Blockade of NFAT activation by the second calcineurin binding site.
Activation of NFAT transcription factors requires their dephosphorylation by the phosphatase calcineurin (CN). NFATs contain two CN binding motifs: PxIxIT and CnBP-B/CNBR2 (which we call LxVP). Here we carry out a detailed comparative analysis of the CN binding activity displayed by the PxIxIT and LxVP sites from different NFATs. Dose-response CN binding experiments with GST fusion proteins of NFATc1 and NFATc2 showed that NFATc1 binds CN in vitro more efficiently than does NFATc2. This difference in binding appears to be caused by the different CN binding potencies of the corresponding LxVP sites; thus while the LxVPc2 peptide fused to GST did not bind CN, GST-LxVPc1 bound it more efficiently than did GST-PxIxITc1 or GST-PxIxITc2. Furthermore, an NFATc2 chimera protein containing the LxVP motif from NFATc1 interacted with CN much more potently than did wild-type NFATc2. Free peptides spanning the LxVP motifs from NFATc1, c3 or c4 displaced CN from GST-NFATc1 and GST-NFATc2 more efficiently than any PxIxIT peptide. PxIxITc2 and LxVPc1 peptides were each able to cross-compete GST-LxVPc1-CN and GST-PxIxITc2-CN binding. In contrast with PxIxITc2, the LxVP peptide not only blocked CN-NFAT binding but also inhibited CN phosphatase activity in vitro. Furthermore, exogenous LxVPc1 blocked NFATc2 phosphorylation and nuclear translocation in vivo. These results suggest a model in which the different CN binding characteristics of the PxIxIT and LxVP sites enable different NFAT members to influence each others activities in cells where they are co-expressed.
Martínez-Martínez
Sara
S
Centro Nacional de Investigaciones Cardiovasculares (CNIC), Melchor Fernández Almagro 3, 28029 Madrid, Spain.
Rodríguez
Antonio
A
López-Maderuelo
Maria Dolores
MD
Ortega-Pérez
Inmaculada
I
Vázquez
Jesús
J
Redondo
Juan Miguel
JM
D020816:Amino Acid Motifs; D001483:Base Sequence; D001667:Binding, Competitive; D019703:Calcineurin / Q000378:metabolism*; D065095:Calcineurin Inhibitors; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D050778:NFATC Transcription Factors / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D010446:Peptide Fragments / Q000502:physiology; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors; D011485:Protein Binding; D017434:Protein Structure, Tertiary
D065095:Calcineurin Inhibitors; D050778:NFATC Transcription Factors; D010446:Peptide Fragments; D019703:Calcineurin; D010744:Phosphoric Monoester Hydrolases