Novel Ca2+/Calmodulin-dependent Protein Kinase II γ-Subunit Variants Expressed in Vascular Smooth Muscle, Brain, and Cardiomyocytes

Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) γ-subunits were cloned from a porcine aortic smooth muscle cDNA library resulting in identification of alternatively spliced CaM kinase II γB- and γC-subunits and a novel γ-subunit variant predicted to encode a 60.2-kDa polypeptide, which was designated the γG-subunit. A clone predicted to encode a 62.2-kDa γ-subunit, designated as γE, was isolated with a variable domain structure similar to a γB-subunit but with a 114-nucleotide insertion in the conserved “association” domain of CaM kinase II subunits. A full-length γE-subunit construct expressed in COS cells resulted in multimeric CaM kinase II holoenzymes (470 kDa) with activation and autoregulatory properties similar to expressed holoenzymes composed of γB-, γC-, or γG-subunits. Expression of γE and related γ-subunit mRNAs containing the 114-base insertion was documented in porcine tissues by reverse transcriptase-polymerase chain reaction. CaM kinase II subunits containing the 38-amino acid insert were identified by Western analysis of partially purified CaM kinase II from carotid arterial smooth muscle and brain using a sequence-specific anti-peptide antibody. Immunoprecipitations of tissue homogenates indicated a comparatively high level of expression of subunits containing the insert in brain and provided evidence for their co-assembly with other more abundant subunits into CaM kinase II heteromultimers. Our analyses indicate the following patterns of γ-subunit expression: vascular smooth muscle, γB > γC > γE,G; heart, γB > γE,C > γG; brain, γE and related subunits > γA,B,C,G. Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) γ-subunits were cloned from a porcine aortic smooth muscle cDNA library resulting in identification of alternatively spliced CaM kinase II γB- and γC-subunits and a novel γ-subunit variant predicted to encode a 60.2-kDa polypeptide, which was designated the γG-subunit. A clone predicted to encode a 62.2-kDa γ-subunit, designated as γE, was isolated with a variable domain structure similar to a γB-subunit but with a 114-nucleotide insertion in the conserved “association” domain of CaM kinase II subunits. A full-length γE-subunit construct expressed in COS cells resulted in multimeric CaM kinase II holoenzymes (470 kDa) with activation and autoregulatory properties similar to expressed holoenzymes composed of γB-, γC-, or γG-subunits. Expression of γE and related γ-subunit mRNAs containing the 114-base insertion was documented in porcine tissues by reverse transcriptase-polymerase chain reaction. CaM kinase II subunits containing the 38-amino acid insert were identified by Western analysis of partially purified CaM kinase II from carotid arterial smooth muscle and brain using a sequence-specific anti-peptide antibody. Immunoprecipitations of tissue homogenates indicated a comparatively high level of expression of subunits containing the insert in brain and provided evidence for their co-assembly with other more abundant subunits into CaM kinase II heteromultimers. Our analyses indicate the following patterns of γ-subunit expression: vascular smooth muscle, γB > γC > γE,G; heart, γB > γE,C > γG; brain, γE and related subunits > γA,B,C,G.