Overexpression, purification and characterization of Dictyostelium calcineurin A

• Published in: Research in microbiology Vol. 148; no. 4; pp. 335 - 343
• Main Authors: Hellstern, S., Dammann, H., Husain, Q., Mutzel, R.
• Format: Journal Article
• Language: English
• Published: Paris Elsevier SAS 01-05-1997; Elsevier
• Subjects: Affinity chromatography; Aniline Compounds - metabolism; Animals; Biochemistry. Physiology. Immunology. Molecular biology; Biological and medical sciences; Calcineurin - genetics; Calcineurin - isolation & purification; Calcineurin - metabolism; Calcineurin A; Calcineurin Inhibitors; Calcineurine A; Calcium; Calmodulin; Calmodulin - pharmacology; Calmoduline; Caseins - metabolism; Cations, Divalent; Cattle; Chlorides - pharmacology; Chromatography, Affinity; Chromatography, DEAE-Cellulose; Chromotographie d'affinité; Dictyostelium - enzymology; Dictyostelium - genetics; Dictyostelium discoideum; Dithiothreitol - pharmacology; DNA, Complementary; Enzyme Inhibitors - pharmacology; Fundamental and applied biological sciences. Psychology; Gene Expression; Magnesium Chloride - pharmacology; Manganese Compounds - pharmacology; Okadaic Acid - pharmacology; Organophosphorus Compounds - metabolism; Phosphatase; Phosphopeptides - metabolism; Phosphorylation; Protein; Protozoa; Protozoan Proteins - antagonists & inhibitors; Protozoan Proteins - genetics; Protozoan Proteins - isolation & purification; Protozoan Proteins - metabolism; Protéine; Recombinant Fusion Proteins; Signal transduction; Sulfhydryl Reagents - pharmacology; Transduction de signal; Transformation, Genetic; Phosphorylation; Protéine; Calcium; Phosphatase; Calcineurin A; Protein; Chromotographie d'affinité; Dictyostelium discoideum; Signal transduction; Transduction de signal; Affinity chromatography; Calcineurine A; Calmoduline; Calmodulin; Protozoa; calmodulin-dependent protein kinase; Purification; Enzyme; Transferases; Gene overexpression; Gene expression; Binding protein; Enzymatic activity; Ca; Mycetozoa
• ISSN: 0923-2508; 1769-7123
• DOI: 10.1016/S0923-2508(97)81589-6

The catalytic subunit of Ca
2+/calmodulin-dependent protein phosphatase (calcineurin A) was overexpressed about 50-fold in
Dictyostelium discoideum cells transformed with a vector containing the cDNA for
D. discoideum calcineurin A under control of the actin-6 promoter. In crude lysates from the overexpressing cell line, high Ca
2+/calmodulin-stimulated phosphatase activity was detected. Calcineurin A was purified by anion exchange chromatography and calmodulin-Sepharose affinity chromatography, and the enzymatic activity of the isolated protein was characterized. Its phosphatase activity was strictly dependent on the addition of divalent metal ions such as Mg
2+ or Mn
2+. Disulphide-reducing agents increased the activity more than 10-fold. Ca
2+/calmodulin stimulated the activity by a factor of 2.5–5. Despite the high extra Ca
2+/calmodulin-dependent phosphatase activity, the overexpressing cell line showed no phenotypic aberrations.
La sous-unité catalytique de la protéine-phosphatase Ca
2+/calmoduline-dépendante (calcineurine A) a été surexprimée 50 fois dans des cellules de
Dictyostelium discoideum transformées avec un vecteur contenant l'ADNc pour la calcineurine A de
D. discoideum sous le contrôle du promoteur actine-6. Dans des lysats de la lignée cellulaire de surexpression, une activité phosphatasique fortement stimulée par la Ca
2+/calmoduline est décelée. La calcineurine A a été purifiée par chromatographie et l'activité enzymatique de la protéine isolée a été caractérisée. Son activité phosphatasique dépend strictement de l'addition d'ions divalents tels Mg
2+ ou Mn
2+. Des agents disulfuresréducteurs accroissent plus de 10 fois cette activité. La Ca
2+/calmoduline la stimule 2,5 à 5 fois. Malgré la forte activité phosphatasique ≪extra≫ Ca
2+/calmoduline-dépendante, la lignée cellulaire de surexpression ne montre pas d'aberrations phénotypiques.