The N-terminal coiled coil domain of the cytohesin/ARNO family of guanine nucleotide exchange factors interacts with the scaffolding protein CASP.
Cytohesin is a guanine nucleotide exchange factor that regulates members of the ADP-ribosylation factor (ARF) family of small GTPases. All of the members of the cytohesin family (including ARNO, ARNO3, and the newly characterized cytohesin-4) have a similar domain distribution consisting of a Sec7 homology domain, a pleckstrin homology domain, and an N-terminal coiled coil. In this study, we attempt to identify proteins that interact specifically with the coiled coil motif of cytohesin. Yeast two-hybrid screening of a B cell library using the cytohesin N terminus as bait, identified CASP, a scaffolding protein of previously unknown function, as a binding partner. CASP contains an internal coiled coil motif that is required for cytohesin binding both in vitro and in COS-1 cells. The specificity of the coiled coil of CASP is not restricted to cytohesin, however, because it is also capable of interacting with other members of the cytohesin/ARNO family, ARNO and ARNO3. In immunofluorescence experiments, CASP localizes to perinuclear tubulovesicular structures that are in close proximity to the Golgi. These structures remain relatively undisturbed when the cells are treated with brefeldin A. In epidermal growth factor-stimulated COS-1 cells overexpressing cytohesin and CASP, cytohesin recruits CASP to membrane ruffles, revealing a functional interaction between the two proteins. These observations collectively suggest that CASP is a scaffolding protein that facilitates the function of at least one member of the cytohesin/ARNO family in response to specific cellular stimuli.
Mansour
Marc
M
Biology Department, Dalhousie University, Halifax, Nova Scotia B3H 4J1, Canada.
Lee
Stella Y
SY
Pohajdak
Bill
B
D000595:Amino Acid Sequence; D000818:Animals; D001402:B-Lymphocytes / Q000502:physiology; D001665:Binding Sites; D019556:COS Cells; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism; D002460:Cell Line; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D018388:Codon, Terminator; D003598:Cytoskeletal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins; D004815:Epidermal Growth Factor / Q000494:pharmacology; D020690:GTPase-Activating Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation; D015723:Gene Library; D006801:Humans; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors; D014162:Transfection
D002352:Carrier Proteins; D018388:Codon, Terminator; C088424:Cux1 protein, rat; D003598:Cytoskeletal Proteins; D004268:DNA-Binding Proteins; D020690:GTPase-Activating Proteins; D008565:Membrane Proteins; D011994:Recombinant Proteins; D014157:Transcription Factors; C102474:cytohesin-2; C410230:tamalin protein, human; D004815:Epidermal Growth Factor