Identification of novel isoforms of the delta subunit of Ca2+/calmodulin-dependent protein kinase II. Differential expression in rat brain and aorta

• Published in: The Journal of biological chemistry Vol. 268; no. 19; pp. 14443 - 14449
• Main Authors: SCHWORER, C. M, ROTHBLUM, L. I, THEKKUMKARA, T. J, SINGER, H. A
• Format: Journal Article
• Language: English
• Published: Bethesda, MD American Society for Biochemistry and Molecular Biology 05-07-1993
• Subjects: Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Animals; Aorta - enzymology; Base Sequence; Biological and medical sciences; Blotting, Southern; Blotting, Western; Brain - enzymology; Calcium-Calmodulin-Dependent Protein Kinases; Cells, Cultured; Conserved Sequence; DNA - genetics; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Gene Expression; Isoenzymes - biosynthesis; Isoenzymes - genetics; Isoenzymes - isolation & purification; Macromolecular Substances; Molecular Sequence Data; Muscle, Smooth, Vascular - enzymology; Mutagenesis; Oligodeoxyribonucleotides; Organ Specificity; Polymerase Chain Reaction; Protein Kinases - biosynthesis; Protein Kinases - genetics; Protein Kinases - isolation & purification; Rats; Sequence Deletion; Sequence Homology, Amino Acid; Sequence Homology, Nucleic Acid; Transferases; Alternative splicing; Vertebrata; Molecular form; Mammalia; Rat; Enzyme; Rodentia; Immunoblotting assay; Tissue specificity; Molecular cloning; Subunit
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/s0021-9258(19)85259-6

Two novel isoforms of the Ca2+/calmodulin-dependent protein kinase II delta subunit were detected in rat aorta. Identification
of the subunits was based on two independent lines of evidence, i.e. detection by immunoblotting of differently sized delta
subunits and DNA sequence analysis of partial cDNA clones of the kinase. Cytosolic extracts from rat brain, aorta, and cultured
aortic cells were analyzed by Western blotting using a delta subunit-specific antipeptide antibody. Aortic extracts demonstrated
a single 53-kDa cross-reactive band approximately 7 kDa smaller than the cross-reactive band seen in brain. To ascertain the
structural basis for this difference, reverse-transcribed RNAs from rat aorta and brain were analyzed by polymerase chain
reaction (PCR), and the PCR fragments were cloned and sequenced. When aortic cDNA was analyzed with a primer pair that spanned
the known variable region of the brain kinase subunit, the amplified PCR products were smaller than the major product obtained
from brain cDNA. The aortic PCR product was cloned and sequenced and found to represent two novel subunit sequences, designated
delta 2 and delta 3 to distinguish them from the previously described delta sequence (now called delta 1) from brain. delta
2 was identical to the predicted delta 1 sequence except for a deletion of 102 base pairs (bp). This deletion corresponded
to nearly the entire variable domain. In the sequence of delta 3, this 102-bp region was replaced by a sequence of 33 bp that
had 79% nucleotide sequence identity to a portion of the gamma subunit variable domain. A fourth form of the delta subunit
(delta 4) was identified in rat skeletal muscle. The delta 4 isoform was characterized by the deletion of a 42-bp sequence
identical to the 42 bp at the 3' end of the 102-bp deletion of delta 2. Reverse-transcription PCR analysis of additional rat
tissues indicated that alternatively spliced variants of the delta subunit of Ca2+/calmodulin-dependent protein kinase II
are expressed in a tissue-specific pattern.