Discriminatory aptamer reveals serum response element transcription regulated by cytohesin-2.
Cytohesins are a family of highly homologous guanine nucleotide exchange factors (GEFs) that act on ADP-ribosylation factors (ARFs). The small ARF-GEFs are involved in integrin signaling, actin cytoskeleton remodeling, and vesicle transport. Here, we selected and applied a specific inhibitor for ARF nucleotide-binding site opener (ARNO)/cytohesin-2, an RNA aptamer that clearly discriminates between cytohesin-1 and cytohesin-2. This reagent bound to an N-terminal segment of cytohesin-2 and did not inhibit ARF-GEF function in vitro. When transfected into HeLa cells, it persisted for at least 6 h without requiring stabilization. Its effect in vivo was to down-regulate gene expression mediated through the serum-response element and knockdown mitogen-activated protein kinase activation, indicating that cytohesin-2 acts by means of mitogen-activated protein kinase signaling. We conclude that the N-terminal coiled-coil and parts of the Sec7 domain of cytohesin-2 are required for serum-mediated transcriptional activation in nonimmune cells, whereas cytohesin-1 is not. Our results indicate that intramer technology can be used not only for assigning novel biological functions to proteins or protein domains but also to prove nonredundancy of highly homologous proteins.
Kekulé Institut für Organische Chemie und Biochemie, University of Bonn, Gerhard-Domagk-Strasse 1, 53121 Bonn, Germany.
Mirko G
MG
Theis
Alexander
A
Knorre
Bettina
B
Kellersch
Jörg
J
Moelleken
Felix
F
Wieland
Waldemar
W
Kolanus
Michael
M
Famulok
D020727:ADP-Ribosylation Factors / Q000378:metabolism; D001483:Base Sequence; D015815:Cell Adhesion Molecules / Q000235:genetics / Q000378:metabolism; D020690:GTPase-Activating Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D015870:Gene Expression; D020662:Guanine Nucleotide Exchange Factors / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D020935:MAP Kinase Signaling System / Q000502:physiology; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D012313:RNA / Q000378:metabolism; D027602:Serum Response Element / Q000235:genetics*; D014158:Transcription, Genetic / Q000502:physiology; D015533:Transcriptional Activation / Q000502:physiology*
D015815:Cell Adhesion Molecules; D020690:GTPase-Activating Proteins; D020662:Guanine Nucleotide Exchange Factors; C066755:Sec7 guanine nucleotide exchange factors; C100723:cytohesin-1; C102474:cytohesin-2; D012313:RNA; D020727:ADP-Ribosylation Factors