Molecular cloning and characterization of novel isoforms of calmodulin-dependent protein kinase II

Multifunctional Ca{dollar}{bsol}sp{lcub}2+{rcub}{dollar} calmodulin dependent protein kinase II (CaMPKII) shows a wide tissue distribution and broad substrate specificity. It was originally isolated from rat brain, and five isoforms have been identified in neuronal tissues since then. Autophosphorylation of CaMPKII in the presence of Ca{dollar}{bsol}sp{lcub}2+{rcub}{dollar}/calmodulin converts the kinase itself to a Ca{dollar}{bsol}sp{lcub}2+{rcub}{dollar}-independent form. Most of the biochemical characterization of CaMPKII has been performed with {dollar}{bsol}alpha{dollar} and {dollar}{bsol}beta{dollar}-isoforms whose expression is limited to neuronal tissues. In smooth muscle, CaMPKII was first isolated as caldesmon kinase, which showed similar calmodulin affinity and substrate spectrum. To further address the structural and biochemical characteristics of smooth muscle CaMPKII, we have identified four novel CaMPKII isoforms ({dollar}{bsol}gamma{dollar}-b, {dollar}{bsol}gamma{dollar}-c, {dollar}{bsol}delta{dollar}-b, and {dollar}{bsol}delta{dollar}-c) from rat aorta cDNA library. The only difference in the nucleotide sequence of these variants from brain {dollar}{bsol}gamma{dollar}- and {dollar}{bsol}delta{dollar}-isoforms was centered in the variable region at the end of the calmodulin binding domain, strongly suggesting that they are alternatively spliced products of the {dollar}{bsol}gamma{dollar}- and {dollar}{bsol}delta{dollar}CaMPKII genes. We have also characterized chicken gizzard smooth muscle CaMPKII with particular focus on its autophosphorylation. The autophospho rylation of smooth muscle CaMPKII produced a partially constitutively active enzyme, as occurs with {dollar}{bsol}alpha{dollar}- and {dollar}{bsol}beta{dollar}-isoforms, but the autophosphorylation kinetics were significantly slower. The phosphorylation during the initial rapid phase coincided with the production of constitutively active enzyme, which is distinct from the brain enzyme where Thr phosphorylation is much faster and predominant than Ser phosphorylation. The identified major autophosphorylation sites were different from the known autophosphorylation sites of {dollar}{bsol}alpha{dollar}- and {dollar}{bsol}beta{dollar}-isoforms. By comparing the phosphopeptide sequence with the known sequences of various isoforms, we concluded that {dollar}{bsol}gamma{dollar}-b, which contains a unique insertion and two deletions at the C-terminal side of the calmodulin binding domain, is the dominant CaMPKII isoform in smooth muscle cell. The molecular mass of smooth muscle CaMPKII was estimated to be 240 kDa which would comprise four subunits, fewer than {dollar}{bsol}alpha{dollar}- and {dollar}{bsol}beta{dollar}-isoforms. To compare major smooth muscle isoforms with their brain counterparts in biochemical properties, we expressed two smooth muscle {dollar}{bsol}gamma{dollar}-variants ({dollar}{bsol}gamma{dollar}-b, {dollar}{bsol}gamma{dollar}-c) and brain {dollar}{bsol}gamma{dollar}-a using pET-E.coli expression system. All three {dollar}{bsol}gamma{dollar}-isoforms showed Ca{dollar}{bsol}sp{lcub}2+{rcub}{dollar}/calmodulin dependent kinase activity and were autophosphorylated in a Ca{dollar}{bsol}sp{lcub}2+{rcub}{dollar}/calmodulin dependent manner. {dollar}{bsol}gamma{dollar}-a showed fastest autophosphorylation kinetics, while {dollar}{bsol}gamma{dollar}-c had the slowest rate. Gel filtration analysis showed that all {dollar}{bsol}gamma{dollar}-variants ({dollar}{bsol}gamma{dollar}a, {dollar}{bsol}gamma{dollar}-b, {dollar}{bsol}gamma{dollar}-c) formed a multimeric structure, but with fewer subunits in {dollar}{bsol}gamma{dollar}-b. Mutagenesis of Thr-286 to Ala preserved Ca{dollar}{bsol}sp{lcub}2+{rcub}{dollar}/calmodulin-independent activity in {dollar}{bsol}gamma{dollar}-b. Estimated molecular mass of {dollar}{bsol}gamma{dollar}-a, {dollar}{bsol}gamma{dollar}-b, {dollar}{bsol}gamma{dollar}-c were 487 kDa (8-9mer), 258 kDa (4-5mer), and 407 kDa (7-8mer)