Functional implications of the subunit composition of neuronal CaM kinase II

Abstract

The assembly of 6-12 subunits of Ca2+/calmodulin-dependent kinase II (CaM kinase II) into holoenzymes is an important structural feature of the enzyme and its postulated role as a molecular detector of Ca2+ oscillations. Using single cell reverse transcriptase-polymerase chain reaction, we show that α- and β-CaM kinase II mRNAs are simultaneously present in the majority of hippocampal neurons examined and that co-assembly of their protein products into heteromers is therefore possible. The subunit composition of CaM kinase II holoenzymes was analyzed by immunoprecipitation with subunit-specific monoclonal antibodies. Rat forebrain CaM kinase II consists of heteromers composed of α and β subunits at a ratio of 2:1 and homomers composed of only α subunits. We examined the functional effect of the heteromeric assembly by analyzing the calmodulin dependence of autophosphorylation. Recombinant homomers of α- or β-CaM kinase II, as well as of alternatively spliced β isoforms, have distinct calmodulin dependences for autophosphorylation based on differences in their calmodulin affinities. Half-maximal autophosphorylation of α is achieved at 130 nM calmodulin, while that for β occurs at 15 nM calmodulin. In CaM kinase II isolated from rat forebrain, however, the calmodulin dependence for autophosphorylation of the β subunits is shifted toward that of α homomers. This suggests that Thr287 in β subunits is phosphorylated by subunits present in the same holoenzyme. Once autophosphorylated, β-CaM kinase II traps calmodulin by reducing the rate of calmodulin dissociation.