Cloning and expression in Escherichia coli of a rat brain cDNA encoding a Ca2+/calmodulin-sensitive inositol 1,4,5-trisphosphate 3-kinase

Biochem J. 1990 Nov 15;272(1):107-12. doi: 10.1042/bj2720107.


Inositol 1,4,5-trisphosphate (InsP3) 3-kinase catalyses the phosphorylation of InsP3 to inositol 1,3,4,5-tetrakisphosphate (InsP4). InsP3 3-kinase activity was stimulated by Ca2+ in the presence of calmodulin (CaM) and the protein was associated with two silver-stained bands which migrated with an apparent Mr of approx. 50,000 on SDS/polyacrylamide gels. Upon limited proteolysis with trypsin, the native InsP3 3-kinase was converted into polypeptides of Mr 44,000 and 36,000. Both tryptic fragments displayed InsP3 3-kinase activity that was Ca2+/CaM-sensitive. A cDNA clone, C5, that encodes the C-terminal part of the InsP3 3-kinase, was isolated by immunoscreening of a rat brain cDNA library. The 5' end of this clone was used in turn to probe the same library, yielding a clone (CP16) containing the entire coding sequence of InsP3 3-kinase. The encoding protein of 459 amino acids (calculated Mr 50,868) has several putative phosphorylation sites for cyclic AMP-dependent protein kinase, protein kinase C and CaM-dependent protein kinase II. When clone C5 was expressed in Escherichia coli, the truncated fusion protein showed Ca2+/CaM-sensitive InsP3 3-kinase activity. Our data demonstrate that the N-terminal part of the protein is not essential for either enzymic or CaM-regulatory properties.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • Blotting, Northern
  • Brain / enzymology*
  • Calcium / pharmacology
  • Calmodulin / pharmacology
  • Chromatography, Affinity
  • Cloning, Molecular
  • DNA / genetics*
  • Escherichia coli / genetics*
  • Gene Library
  • Kinetics
  • Molecular Sequence Data
  • Molecular Weight
  • Phosphotransferases (Alcohol Group Acceptor)*
  • Phosphotransferases / genetics*
  • Phosphotransferases / isolation & purification
  • Phosphotransferases / metabolism
  • Rats
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism


  • Calmodulin
  • Recombinant Proteins
  • DNA
  • Phosphotransferases
  • Phosphotransferases (Alcohol Group Acceptor)
  • Inositol 1,4,5-trisphosphate 3-kinase
  • Calcium