane ruffling. The formation of active ARF6GTP is stimulated by guanine nucleotide exchange factors (GEFs) such as cytohesins, which translocate to the plasma membrane in agonist-stimulated cells by binding the lipid second messenger phosphatidylinositol 3,4,5-trisphosphate through the pleckstrin homology domain with subsequent ARF6 activation. Using cytohesin 2 as bait in yeast two-hybrid screening, we have isolated a cDNA encoding a protein termed interaction protein for cytohesin exchange factors 1 (IPCEF1). Using yeast two-hybrid and glutathione S-transferase pull-down assays coupled with deletion mutational analysis, the specific domains required for the cytohesin 2-IPCEF1 interaction were mapped to the coiled-coil domain of cytohesin 2 and the C-terminal 121 amino acids of IPCEF1. IPCEF1 also interacts with the other members of the cytohesin family of ARF GEFs, suggesting that the interaction with IPCEF1 is highly conserved among the cytohesin family of ARF GEFs. The interaction of cytohesin 2 and IPCEF1 in mammalian cells was demonstrated by immunoprecipitation. Immunofluorescence analysis revealed that IPCEF1 co-localizes with cytohesin 2 to the cytosol in unstimulated cells and translocates to the plasma membrane via binding to cytohesin 2 in epidermal growth factor-stimulated cells. However, a deletion mutant of IPCEF1 that lacks the cytohesin 2 binding site failed to co-migrate with cytohesin 2 to the membrane in stimulated cells. The functional significance of the IPCEF1-cytohesin 2 interaction is demonstrated by showing that IPCEF1 increases the in vitro and in vivo stimulation of ARFGTP formation by cytohesin 2.

Highlights

The ADP-ribosylation factor (ARF)1 family of small GTPases regulate membrane trafficking at multiple sites within the cell
The formation of active ARF6GTP is stimulated by guanine nucleotide exchange factors (GEFs) such as cytohesins, which translocate to the plasma membrane in agonist-stimulated cells by binding the lipid second messenger phosphatidylinositol 3,4,5-trisphosphate through the pleckstrin homology domain with subsequent ADP-ribosylation factor 6 (ARF6) activation
The Interaction with interaction protein for cytohesin exchange factors 1 (IPCEF1) Is Highly Conserved among Cytohesin Family ARF GEFs—Since all members of the cytohesin family contain the N-terminal CC domain, we examined whether the other members of this family interact with IPCEF1 using both the yeast two-hybrid and glutathione S-transferase (GST) pull-down assays

Summary

EXPERIMENTAL PROCEDURES

Chemicals—All chemicals were obtained from Sigma unless otherwise specified. DNA restriction enzymes were from Roche Applied Science. GST Fusion Protein Pull-down Assay—COS cells were plated into 100-mm Petri dishes and allowed to grow to 70 – 80% confluency They were transfected with FLAG-tagged full-length IPCEF1 or its deletion mutant-encoding plasmids (5 ␮g of DNA). COS cells transfected with ARF6-HA, GFP, or GFP-cytohesin 2 and FLAG or FLAG-tagged full-length IPCEF1 plasmids (3:1:1 ratio, 10 ␮g of total DNA) were serum-starved for 2 h and incubated for 5 min with or without 200 ng/ml EGF. After 1 h of mixing at 4 °C, the beads were washed three times with wash buffer (50 mM Tris-HCl, pH 7.5, 0.15 M NaCl, 10 mM MgCl2, 1% Triton X-100, 2 mM ZnCl2, and 0.1% protease inhibitors), boiled in SDS-PAGE sample buffer, and analyzed by immunoblotting using a monoclonal anti-HA antibody (Covance). All images presented are single sections in the z-plane

RESULTS

DISCUSSION

Kanamarlapudi Venkateswarlu