Abstract
The calcium/calmodulin-regulated phosphatase calcineurin (CN) is the site of action of the immunosuppressive drugs cyclosporin A (CsA) and FK506. CN has recently been established as a key signaling enzyme in the T cell signal transduction cascade and an important regulator of transcription factors such as NF-AT and OAP/Oct-1, which are involved in the expression of a number of important T cell early genes. CsA and FK506 act by forming complexes with their respective intracellular receptors cyclophilin and FKBP (immunophilins), which can then bind to CN, inhibiting its enzymatic activity and thereby preventing early gene expression. CN is comprised of two subunits: a 59-kDa catalytic subunit (CNA), which contains a calmodulin binding domain and autoinhibitory region, and a 19-kDa intrinsic calcium binding regulatory subunit (CNB). In this study, we have utilized a series of deletion mutants of the CNA subunit to investigate the subunit and molecular requirements that govern the interaction of CN with drug-immunophilin complexes. The calmodulin binding and autoinhibitory domains of the CNA subunit were found to be dispensable for the binding of CN to drug-immunophilin complexes. In contrast, we found that the regulatory CNB subunit appears to play an obligatory role in this interaction and have defined an amino acid sequence of the CNA subunit which forms the binding site for CNB. Although necessary, the CNB subunit per se is not sufficient to mediate an interaction with drug-immunophilin complexes; amino acid residues of the CNA subunit, specifically a region located within the putative catalytic domain, are also required for the interaction of CN with both FKBP-FK506 and cyclophilin A-CsA.
Highlights
The calcidcalmodulin-regulated phosphatase cal- ceptors, cyclophilin and FKBP(8-101, respectively, collectively cineurin (CN)is the siotfeaction of the immunosuppres- known as the immunophilin(1s 1).The bindingof each drugto sive drugs cyclosporin A (CsA)and FK506
The calmodulinbinding and autoinhibi- Second, in vivo studies demonstrated that overexpression of tory domainsof the CNA subunit were foundto be dis- CNA and calmodbinding regulatory subunit (CNB) subunits was able to render T cells markedly pensable for the binding of CN to drug-immunophilin resistant t o the inhibitory effects of Cyclosporin A (CsA) and FK506 and to complexes
Interaction and have definedan amino acid sequencoef a calcium-independent, constitutivelyactive mutant the CNA subunit which formsthe binding site for CNB. of CN was able to synergize with phorbol ester and activate necessary,the CNB subunit per se is not suffi- these latter promoters, replacing the normal requirement for cient to mediate an interaction with drug-immunophilin an increase in intracellular calcium [18, 19]
Summary
Plasmid Construction-For carboxyl-terminal CN deletion constructs, the influenza virus hemagglutinin epitope (EFYPYDVPDYA) [29]was fused in-frame to the amino terminus of murine CN with the polymerase chain reaction (PCR). GAL-FKBPwas generated by making a translational fusion between the human FKBP-12 open reading frame and the GAL4 DNA binding domain [1-147] contained within plasmid pASl[26]. Drug-Immunophilin Binding and Immunoprecipitation-Glutathione S-transferase immunophilin fusion proteins were prepared as describedpreviously [25].Preformed immobilized GST-FKBP-12fusion protein.FK506 or GST-cyclophilinA fusion protein.CsA complexes (10 pg) were mixedwith aliquots (50pl) of extracts derived fromtransiently transfected COS-7 cellsin a total volume of 200 pl of lysis buffer. Yeast Strains, Manipulations, and lho-hybridScreening"Y153 [26], MATaleu2-3,112u, ra, trpl-901, his3-A200a, de2-101g, a14A, gal8OA, URA3::GAL-lacZ, LYS2::GAL-HISS.To generate Y153ACNB, a CNB disruption vector (cnbA:ADEZ)was created by cloning a 4.7-kilobase genomic BamHI fragment containing the ADE-2 gene into BgZII-. Transformed colonies were selected by growth at 30 "C on SC plates lacking the appropriate nutrients; interaction in the two-hybrid system was observed by growth in the absence of histidine and confirmed by p-galactosidase assay [26]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
