Characterization of a cDNA Clone Encoding the Calmodulin-Binding Domain of Mouse Brain Calcineurin

• Published in: Proceedings of the National Academy of Sciences - PNAS 1988-12, Vol.85 (23), p.8983-8987
• Main Authors: Kincaid, Randall L., Nightingale, Maria S., Martin, Brian M.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Amino acids; Animals; Antibodies; Base Sequence; Biological and medical sciences; Brain - enzymology; Calcineurin; Calmodulin - metabolism; Calmodulin-Binding Proteins - genetics; Calmodulin-Binding Proteins - metabolism; Cloning, Molecular; Complementary DNA; DNA; DNA - genetics; DNA - isolation & purification; Enzymes; Fundamental and applied biological sciences. Psychology; Generally accepted auditing standards; Genes; Genes. Genome; Genetic Vectors; Mice; Molecular and cellular biology; Molecular genetics; Molecular Sequence Data; Phosphatases; Phosphoprotein Phosphatases - genetics; Phosphoprotein Phosphatases - metabolism; Proteins; Regional identity; Restriction Mapping; Sequence Homology, Nucleic Acid; Sequencing
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.85.23.8983

A cDNA clone corresponding to a portion of the catalytic subunit of calmodulin (CaM)-dependent phospho-protein phosphatase (calcineurin) was isolated from a murine brain library by expression vector immunoscreening. A β -galactosidase fusion protein that reacted on Western blots with anti-calcineurin antibodies and biotinylated CaM was purified in preparative amounts using CaM-Sepharose affinity chromatography. Partial digestion of the hybrid protein with Staphylococcus aureus V-8 protease produced several immunoreactive peptides that appeared identical to fragments generated from authentic brain calcineurin. The 1111-base-pair (bp) EcoRI insert contained an open reading frame encoding a protein of 35 kDa followed by a 190-bp 3′ noncoding region; seven peptides obtained by partial amino acid sequencing of the bovine brain enzyme were found in the deduced sequence. A domain ≈ 12 kDa from the carboxyl terminus was deduced to be the CaM-binding site based on consensus structural features and a sequence of seven amino acids highly related to smooth muscle myosin light-chain kinase. Two regions with identity to protein phosphatases 1 and 2A were found in the amino half of the cloned sequence; however, the intervening sequence contained apparent insertions, suggesting splicing of subdomains. Thus, the structure of calcineurin is chimeric, consisting of conserved catalytic elements and a regulatory CaM-binding domain.