SUBSTRATE-DIRECTED FUNCTION OF CALMODULIN IN AUTOPHOSPHORYLATION OF CA2+ CALMODULIN-DEPENDENT PROTEIN-KINASE-II/

Autophosphorylation of Thr(286) in Ca2+/calmodulin-dependent protein k inase II occurs within each holoenzyme by an intersubunit reaction and is essential for kinase function in vivo, In addition to a kinase-dir ected function of calmodulin to activate the kinase, a second calmodul in is required for the autophosphorylation of each Thr(286) (Hanson, P . I., Meyer, T., Stryer, L., and Schulman, H. (1994) Neuron 12, 943-95 6), We have engineered heteromeric holoenzymes comprising distinct ''k inase'' and ''substrate'' subunits to test for kinase- and substrate-d irected functions of calmodulin, The obligate kinase subunits have asp artate residues substituted for threonine at positions 286, 305, and 3 06 (the autophosphorylation and calmodulin-binding sites), making it c onstitutively active but unable to bind calmodulin, Obligate substrate subunits are catalytically inactive (K42M mutation) but are able to b ind calmodulin, Phosphorylation of substrate subunits occurs specifica lly at Thr(286) and is completely dependent upon the presence of calmo dulin, Blocking the ability of the substrate subunit to bind calmoduli n, either with inhibitor KN-93 or by mutagenesis of the calmodulin-bin ding domain of the substrate subunit, prevents its phosphorylation, co nsistent with a substrate-directed function of calmodulin that require s its direct binding to the subunit being phosphorylated.