Modulation of calcineurin phosphotyrosyl protein phosphatase activity by calmodulin and protease treatment

Biochem Biophys Res Commun. 1986 Oct 15;140(1):320-8. doi: 10.1016/0006-291x(86)91093-4.

Abstract

Calcineurin (CN) dephosphorylated [32P] phosphotyrosyl glutamine synthetase, a model phosphoprotein substrate containing approximately 1 mol of phosphotyrosine per mol subunit. Phosphatase activity with and without calmodulin (CaM) was greatly stimulated by Mn2+; with Ca2+, even in the presence of CaM, activity was very low. CaM-stimulated phosphatase activity exhibited deactivation with time; initial rates declined markedly after 2-3 min. The Michaelis constant for substrate (3 microM) was identical whether 2 or 12 min assays (with CaM) were used suggesting that the decreased rate of hydrolysis did not result from a decrease in affinity for the phosphoprotein substrate. Limited proteolysis of CN by chymotrypsin increased phosphatase activity 2-3 times that of CaM-supported activity; however, addition of CaM to assays with protease-activated CN reduced activity to that observed for non-proteolyzed enzyme. These data suggest that, in addition to stimulation, CaM can inhibit certain activated conformations of the phosphatase.

MeSH terms

  • Animals
  • Calcium / pharmacology
  • Calmodulin / pharmacology*
  • Calmodulin-Binding Proteins / metabolism*
  • Cattle
  • Chymotrypsin / pharmacology
  • In Vitro Techniques
  • Kinetics
  • Manganese / pharmacology
  • Peptide Hydrolases / pharmacology*
  • Phosphoprotein Phosphatases / analysis*
  • Protein Tyrosine Phosphatases

Substances

  • Calmodulin
  • Calmodulin-Binding Proteins
  • Manganese
  • Phosphoprotein Phosphatases
  • Protein Tyrosine Phosphatases
  • Peptide Hydrolases
  • Chymotrypsin
  • Calcium