Interaction protein for cytohesin exchange factors 1 (IPCEF1) binds cytohesin 2 and modifies its activity.
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.
Venkateswarlu
Kanamarlapudi
K
Department of Pharmacology, The University of Bristol, Bristol BS8 1TD, United Kingdom. k.venkateswarlu@bristol.ac.uk
D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors / Q000378:metabolism; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D019556:COS Cells; D015815:Cell Adhesion Molecules / Q000737:chemistry* / Q000378:metabolism* / Q000502:physiology*; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D020690:GTPase-Activating Proteins / Q000737:chemistry / Q000378:metabolism*; D017353:Gene Deletion; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D050356:Lipid Metabolism; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009154:Mutation; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013997:Time Factors; D014162:Transfection; D020798:Two-Hybrid System Techniques; D047908:Intracellular Signaling Peptides and Proteins
D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors; D000596:Amino Acids; D015815:Cell Adhesion Molecules; D020690:GTPase-Activating Proteins; D005982:Glutathione Transferase; D018129:Phosphatidylinositol Phosphates; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; C479748:IPCEF1 protein, rat; D047908:Intracellular Signaling Peptides and Proteins; C102474:cytohesin-2; C060974:phosphatidylinositol 3,4,5-triphosphate; C000715348:ARF6 protein, human; C000715349:Arf6 protein, rat