Biochemical and cellular characteristics of the four splice variants of protein kinase CK1α from zebrafish (Danio rerio)

Abstract

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.

The first armadillo repeat is involved in the recognition and regulation of β-catenin phosphorylation by protein kinase CK1

“…CK1␣ from zebrafish with N-terminal His tags was expressed in Escherichia coli BL21(DE3) and purified on Ni 2ϩ -nitrilotriacetic acid-agarose as described in ref. 5. GST fusion proteins were purified by using glutathione-agarose beads and elution with 20 mM glutathione dissolved in PBS.…”

“…The assay has been described in detail in ref. 5. Brief ly, a range of concentrations of substrate were phosphorylated by incubation in a 30-l volume containing 50 mM Tris⅐HCl buffer (pH 7.5), 10 mM MgCl 2 , 100 mM NaCl, and 100 M [␥-32 P]ATP (specific radioactivity, 500 -1,000 cpm/pmol).…”

“…In vertebrates, there are genes encoding for seven CK1 isoforms (␣, ␤, ␥1, ␥2, ␥3, ␦, and ), which also generate different proteins through alternative splicing (2)(3)(4)(5). All CK1 isoforms display a high degree of sequence identity within their kinase domains (Ͼ50%), but they differ significantly in their N-and C-terminal extensions.…”

A noncanonical sequence phosphorylated by casein kinase 1 in β-catenin may play a role in casein kinase 1 targeting of important signaling proteins

“…Recombinant CK1␣ and CK1␣L from zebrafish (Danio rerio) with amino-terminal (His) 6 tags were expressed in Escherichia coli BL21 (DE3) and purified through Ni 2ϩ -NTA-agarose columns as described (15).…”

“…Within the CK1 family, there are several genes that code for related kinases in all species. In mammals and vertebrates, there are seven CK1 genes coding for ␣, ␤, ␥1, ␥2, ␥3, ␦, and isoforms (14), and some of these genes generate several proteins through alternative splicing (15). The CK1 isoforms may have different functions; for instance, CK1 is involved in regulating circadian rhythm through phosphoration of period (per) proteins (16), and it has also been linked to the activity of group I metabotropic glutamate receptors (17).…”