Catherine C. Allende

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

Protein kinase casein kinase 1 (CK1) phosphorylates Ser-45 of β-catenin, “priming” the subsequent phosphorylation by glycogen synthase-3 of residues 41, 37, and 33. This concerted phosphorylation of β-catenin signals its degradation and prevents its function in triggering cell division. The sequence around Ser-45 does not conform to the canonical consensus for CK1 substrates, which prescribes either phosphoamino acids or acidic residues in position n-3 from the target serine. However, the β-catenin sequence downstream from Ser-45 is very similar to a sequence recognized by CK1 in nuclear factor for activated T cells 4. The common features include an SLS motif followed two to five residues downstream by a cluster of acidic residues. Synthetic peptides reproducing residues 38-65 of β-catenin were assayed with purified rat liver CK1 or recombinant CK1α and CK1αL from zebrafish. The results demonstrate that SLS and acidic cluster motifs are crucial for CK1 recognition. Pro-44 and Pro-52 are also important for efficient phosphorylation. Similar results were obtained with the different isoforms of CK1. Phosphorylation of mutants of full-length recombinant β-catenin from zebrafish confirmed the importance of the SLS and acidic cluster motifs. A search for proteins with similar motifs yielded, among other proteins, adenomatous polyposis coli, previously found to be phosphorylated by CK1. There is a strong correlation of β-catenin mutations found in thyroid tumors with the motifs recognized by CK1 in this protein.

Promiscuous subunit interactions: A possible mechanism for the regulation of protein kinase CK2

Protein kinase CK2 is a ubiquitous eukaryotic ser/thr protein kinase. The active holoenzyme is a heterotetrameric protein composed of catalytic (α and α′) and regulatory (β) subunits that phosphorylates many different protein substrates and appears to be involved in the regulation of cell division. Despite important structural studies, the intimate details of the interactions of the α catalytic subunits with the β regulatory subunits are unknown. Recent evidence that indicates that both CK2 subunits can interact promiscuously with other proteins in a manner that excludes the binding of their complementary CK2 partners has opened the possibility that the phosphorylating activity of this enzyme may be regulated in a novel way. These alternative interactions could limit the in vivo availability of CK2 subunits to generate fully active holoenzyme CK2 tetramers. Likewise, variations in the ratio of α‐ and β‐subunits could determine the activity of several phosphorylating and dephosphorylating activities. The promiscuity of the CK2 subunits can be extrapolated to a more widespread phenomenon in which “wild‐card” proteins could act as general switches by interacting and regulating several catalytic activities. J. Cell. Biochem. Suppls. 30/31:129–136, 1998.

Oocyte adenylyl cyclase contains Ni, yet the guanine nucleotide-dependent inhibition by progesterone is not sensitive to pertussis toxin

The possible susceptibility to pertussis toxin of the guanine nucleotide‐dependent inhibition of oocyte adenylyl cyclase by progesterone was investigated. This action of progesterone is mediated by a membrane bound receptor as opposed to a receptor of cytosolic or nuclear localization. However, the inhibitory effect of progesterone was unaffected by pertussis toxin, even though the oocyte membrane Ni was fully ADP‐ribosylated with pertussis toxin, as revealed by lack of further [32P]ADP‐ribosylation on subsequent re‐incubation with pertussis toxin. These results indicate that the action of progesterone, in spite of being nucleotide‐dependent, is either not mediated by Ni, suggesting the existence of an additional nucleotide regulatory component, or if mediated by Ni, involves a mode of regulation of this coupling protein that is different from that by which all other inhibitory hormones act on adenylyl cyclase.

Cell cycle regulatory protein p27KIP1 is a substrate and interacts with the protein kinase CK2.

The protein kinase CK2 is constituted by two catalytic (α and/or α′) and two regulatory (β) subunits. CK2 phosphorylates more than 300 proteins with important functions in the cell cycle. This study has looked at the relation between CK2 and p27KIP1, which is a regulator of the cell cycle and a known inhibitor of cyclin‐dependent kinases (Cdk). We demonstrated that in vitro recombinant Xenopus laevis CK2 can phosphorylate recombinant human p27KIP1, but this phosphorylation occurs only in the presence of the regulatory β subunit. The principal site of phosphorylation is serine‐83. Analysis using pull down and surface plasmon resonance (SPR) techniques showed that p27KIP1 interacts with the β subunit through two domains present in the amino and carboxyl ends, while CD spectra showed that p27KIP1 phosphorylation by CK2 affects its secondary structure. Altogether, these results suggest that p27KIP1 phosphorylation by CK2 probably involves a docking event mediated by the CK2β subunit. The phosphorylation of p27KIP1 by CK2 may affect its biological activity.

The cDNAs coding for the α‐ and β‐subunits of Xenopus laevis casein kinase II

Using a λgt10 cDNA library obtained from Xenopus laevis oocytes and probes derived from the known sequences of the human and Drosophila genes, a cDNA coding for the α‐subunit of the X. laevis casein kinase II was isolated. The coding sequence of this clone determines a polypeptide of 350 amino acids. The X. laevis sequence is 98% identical to the human and rat proteins in the first 323 amino acids. Using the polymerase chain reaction to generate a 370‐nucleotide‐long probe, it was possible to clone and sequence a cDNA of 900 nucleotides that coded for the X. laevis β‐subunit of casein kinase II. The derived protein sequence is 215 amino acids long and again shows an extraordinary degree of conservation with other species.

Biochemical and cellular characteristics of the four splice variants of protein kinase CK1α 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 α, β, γ1, γ2, γ3, δ, and ε. The CK1α gene can generate four variants (CK1α, CK1αS, CK1αL, and CK1αLS) 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 CK1α 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 CK1α: (a) it increases the apparent Km for ATP twofold, from ∼30 to ∼60 μM; (b) it decreases the sensitivity to the CKI‐7 inhibitor, raising the I50 values from 113 to ∼230 μM; (c) it greatly decreases the heat stability of the enzyme at 40°C. In addition, the insertion of the “L” fragment exerts very important effects on some cellular properties of the enzyme. CK1αL concentrates in the cell nucleus, excluding nucleoli, while the CK1α 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 CK1α variant (half‐life of 400 min) in transfected Cos‐7 cells. J. Cell. Biochem. 86: 805–814, 2002.

Oncogenic ras protein induces meiotic maturation of amphibian oocytes in the presence of protein synthesis inhibitors

Microinjection of the activated ras oncogenic protein can induce the meiotic maturation of Xenopus laevis oocytes, a process that can also be triggered by progesterone or high concentrations of insulin. Cycloheximide and puromycin, well‐known inhibitors of protein synthesis, block the maturation process induced by progesterone and insulin but do not affect the maturation caused by H‐ras lys 12 protein microinjection. Theophylline, an inhibitor of cAMP phosphodiesterase that also affects oocyte protein synthesis, does cause a partial inhibition of ras protein‐induced maturation. These findings indicate that ras protein acts on the oocyte maturation process at a point that is downstream of the protein synthesis requirement, a characteristic shared with the maturation promoting factor, an activity that appears in oocytes and mitotic cells at the onset of cell division.

Effects of phosphorylation by protein kinase CK2 on the human basal components of the RNA polymerase II transcription machinery

We have investigated the role of phosphorylation by vertebrate protein kinase CK2 on the activity of the General Transcription Factors TFIIA, TFIIE, TFIIF, and RNAPII. The largest subunits of TFIIA, TFIIE, and TFIIF were phosphorylated by CK2 holoenzyme. Also, RNA polymerase II was phosphorylated by CK2 in the 214,000 and 20,500 daltons subunits. Our results show that phosphorylation of TFIIA, TFIIF, and RNAPII increase the formation of complexes on the TATA box of the Ad‐MLP promoter. Also, phosphorylation of TFIIF increases the formation of transcripts, where as phosphorylation of RNA polymerase II dramatically inhibits transcript formation. Furthermore, we demonstrate that CK2β directly interacts with RNA polymerase II, TFIIA, TFIIF, and TBP. These results strongly suggest that CK2 may play a role in regulating transcription of protein coding genes.

Cloning and chromosomal localization of the gene coding for human protein kinase CK1

A cDNA clone coding for human protein kinase CK1 (casein kinase 1) has been isolated and sequenced demonstrating that it corresponds to a homolog of the CK1α form found in bovine brain. The derived amino acid sequence of the human CK1α is identical to the bovine counterpart except that it contains 12 extra amino acids at the carboxyl end. Using this cDNA sequence and PCR amplification, YAC genomic clones that contain this human CK1α sequence have been isolated. These YACs have been used for fluorescent in situ hybridization in order to localize the human CK1α gene to chromosome 13q13.

Effect of metal ions on the activity of cascein kinase II from Xenopus laevis

CaSein kinase II purified from the nuclei of Xenopus laevis oocytes as well as the recombinant α and β subunits of the X. laevis CKII, produced in E. coli from the cloned cDNA genes, were tested with different divalent metal ions. The enzyme from both sources was active with either Mg2+, Mn2+, or Co2+. Optimal concentrations were 7–10 mM for Mg2+, 0.5–0.7 mM for Mn2+ and 1–2 mM for Co2+. In the presence of Mn2+ or Co2+ the enzyme used GTP more efficiently than ATP as a phosphate donor while the reverse was true in the presence ofMg2+. The apparent K m values for both nucleotide triphosphates were greatly decreased in the presence of Mn2+ as compared with Mg2+. Addition of Zn2+ (above 150 μM) to an assay containing the optimal Mg2+ ion concentration caused strong inhibition of both holoenzyme and α subunit. Inhibition of the holoenzyme by 400 μM Ni2+ could be reversed by high concentrations of Mg2+ but no reversal of this inhibition was observed with the α subunit.