Biochemical and cellular characteristics of the four splice variants of protein kinase CK1alpha from zebrafish (Danio rerio).
Protein kinase CK1 (previously known as casein kinase I) conforms to a subgroup of the great protein kinase family found in eukaryotic organisms. The CK1 subgroup of vertebrates contains seven members known as alpha, beta, gamma1, gamma2, gamma3, delta, and epsilon. The CK1alpha gene can generate four variants (CK1alpha, CK1alphaS, CK1alphaL, and CK1alphaLS) through alternate splicing, characterized by the presence or absence of two additional coding sequences. Exon "L" encodes a 28-amino acid stretch that is inserted after lysine 152, in the center of the catalytic domain. The "S" insert encodes 12 amino acid residues and is located close to the carboxyl terminus of the protein. This work reports some biochemical and cellular properties of the four CK1alpha variants found to be expressed in zebrafish (Danio rerio). The results obtained indicate that the presence of the "L" insert affects several biochemical properties of CK1alpha: (a) it increases the apparent Km for ATP twofold, from approximately 30 to approximately 60 microM; (b) it decreases the sensitivity to the CKI-7 inhibitor, raising the I50 values from 113 to approximately 230 microM; (c) it greatly decreases the heat stability of the enzyme at 40 degrees C. In addition, the insertion of the "L" fragment exerts very important effects on some cellular properties of the enzyme. CK1alphaL concentrates in the cell nucleus, excluding nucleoli, while the CK1alpha variant is predominantly cytoplasmic, although some presence is observed in the nucleus. This finding supports the thesis that the basic-rich region found in the "L" insert acts as a nuclear localization signal. The "L" insert-containing variant was also found to be more rapidly degraded (half-life of 100 min) than the CK1alpha variant (half-life of 400 min) in transfected Cos-7 cells.
Burzio
Veronica
V
Programa de Biología Celular y Molecular, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Casilla 70086, Santiago 7, Chile.
Antonelli
Marcelo
M
Allende
Catherine C
CC
Allende
Jorge E
JE
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D047388:Casein Kinases; D003001:Cloning, Molecular; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005091:Exons; D005455:Fluorescent Antibody Technique; D007438:Introns; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D010455:Peptides / Q000494:pharmacology; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013379:Substrate Specificity; D014162:Transfection; D015027:Zebrafish / Q000378:metabolism*
D004791:Enzyme Inhibitors; D007527:Isoenzymes; D010455:Peptides; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases; D047388:Casein Kinases