Jorge E. 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.

p53 Codon 72 Polymorphism and Risk of Cervical Cancer

Storey et al. (1998) implicated the proline/argine polymorphism of the codon 72 of the tumor-suppressor gene p53 in the development of cervical cancer (CC) with the observation that the p53 protein is more efficiently inactivated by the E6 oncoprotein of human papillomavirus in p53 arginine as compared with its proline isoform. These authors further noted that in the United Kingdom, individuals homozygous for the arginine allele were several times more susceptible to HPV-associated tumorigenesis that proline/arginine heterozygotes. Subsequent studies in different countries failed to unanimously confirm this association. Motivated by the high incidence of CC in Chile, we undertook a case control study obtaining the following frequencies for genotypes PP, AP and AA in 60 ICC cases and 53 carefully selected controls: 0.067, 0.250, 0.683 and 0.075, 0.453, 0.472 respectively. A significant difference (X2 = 3.19 p < 0.02) and an odds ratio of 2.62 supported Storey et al (1998)s results. In addition, rejecting previous hypotheses about the world distribution of the p53 codon 72 polymorphism, we conclude that this distribution most likely represents ancient human dispersal routes. Several methodological and biological explanations for the results obtained in previous negative association studies are briefly discussed.

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.

Molecular cloning and sequence determination of a cDNA coding for the α-subunit of a Go-type protein of Xenopus laevis oocytes

Xenopus laevis oocytes are cells ideally suited to the study of signal transduction and of the G‐proteins that are involved in this process. A X. laevis cDNA library in λgt10 has been screened with a mixture of three oligonucleotide probes designed to detect sequences found in various mammalian α‐subunits of G‐proteins. One of these clones has been purified through tertiary screening and the DNA insert has been sequenced. This clone was found to include the total sequence coding for a 354 amino acid protein that is 89% identical to the sequence of α‐subunit of rat Go. The differences with the mammalian protein were clustered in amino acids 290–315, which have been postulated to define the region interacting with the receptor and effector molecule. The homology with the α‐subunits of other mammalian G‐proteins is lower (65–70% to Gi and 42% to Gs). On this basis, this clone can be classified as Go‐like.

Molecular cloning and sequence determination of four different cDNA species coding for α-subunits of G proteins from Xenopus laevis oocytes

A cDNA library preprared from Xenopus laevis oocytes in λgt10 was screened with a mixture of three oligonucleotide probes designed to detect sequences found in different mammalian genes coding for a‐subunits of G‐proteins. In addition to a clone coding for a Gαo‐type subunit previously reported [(1989) FEBS Lett. 244, 188‐192] four additional clones have been found coding for different Gα protein subunits. By comparison with mammalian α‐subunits, these oocyte cDNAs correspond to two closely related Gas‐la, to a Gαi‐1 and to a Gαi‐3 species. The derived amino acid sequences showed that both Gαs species contain 379 residues, corresponding to the short species without the serine residue and with a calculated M r of 42720. The Gαi‐1 gene encodes a 354 amino acid protein with an M r, of 39000 and the Gαi‐3 encodes an incomplete open reading frame of 345 residues, lacking the first 9 amino acid residues at the NH2, terminus. All these Gα‐subunits showed high identity with their respective mammalian counterparts (75–80%), indicating a great degree of conservation through the evolution and the important cellular regulatory function that they play.

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.

Induction of amphibian oocyte maturation by polyvalent cations and alkaline pH in the absence of potassium ions

Abstract The maturation of Xenopus laevis oocytes was studied in media free of added potassium salts. Under these conditions maturation could be triggered by 1 mM Mn2+ and La3+ and, to a lesser extent, by 2–4 mM Ca2+ and Mg2+. Maturation induced by 1.5 mM Mn2+ was inhibited by K+ concentrations above 0.25 mM. Potassium was inhibitory when added up to 2 hr before germinal vesicle breakdown occurred. In potassium-free media, maturation could be induced by incubation of oocytes under mild alkaline media (pH 8.5–9). A high percentage of medium-sized oocytes (stage IV according to Dumont) was induced to mature by progesterone in the absence of potassium. Maturation of oocytes in potassium-free media was normal by the criteria of germinal vesicle breakdown, production of maturation promoting factor, vitelline membrane activation, and inhibition by known maturation inhibitors.

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

Multiple phosphorylation of β-catenin by glycogen synthase kinase 3 (GSK3) in the Wnt pathway is primed by CK1 through phosphorylation of Ser-45, which lacks a typical CK1 canonical sequence. Synthetic peptides encompassing amino acids 38–64 of β-catenin are phosphorylated by CK1 on Ser-45 with low affinity (Km ≈1 mM), whereas intact β-catenin is phosphorylated at Ser-45 with very high affinity (Km ≈200 nM). Peptides extended to include a putative CK1 docking motif (FXXXF) at 70–74 positions or a F74AA mutation in full-length β-catenin had no significant effect on CK1 phosphorylation efficiency. β-Catenin C-terminal deletion mutants up to residue 181 maintained their high affinity, whereas removal of the 131–181 fragment, corresponding to the first armadillo repeat, was deleterious, resulting in a 50-fold increase in Km value. Implication of the first armadillo repeat in β-catenin targeting by CK1 is supported in that the Y142E mutation, which mimics phosphorylation of Tyr-142 by tyrosine kinases and promotes dissociation of β-catenin from α-catenin, further improves CK1 phosphorylation efficiency, lowering the Km value to <50 nM, approximating the physiological concentration of β-catenin. In contrast, α-catenin, which interacts with the N-terminal region of β-catenin, prevents Ser-45 phosphorylation of CK1 in a dose-dependent manner. Our data show that the integrity of the N-terminal region and the first armadillo repeat are necessary and sufficient for high-affinity phosphorylation by CK1 of Ser-45. They also suggest that β-catenin association with α-catenin and β-catenin phosphorylation by CK1 at Ser-45 are mutually exclusive.