Ana Jedlicki

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.

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.

Activity of the E75E76 mutant of the α subunit of casein kinase II from Xenopus laevis

The cDNA gene coding for the α subunit of Xenopus laevis casein kinase II was mutated using the overlap extension PCR method. The mutation substituted glutamic acids for Lys75 and Lys76, changing the charge distribution of a very basic sequence found in the α subunit. Expression of the mutated cDNA in a pT7‐7 vector in E. coli yielded an active mutant recombinant protein that was extensively purified. This mutant was not significantly affected in its app. K m for casein or a model peptide substrate, nor in its interaction with the activating β subunit. Inhibition by quercetin and by 5,6‐dichloro‐1‐β‐d‐ribofuranosyl benzimidazole was also the same for mutant and wild type subunits. However, the CKII αE75E76 mutant was at least one order of magnitude less sensitive to inhibition by polyanionic inhibitors such as heparin, poly U, copolyglutamic acid:tyrosine (4:1) and 2,3 diphosphoglycerate.

Autophosphorylation of carboxy‐terminal residues inhibits the activity of protein kinase CK1α

CK1 constitutes a protein kinase subfamily that is involved in many important physiological processes. However, there is limited knowledge about mechanisms that regulate their activity. Isoforms CK1δ and CK1ε were previously shown to autophosphorylate carboxy‐terminal sites, a process which effectively inhibits their catalytic activity. Mass spectrometry of CK1α and splice variant CK1αL has identified the autophosphorylation of the last four carboxyl‐end serines and threonines and also for CK1αS, the same four residues plus threonine‐327 and serine‐332 of the S insert. Autophosphorylation occurs while the recombinant proteins are expressed in Escherichia coli. Mutation of four carboxy‐terminal phosphorylation sites of CK1α to alanine demonstrates that these residues are the principal but not unique sites of autophosphorylation. Treatment of autophosphorylated CK1α and CK1αS with λ phosphatase causes an activation of 80–100% and 300%, respectively. Similar treatment fails to stimulate the CK1α mutants lacking autophosphorylation sites. Incubation of dephosphorylated enzymes with ATP to allow renewed autophosphorylation causes significant inhibition of CK1α and CK1αS. The substrate for these studies was a synthetic canonical peptide for CK1 (RRKDLHDDEEDEAMS*ITA). The stimulation of activity seen upon dephosphorylation of CK1α and CK1αS was also observed using the known CK1 protein substrates DARPP‐32, β‐catenin, and CK2β, which have different CK1 recognition sequences. Autophosphorylation effects on CK1α activity are not due to changes in Kmapp for ATP or for peptide substrate but rather to the catalytic efficiency per pmol of enzyme. This work demonstrates that CK1α and its splice variants can be regulated by their autophosphorylation status. J. Cell. Biochem. 106: 399–408, 2009.

Basic region of residues 228-231 of protein kinase CK1α is involved in its interaction with axin: Binding to axin does not affect the kinase activity

Protein kinase CK1, also known as casein kinase 1, participates in the phosphorylation of β‐catenin, which regulates the functioning of the Wnt signaling cascade involved in embryogenesis and carcinogenesis. β‐catenin phosphorylation occurs in a multiprotein complex assembled on the scaffold protein axin. The interaction of CK1α from Danio rerio with mouse‐axin has been studied using a pull‐down assay that uses fragments of axin fused to glutathione S transferase, which is bound to glutathione sepharose beads. The results indicate that the three lysines present in the basic region of residues 228–231 of CK1α are necessary for the binding of CK1 to axin. Lysine 231 is particularly important in this interaction. In order to define the relevance of the axin‐CK1α interaction, the effect of the presence of axin on the phosphorylating activity of CK1α was tested. It is also evident that the region of axin downstream of residues 503–562 is required for CK1α interaction. The binding of CK1α to axin fragment 292–681 does not facilitate the phosphorylation of β‐catenin despite the fact that this axin fragment can also bind β‐catenin. Binding of CK1α to axin is not required for the phosphorylation of axin itself and, likewise, axin does not affect the kinetic parameters of the CK1α towards casein or a specific peptide substrate.

Assessing footprints of selection in commercial Atlantic salmon populations using microsatellite data

Relatively large rates of response to traits of economic importance have been observed in different selection experiments in salmon. Several QTL have been mapped in the salmon genome, explaining unprecedented levels of phenotypic variation. Owing to the relatively large selection intensity, individual loci may be indirectly selected, leaving molecular footprints of selection, together with increased inbreeding, as its likely relatives will share the selected loci. We used population differentiation and levels of linkage disequilibrium in chromosomes known to be harbouring QTL for body weight, infectious pancreatic necrosis resistance and infectious salmon anaemia resistance to assess the recent selection history at the genomic level in Atlantic salmon. The results clearly suggest that the marker SSA0343BSFU on chromosome 3 (body weight QTL) showed strong evidence of directional selection. It is intriguing that this marker is physically mapped to a region near the coding sequence of DVL2 , making it an ideal candidate gene to explain the rapid evolutionary response of this chromosome to selection for growth in Salmo salar. Weak evidence of diversifying selection was observed in the QTL associated with infectious pancreatic necrosis and infectious salmon anaemia resistance. Overall, this study showed that artificial selection has produced important changes in the Atlantic salmon genome, validating QTL in commercial salmon populations used for production purposes according to the recent selection history.

Role of sulfatide on phosphoenzyme formation and ouabain binding of the (Na+ + K+) ATPase☆

A microsomal fraction rich in (Na+ + K+)ATPase activity has been isolated from the outer medulla of pig kidney. The ability of this preparation to form phosphoenzyme on incubation with [gamma-32P]ATP and to bind [3H]ouabain was studied when its sulfatide was hydrolyzed by arylsulfatase treatment. The K+-dependent hydrolysis of the Na+-dependent phosphorylated intermediate as well as the ouabain binding were inactivated in direct relation to the breakdown of sulfatide. Both characteristics of the (Na+ + K+)ATPase preparation, lost by arylsulfatase treatment, were partially restored by the sole addition of sulfatide. These experiments indicate that sulfatide may play a role in sodium ion transport either in the conformational transition of the K+-insensitive phosphointermediate, E1P, to the K+-sensitive intermediate, E2P, or in the configuration of the high-affinity binding site for K+ of the E2P form. In addition, this glycolipid may have a specific role in the proteolipidic subunit that binds ouabain.

Molecular detection and species identification of Alexandrium (Dinophyceae) causing harmful algal blooms along the Chilean coastline.

Misleading morphological observations assigned Alexandrium catenella as local dinoflagellate responsible for HABs in Southern Chilean coasts. Our work based on molecular methods found that local Alexandrium belongs to group I of the tamarensis complex composed mainly of A. tamarense.

Genomic Predictions and Genome-Wide Association Study of Resistance Against Piscirickettsia salmonis in Coho Salmon (Oncorhynchus kisutch) Using ddRAD Sequencing

Piscirickettsia salmonis is one of the main infectious diseases affecting coho salmon (Oncorhynchus kisutch) farming, and current treatments have been ineffective for the control of this disease. Genetic improvement for P. salmonis resistance has been proposed as a feasible alternative for the control of this infectious disease in farmed fish. Genotyping by sequencing (GBS) strategies allow genotyping of hundreds of individuals with thousands of single nucleotide polymorphisms (SNPs), which can be used to perform genome wide association studies (GWAS) and predict genetic values using genome-wide information. We used double-digest restriction-site associated DNA (ddRAD) sequencing to dissect the genetic architecture of resistance against P. salmonis in a farmed coho salmon population and to identify molecular markers associated with the trait. We also evaluated genomic selection (GS) models in order to determine the potential to accelerate the genetic improvement of this trait by means of using genome-wide molecular information. A total of 764 individuals from 33 full-sib families (17 highly resistant and 16 highly susceptible) were experimentally challenged against P. salmonis and their genotypes were assayed using ddRAD sequencing. A total of 9,389 SNPs markers were identified in the population. These markers were used to test genomic selection models and compare different GWAS methodologies for resistance measured as day of death (DD) and binary survival (BIN). Genomic selection models showed higher accuracies than the traditional pedigree-based best linear unbiased prediction (PBLUP) method, for both DD and BIN. The models showed an improvement of up to 95% and 155% respectively over PBLUP. One SNP related with B-cell development was identified as a potential functional candidate associated with resistance to P. salmonis defined as DD.

CK2α/CK1α chimeras are sensitive to regulation by the CK2β subunit

The effect of CK2β on the activity of CK2α and other protein kinases that can bind this regulatory subunit is not fully understood. In an attempt to improve our understanding of this effect, chimeras of CK2α and CK1α have been constructed. These chimeras contain different portions of the CK2α amino terminal region that are involved in the interaction with CK2β to form CK2 tetramers. In the case of chimeras 1 and 2, the portions of CK2α replace the corresponding segments of CK1α. In the case of chimera 3, the fragment of CK2α is added to the whole CK1α molecule with the exception of the initial methionine. Chimera 3 has 8% of the activity of CK1αWT, while chimeras 1 and 2 are 3 orders of magnitude less active than CK1αWT. All three chimeras bind tightly to CK2β, but only chimeras 1 and 2 are significantly stimulated in their capacity to phosphorylate casein and canonical peptide substrates by addition of the regulatory subunit. No stimulation was observed with phosvitin or non-canonical peptides derived from β-catenin. CK2β protects chimeras 1 and 2 from thermal inactivation. Chimera 2 can phosphorylate CK2β and autophosphorylate; however, salt concentrations above 150 mM NaCl eliminate the phosphorylation of CK2β but not the autophosphorylation of chimera 2. Similarly, high salt decrease the stimulatory effect of CK2β on the phosphorylation of casein.