INTERACTION OF CALMODULIN WITH A PUTATIVE CALMODULIN-BINDING DOMAIN OF INOSITOL 1,4,5-TRIPHOSPHATE 3-KINASE - EFFECTS OF SYNTHETIC PEPTIDESAND SITE-DIRECTED MUTAGENESIS OF TRP165

Recombinant rat brain inositol 1,4,5-triphosphate [Ins(1,4,5)P3] 3-kin ase was expressed in Escherichia coli as a beta-galactosidase fusion p roduct. It could be adsorbed onto calmodulin-Sepharose and eluted in C a2+-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) In hibition of Ca2+/calmodulin stimulation by a synthetic peptide based o n a candidate calmodulin-binding domain. The inhibition was mimicked b y a well-characterized peptide derived from the sequence of smooth mus cle myosin light-chain kinase calmodulin-binding site. (b) The constru ction of two mutants by site-directed mutagenesis of Trp165 to Gly or Arg. Both mutants displayed kinase activity but were no longer Ca2+/ca lmodulin sensitive, supporting, therefore, the role of Trp165 in calmo dulin binding.