Interaction of calmodulin with a putative calmodulin-binding domain of inositol 1,4,5-triphosphate 3-kinase. Effects of synthetic peptides and site-directed mutagenesis of Trp165.
Recombinant rat brain inositol 1,4,5-triphosphate [Ins(1,4,5)P3] 3-kinase was expressed in Escherichia coli as a beta-galactosidase fusion product. It could be adsorbed onto calmodulin-Sepharose and eluted in Ca(2+)-free medium as a 48-kDa protein. Purification could be achieved in a single step. Molecular evidence for a calmodulin-binding domain on Ins(1,4,5)P3 3-kinase can be shown by the following approaches. (a) Inhibition of Ca2+/calmodulin stimulation by a synthetic peptide based on a candidate calmodulin-binding domain. The inhibition was mimicked by a well-characterized peptide derived from the sequence of smooth muscle myosin light-chain kinase calmodulin-binding site. (b) The construction of two mutants by site-directed mutagenesis of Trp165 to Gly or Arg. Both mutants displayed kinase activity but were no longer Ca2+/calmodulin sensitive, supporting, therefore, the role of Trp165 in calmodulin binding.
Erneux
C
C
Institut de Recherche Interdisciplinaire (IRIBHN), Université Libre de Bruxelles, Belgium.
Moreau
C
C
Vandermeers
A
A
Takazawa
K
K
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001921:Brain / Q000201:enzymology; D002118:Calcium / Q000494:pharmacology; D002147:Calmodulin / Q000378:metabolism / Q000494:pharmacology*; D004926:Escherichia coli; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010770:Phosphotransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017853:Phosphotransferases (Alcohol Group Acceptor); D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D014364:Tryptophan / Q000235:genetics*; D001616:beta-Galactosidase
D002147:Calmodulin; D011993:Recombinant Fusion Proteins; D014364:Tryptophan; D010770:Phosphotransferases; D017853:Phosphotransferases (Alcohol Group Acceptor); C051038:Inositol 1,4,5-trisphosphate 3-kinase; D001616:beta-Galactosidase; D002118:Calcium