Identification of cyclophilin A as a CD99-binding protein by yeast two-hybrid screening

Immunology Letters
Hyun Jung KimTae Jin Kim

Abstract

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.

References

Apr 15, 1992·Proceedings of the National Academy of Sciences of the United States of America·B SherryA Cerami
Oct 31, 2000·Circulation Research·Z G JinB C Berk
May 17, 2001·Proceedings of the National Academy of Sciences of the United States of America·T PushkarskyM Bukrinsky
Jan 29, 2002·Nature Immunology·Alan R SchenkelWilliam A Muller
Feb 7, 2002·Proceedings of the National Academy of Sciences of the United States of America·Kristine N BrazinAmy H Andreotti
Mar 21, 2002·The Biochemical Journal·Michela CapanoMartin Crompton
May 6, 2003·Nature Structural Biology·Bruce R HowardChristopher P Hill

❮ Previous
Next ❯

Related Concepts

Related Feeds

Apoptosis

Apoptosis is a specific process that leads to programmed cell death through the activation of an evolutionary conserved intracellular pathway leading to pathognomic cellular changes distinct from cellular necrosis