Substrate-directed function of calmodulin in autophosphorylation of Ca2+/calmodulin-dependent protein kinase II.

Autophosphorylation of Thr286 in Ca2+/calmodulin-dependent protein kinase II occurs within each holoenzyme by an intersubunit reaction and is essential for kinase function in vivo. In addition to a kinase-directed function of calmodulin to activate the kinase, a second calmodulin is required for the autophosphorylation of each Thr286 (Hanson, P. I., Meyer, T., Stryer, L., and Schulman, H. (1994) Neuron 12, 943-956). We have engineered heteromeric holoenzymes comprising distinct "kinase" and "substrate" subunits to test for kinase- and substrate-directed functions of calmodulin. The obligate kinase subunits have aspartate residues substituted for threonine at positions 286, 305, and 306 (the autophosphorylation and calmodulin-binding sites), making it constitutively active but unable to bind calmodulin. Obligate substrate subunits are catalytically inactive (K42M mutation) but are able to bind calmodulin. Phosphorylation of substrate subunits occurs specifically at Thr286 and is completely dependent upon the presence of calmodulin. Blocking the ability of the substrate subunit to bind calmodulin, either with inhibitor KN-93 or by mutagenesis of the calmodulin-binding domain of the substrate subunit, prevents its phosphorylation, consistent with a substrate-directed function of calmodulin that requires its direct binding to the subunit being phosphorylated.