Casein kinase I alpha and alpha L: alternative splicing-generated kinases exhibit different catalytic properties.
Casein kinase I (CKI) is a family of serine/threonine protein kinases found in all eukaryotes examined to date. Here, the rat CKI isoforms alpha and alpha L were cloned and expressed in both eukaryotic and prokaryotic systems. Characterization of the genomic DNA flanking the exon unique to CKI alpha L demonstrated that CKI alpha and CKI alpha L arise by the alternative splicing of a common pre-mRNA molecule. To the best of our knowledge, the alpha L isoform is the only known active serine/threonine kinase to contain an insert within its catalytic domain. Tissue distribution of each splicing isoform was examined by RT-PCR, immunoprecipitation, and Western blotting. Both isoforms were expressed in all tissues tested but at different levels. Bacterially expressed CKI alpha isoforms were active and therefore biochemically characterized. CKI alpha and CKI alpha L proteins were demonstrated to have casein kinase I catalytic properties. More importantly, the recombinant isoform proteins exhibited differences in binding and activity toward common CKI substrates. These observations demonstrate that the alpha L insert within the kinase domain modulates substrate kinetics. These kinetic differences suggest that CKI alpha and CKI alpha L may perform different biological roles.
Zhang
J
J
Department of Pharmacology, University of Wisconsin Medical School, Madison 53706, USA.
Gross
S D
SD
Schroeder
M D
MD
Anderson
R A
RA
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001921:Brain / Q000201:enzymology*; D047388:Casein Kinases; D002384:Catalysis; D002417:Cattle; D017931:DNA Primers; D005091:Exons; D005982:Glutathione Transferase / Q000096:biosynthesis; D007438:Introns; D007527:Isoenzymes / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011494:Protein Kinases / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D012322:RNA Precursors / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014158:Transcription, Genetic
D017931:DNA Primers; D007527:Isoenzymes; D012322:RNA Precursors; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D011494:Protein Kinases; D047388:Casein Kinases