Interchangeable associations of calcineurin regulatory subunit isoforms with mammalian and fungal catalytic subunits.
Two mammalian genes for the Ca(2+)-binding regulatory subunit of the calmodulin-dependent protein phosphatase (calcineurin) were identified recently, suggesting unique associations with tissue-specific catalytic subunits. The murine brain (beta 1) and testis (beta 2) isoforms of the regulatory subunit were expressed with poly-histidine carboxyl termini and purified by Ni(2+)-chelate chromatography, each exhibiting high affinity Ca2+ binding on nitrocellulose overlays. Using chromatographic methods to assess complex formation, the beta 1 and beta 2 isoforms appeared indistinguishable in their binding to bacterially expressed forms of the murine brain (alpha 1) or testis (alpha 3) catalytic subunits; this suggests that multiple heterodimeric forms may be present in some tissues. Furthermore, both beta 1 and beta 2 formed complexes with the recombinant catalytic subunit from Neurospora crassa. For this catalytically active fungal enzyme, stoichiometric amounts of mammalian regulatory subunit increased calmodulin-dependent activity without affecting that stimulated by Mn2+ alone. Maximal hydrolysis of rho-nitrophenyl phosphate by the N. crassa catalytic subunit was stimulated 80-120% with the beta 1 isoform and 30-50% by beta 2. Significantly, the incubation time at 4 degrees C required for optimal activation (6-8 h) was much greater than that for association (1 h), indicating that the catalytic subunit undergoes a slow transition to an activated conformation after binding the regulatory subunit. The production of functional heterodimers of mammalian and fungal proteins implies highly conserved interaction domains on the catalytic and regulatory subunits of this phosphatase.
Ueki
K
K
Section on Immunology, National Institute on Alcohol Abuse and Alcoholism, Rockville, Maryland 20852.
Kincaid
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RL
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D019703:Calcineurin; D002148:Calmodulin-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004269:DNA, Bacterial; D004271:DNA, Fungal; D004789:Enzyme Activation; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009492:Neurospora crassa / Q000201:enzymology* / Q000235:genetics; D010749:Phosphoprotein Phosphatases / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism
D002148:Calmodulin-Binding Proteins; D004269:DNA, Bacterial; D004271:DNA, Fungal; D011994:Recombinant Proteins; D019703:Calcineurin; D010749:Phosphoprotein Phosphatases