RCAN carboxyl end mediates calcineurin docking-dependent inhibition via a site that dictates binding to substrates and regulators

• Published in: Proceedings of the National Academy of Sciences - PNAS 2009-04, Vol.106 (15), p.6117-6122
• Main Authors: Martínez-Martínez, Sara, Genescà, Lali, Rodríguez, Antonio, Raya, Alicia, Salichs, Eulàlia, Were, Felipe, López-Maderuelo, María Dolores, Redondo, Juan Miguel, de la Luna, Susana
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Amino acids; Animals; Binding Sites; Biochemistry; Biological Sciences; Calcineurin - chemistry; Calcineurin - genetics; Calcineurin - metabolism; Catalytic activity; Cell Line; Conserved Sequence; Enzymes; Goods and services tax; HEK293 cells; Humans; Hydrophobic and Hydrophilic Interactions; Kinases; Mice; Modulated signal processing; Molecular Sequence Data; Muscle Proteins - chemistry; Muscle Proteins - genetics; Muscle Proteins - metabolism; Mutation; Phosphatases; Phosphoric Monoester Hydrolases - metabolism; Phosphorylation; Physiological regulation; Protein Structure, Secondary; Proteins; Quantification; Sequence Alignment; Signal Transduction; Substrate Specificity
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.0812544106

Specificity of signaling kinases and phosphatases toward their targets is usually mediated by docking interactions with substrates and regulatory proteins. Here, we characterize the motifs involved in the physical and functional interaction of the phosphatase calcineurin with a group of modulators, the RCAN protein family. Mutation of key residues within the hydrophobic docking-cleft of the calcineurin catalytic domain impairs binding to all human RCAN proteins and to the calcineurin interacting proteins Cabin1 and AKAP79. A valine-rich region within the RCAN carboxyl region is essential for binding to the docking site in calcineurin. Although a peptide containing this sequence compromises NFAT signaling in living cells, it does not inhibit calcineurin catalytic activity directly. Instead, calcineurin catalytic activity is inhibited by a motif at the extreme C-terminal region of RCAN, which acts in cis with the docking motif. Our results therefore indicate that the inhibitory action of RCAN on calcineurin-NFAT signaling results not only from the inhibition of phosphatase activity but also from competition between NFAT and RCAN for binding to the same docking site in calcineurin. Thus, competition by substrates and modulators for a common docking site appears to be an essential mechanism in the regulation of Ca²⁺-calcineurin signaling.