Identification of cyclophilin A as a CD99-binding protein by yeast two-hybrid screening.
CD99 is a 32kDa surface glycoprotein, which is involved in the migration of leukocytes and the transport of ganglioside GM1 and transmembrane proteins. To identify signaling mechanisms triggered by CD99 engagement, a LexA-based yeast two-hybrid system was utilized to identify proteins interacting with the cytoplasmic domains of CD99. In seven positive clones, we attempted to ascertain whether cyclophilin A (CypA) was involved in CD99-mediated signaling, since CypA had been implicated as a signaling regulator for kinases and phosphatases. The interaction between CD99 and CypA was confirmed by co-immunoprecipitation and confocal immunofluorescence studies. Interestingly, the amounts of CypA associated with CD99 increased upon CD99 engagement. We prepared an expression plasmid by inserting CypA cDNA into pEGFP, in order to visualize cellular CypA. In HeLa or HEK 293T cells transfected with the pEGFP-CypA plasmid, GFP-tagged CypA was diffusely present in the cytoplasm of untreated cells. However, CypA-GFP moved to the cell periphery and membrane blebbing, and became colocalized with CD99 upon CD99 engagement. These results suggest that CypA may be either a signaling mediator or a signaling regulator for CD99.
Kim
Hyun Jung
HJ
Department of Molecular Cell Biology, Center for Molecular Medicine, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Chunchun-dong 300, Suwon 440746, Republic of Korea.
Chong
Kyung Hee
KH
Kang
Shin Wook
SW
Lee
Jae-Rin
JR
Kim
Ji-Yeon
JY
Hahn
Myong-Joon
MJ
Kim
Tae Jin
TJ
D000071443:12E7 Antigen; D015703:Antigens, CD / Q000235:genetics / Q000378:metabolism*; D001921:Brain / Q000378:metabolism; D015815:Cell Adhesion Molecules / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D021984:Cyclophilin A / Q000235:genetics / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics; D049452:Green Fluorescent Proteins; D006801:Humans; D047468:Immunoprecipitation; D011485:Protein Binding; D021381:Protein Transport; D011993:Recombinant Fusion Proteins; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D020798:Two-Hybrid System Techniques
D000071443:12E7 Antigen; D015703:Antigens, CD; C061828:CD99 protein, human; D015815:Cell Adhesion Molecules; D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D021984:Cyclophilin A