Characterization of binding activity between nuclear factor of activated T cells and calcineurin by amplified luminescent proximity homogeneous assay.
For quantitative evaluation of the relationship between biological binding partners, including protein-protein interactions, a novel analyzing system, amplified luminescent proximity homogeneous assay (ALPHA), has been developed. We here employed ALPHA for accurate assessment of the binding properties between nuclear factor of activated T cells 1 (NFAT1) and calcineurin (CN), which is essential for Ca2+-dependent regulation of immune responses. A recombinant protein of the Ca2+ regulatory domain (CRD) of NFAT1 was prepared and its binding activity with biotinylated CN was determined by ALPHA (Kd = 0.20 microM). The contribution of each CN-binding component involved in the CRD of NFAT1 to CN/NFAT1 binding was next examined by competitive assay. Not only the whole CRD but also the N- and C-terminal CN-binding regions (CNBR1 and CNBR2, respectively) dose-dependently blocked CN/NFAT1 binding and their potency was CRD >> CNBR2 > or = CNBR1. CN/NFAT1-binding properties were further characterized using short inhibitory peptides derived from NFAT1-CNBR1 as well as NFAT4-CNBR2. In conclusion, ALPHA is a useful system to analyze biological signaling cascades, due to its capability of quantitative evaluation of protein-protein interactions.
Kitamura
Noriko
N
Cytokine Project, The Tokyo Metropolitan Institute of Medical Science, 3-18-22, Honkomagome, Bunkyo-ku, Tokyo 113-8613, Japan.
Kaminuma
Osamu
O
Kitamura
Fujiko
F
Miyatake
Shoichiro
S
D001667:Binding, Competitive; D019703:Calcineurin / Q000737:chemistry*; D006801:Humans; D008163:Luminescent Measurements / Q000379:methods*; D050778:NFATC Transcription Factors / Q000737:chemistry* / Q000235:genetics; D025941:Protein Interaction Mapping / Q000379:methods*; D017434:Protein Structure, Tertiary / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics
D050778:NFATC Transcription Factors; D011994:Recombinant Proteins; D019703:Calcineurin