Phosphorylation of Delta Sleep-Inducing Peptide (DSIP) by casein kinase II in vitro

A phosphorylated analogue of DSIP at Ser⁷ has been shown to exist endogenously by immunochemical studies. An enzyme which could phosphorylate DSIP has not yet been identified. In the present study, we examined DSIP as a substrate for in vitro phosphorylation by casein kinase II. DSIP was phosphorylated by the enzyme with apparent K_m and V_max values of 20 mM and 90.9 nmol/min/mg protein, respectively. Both ATP and GTP were utilized as phosphoryl donors. Phosphorylation of DSIP was inhibited by heparin and enhanced by spermine. These results demonstrate that DSIP can serve as a possible substrate for casein kinase II in vitro.     

Cloning of the gene encoding the catalytic subunit of casein kinase II from the yeast Yarrowia lipolytica

Casein kinase II from the yeast Yarrowia lipolytica is a heterotetramer of the form αα′β₂. We report on the cloning and sequencing of a partial cDNA and of the complete genomic DNA coding for the catalytic α subunit of the casein kinase II from this yeast species. The sequence of the gene coding for this enzyme has been analyzed. No intron was found in the gene, which is present in a single copy. The deduced amino acid sequence of the gene shows high similarity with those of α subunit described in other species, although, uniquely, Y. lipolytica CKIIα lacks cysteines. We find that the α subunit sequence of Y. lipolytica CKII is shown greater homology with the corresponding protein from S. pombe than with that from S. cerevisiae. We have analyzed CKIIα expression and CKIIα activity. We show that expression of this enzyme is regulated. The catalytic subunit is translated from a single mRNA, and the enzyme is present at a very low level in Y. lipolytica, as in other yeasts. Received: 20 December1997 / Accepted: 19 June 1997     

Casein kinase II stimulates rat liver mitochondrial glycerophosphate acyltransferase activity

Rat liver mitochondrial glycerophosphate acyltransferase (mtGAT) possesses 14 consensus sites for casein kinase II (CKII) phosphorylation. To study the functional relevance of phosphorylation to the activity of mtGAT, we treated isolated rat liver mitochondria with CKII and found that CKII stimulated mtGAT activity approximately 2-fold. Protein phosphatase-lambda treatment reversed the stimulation of mtGAT by CKII. Labeling of both solubilized and non-solubilized mitochondria with CKII and [gamma-32P]ATP resulted in a 32P-labeled protein of 85kDa, the molecular weight of mtGAT. Our findings suggest that CKII stimulates mtGAT activity by phosphorylation of the acyltransferase. The significance of this observation with respect to hormonal control of the enzyme is discussed.     

Purification and properties of a casein kinase II-like enzyme from Neurospora crassa

A serine/threonine protein kinase was partially purified from Neurospora crassa. Its physical and catalytic properties were typical of casein kinase II. In vitro, the kinase phosphorylated a calpain like protease from Allomyces arbuscula with higher affinity than a mixture of caseins.     

Interaction of casein kinase II with ribosomal protein L22 of Drosophila melanogaster

The ubiquitous eukaryotic protein kinase CKII (casein kinase II) has been found to interact with a number of cellular proteins, either through the catalytic subunit or the regulatory subunit. Using the yeast two-hybrid screening method, we found that the catalytic subunit of Drosophila melanogaster CKII (DmCKII) interacts with Drosophila ribosomal protein L22 (rpL22). This interaction was also observed in vitro with a glutathione-S-transferase (GST)-rpL22 fusion protein. The predicted full-length Drosophila rpL22 protein has an N-terminal extension rich in alanine, lysine, and proline that appears to be unique to Drosophila. Deletion mapping revealed that the conserved core of rpL22 is responsible for the interaction with CKII. Moreover, purified DmCKII can phosphorylate a GST-L22 fusion protein at the C-terminal end, suggesting that this protein may be a substrate of CKII in Drosophila.     

Purification of substrate proteins of casein kinases from the cytosol fraction of AH-66 hepatoma cells

We have attempted to purify endogenous substrate proteins for casein kinases I and II from the cytosol of AH-66 hepatoma cells. Utilizing the fact that only a few substrates are concentrated in the fraction eluted from DEAE-cellulose between 0.3 and 0.6 M NaCl, two substrates were purified from this fraction by DEAE-cellulose chromatography, hydroxyapatite chromatography, and HPLC on a DEAE-5PW column. The purified substrate proteins had molecular masses of 30.5 kDa and 31 kDa. The 31-kDa protein substrate was markedly phosphorylated by casein kinase II, but only slightly by casein kinase I. The radioactive phosphate incorporated into 31-kDa substrate by casein kinase II was 0.2 mol/mol of the protein and phosphorylation occurred on both threonine and serine residues. The 30.5 kDa protein was only slightly phosphorylated by casein kinase II, but not at all by casein kinase I.     

Casein kinase I: another cog in the circadian clockworks

Multiple components of the circadian central clock are phosphoproteins, and it has become increasingly clear that posttranslational modification is an important regulator of circadian rhythm in diverse organisms, from dinoflagellates to humans. Genetic studies in Drosophila have identified double-time (dbt), a serine/threonine protein kinase that is highly homologous to human casein kinase I epsilon (CKIε), as the first kinase linked to behavioral rhythms. Identification of a missense mutation in CKIε as the tau mutation in the Syrian hamster places CKIε within the core clock machinery in mammals. Most recently, identification of a phosphorylation site mutant of hPER2 in a family with an inherited circadian rhythm abnormality strongly suggests that PER2 is a physiologically relevant substrate of CKI. Phosphorylation may regulate multiple properties of clock proteins, including stability and intracellular localization. (Chronobiology International, 18(3), 389-398, 2001)     

Endogenous protein phosphorylation and casein kinase activity during seed development in Araucaria angustifolia

Protein kinases and phosphatases are responsible for several cellular events mediated by protein phosphorylation and dephosphorylation. Among these events are cell growth and differentiation and cellular metabolism. Casein kinase I (CKI) and casein kinase II (CKII) are involved in the phosphorylation of several substrates. Endogenous protein phosphorylation and casein kinase activity were investigated in the megagametophyte of the native Brazilian conifer Araucaria angustifolia, during seed development. It was observed that a number of different polypeptides are phosphorylated in vitro in the three megagametophyte stages of development tested (from globular, cotyledonary and mature embryos, respectively) and the phosphate was incorporated mainly in serine residues. The use of okadaic acid and vanadate in the phosphorylation reactions increased phosphate incorporation in several polypeptides suggesting the presence of serine/threonine as well as tyrosine phosphatases in the megagametophyte. Also, the results obtained in experiments with CKII inhibitor, GTP as phosphate donor, RNA hybridizations, and in-gel kinase assays indicate the presence of CKII in the A. angustifolia megagametophyte.     

Cyclin H is a new binding partner for protein kinase CK2

PTP-FERM is a protein tyrosine phosphatase (PTP) of Caenorhabditis elegans containing a FERM domain and a PDZ domain. Here we report the characterization of PTP-FERM and the essential role of its FERM domain in the localization of PTP-FERM in the worm. There are at least three alternatively spliced PTP-FERM isoforms, all of which contain a band 4.1/FERM domain, a PDZ domain, and a catalytic domain. PTP-FERM possessed phosphatase activity. PTP-FERM was expressed predominantly in neurons in the nerve ring and the ventral nerve cord. PTP-FERM was found in the nerve processes and to be enriched in the peri-membrane region. Studies using various deletion mutants revealed that the FERM domain was essential and sufficient for the subcellular localization. These results suggest the essential role of the FERM domain in the function of PTP-FERM in the neurons of C. elegans.     

Serum-stimulated cell growth causes oscillations in casein kinase II activity

We have tested the effects of serum-stimulated growth of quiescent WI38 human lung fibroblasts on cellular casein kinase II (CK-II) activity. Using the casein kinase II synthetic substrate RRREEETEEE we find a transient 6-fold elevation in CK-II activity in cell homogenates within 30 min following serum stimulation. Additional cycles of CK-II activation and inactivation are seen at 12 and 24 h after stimulation. The oscillations in CK-II activity are largely independent of de novo protein synthesis, and, thus, are likely to reflect cycles of post-translational activation and inhibition of the cellular kinase pool. In contrast to the activity profile of CK-II, we find that cyclic AMP-dependent protein kinase is rapidly inhibited upon serum-stimulation of WI38 cells. These results demonstrate that CK-II activity is subject to unique cellular regulation during proliferation and are consistent with the postulate that CK-II plays an important role in cell growth.     

Cytosolic thymidine kinase activity in cultured human fibroblasts from individuals with galactokinase deficiency

The structural genes for human galactokinase (GALK) and the human cytosolic form of thymidine kinase (TK1) are located on 17q21–q22. These two loci are tightly linked, and studies on Chinese hamster cell lines have shown that the expression of TK1 and GALK genes may alter simultaneously. We investigated the possibility of a dependent mutation of TK1 and GALK genes in cultured fibroblasts obtained from two patients homozygous for the GALK^G-deficient gene. Since we showed that the TK1 level varies as a function of the passage and the growth rate of a given strain, our experiments were performed on nonstored skin fibroblasts, between the third and the fifth passage for both controls and patients. We found that TK1 levels in GALK-deficient cells were almost 75% of those observed in control strains with a similar growth rate. Previous results in the literature have shown a pronounced decrease in TK1 activity in three GALK-deficient fibroblastic strains. We suggest that these disparities of TK1 levels in GALK-deficient fibroblasts may be related either to genetic heterogeneity of GALK deficiency or to differences in culture conditions. This work was supported in part by grants from La CNAMTS and l’Université de Paris-Sud (AI 86 10).     

Protein kinase C and casein kinase II activities in two human colon carcinoma cell lines,HT-29 and CaCo-2: Possible correlation with differentiation

Protein kinase C (PK-C) and casein kinase II (CK-II) activities were studied in two human colon carcinoma cell lines (HT-29 and CaCO-2) undergoing differentiationin vitro resulting, in small-intestine-like cells. CaCo-2 cells, when grown under standard conditions, appear to undergo spontaneous differentiation. In these cells PK-C and CK-II activities were determined on day 5, 10 and 15. No significant differences in activities were seen either in PK-C or CK-II activity. HT-29 cells, when grown in glucose-free medium can be stimulated to undergo differentiation which is completed within 20 days. PK-C and CK-II activities were determined after 5, 10, 15, 20 and 25 days, respectively. PK-C activity rose from 7.9±3.5 pmole³²P/mg protein/min at day 5 to 37.5±14.8 pmole³²P/mg protein/min at day 20. After 25 days the activity was reduced to 20.0±7.8 pmole³²P/mg protein/min. CK-II activity did not change significantly during day 5 to 20, but on day 25 there was a significant decrease in CK-II activity from 94.9±6.4 pmole³²P/mg protein/min (day 20) to 62.6±3.9 pmole³²P/mg protein/min (day 25) p=0.003. The results in this study indicate a role for PK-C and CK-II in cell growth and differentiation.     

Crosstalk between casein kinase II and Ste20-related kinase Nak1

Although the sterile 20 (Ste20) serine/threonine protein kinase was originally identified as a component of the S. cerevisiae mating pathway, it has homologs in higher eukaryotes and is part of a larger family of Ste20-like kinases. Ste20-like kinases are involved in multiple cellular processes, such as cell growth, morphogenesis, apoptosis and immune response. Carrying out such a diverse array of biological functions requires numerous regulatory inputs and outputs in the form of protein-protein interactions and post-translational modifications. Hence, a thorough knowledge of Ste20-like kinase binding partners and phosphorylation sites will be essential for understanding the various roles of these kinases. Our recent study revealed that Schizosaccharomyces pombe Nak1 (a conserved member of the GC-kinase sub-family of Ste20-like kinases) is in a complex with the leucine-rich repeat-containing protein Sog2. Here, we show a novel and unexpected interaction between the Nak1-Sog2 kinase complex and Casein kinase 2 (Cka1, Ckb1 and Ckb2) using tandem-affinity purification followed by mass spectrometric analysis. In addition, we identify unique phosphosites on Nak1, Sog2 and the catalytic subunit of casein kinase 2, Cka1. Given the conserved nature of these kinases, we expect this work will shed light on the functions of these proteins both in yeast and higher eukaryotes.     

Expression and regulation of casein kinase 2 during heat shock in Verticillium dahliae.

A homogeneous preparation of casein kinase 2 has been isolated from the phytopathogenic fungus Verticillium dahliae (the parasite of cotton). The enzyme consists of three subunits with molecular masses of 53, 41, and 38 kDa. Highly specific immune serum against casein kinase 2 has been obtained. By means of immunoblotting, enzyme-linked immunosorbent assay (ELISA), and immunochemical isolation on protein A-Sepharose, it is shown that the amount of casein kinase 2 increases under heat shock conditions (at least in part due to the synthesis de novo), while the synthesis of the majority of other proteins falls. The activity of casein kinase 2 is supressed during heat shock and so does not correlate with its content. The results give an evidence for the two-step model of casein kinase 2 regulation during heat shock.     

Golgi apparatus casein kinase phosphorylates bioactive Ser-6 of bone morphogenetic protein 15 and growth and differentiation factor 9

Bone morphogenetic protein-15 (BMP-15) and growth and differentiation factor-9 (GDF-9) are oocyte-secreted factors that play essential roles in human folliculogenesis and ovulation. Their bioactivity is tightly regulated through phosphorylation, likely to occur within the Golgi apparatus of the secretory pathway. Here we show that Golgi apparatus casein kinase (G-CK) catalyzes the phosphorylation of rhBMP-15 and rhGDF-9. rhBMP-15, in particular, is an excellent substrate for G-CK. In each protein a single residue is phosphorylated by G-CK, corresponding to the serine residue at the sixth position of the mature region of both rhBMP-15 and rhGDF-9, whose phosphorylation is required for biological activity.     

Protein kinase activity on the cell surface of a macrophage-like cell line,J774.1 cells

Protein kinase activity was demonstrated on the cell surface of a murine macrophage-like cell line, J774.1 cells, and was characterized in detail. When intact cells were incubated with [γ-³²P]ATP, a transfer of [³²P]phosphate into acid-insoluble materials of the cells occurred. This reaction was Mg²⁺-dependent but cAMP-independent, and Mg²⁺ could be substituted for by Mn²⁺. The reaction products were found to be proteins, as revealed by SDS-polyacrylamide gel electrophoresis and autoradiography, with phosphomonoester linkages to serine and threonine residues, but not to tyrosine. The results of experiments with chemical and enzymatic treatments as well as Con A-Sepharose column chromatography ruled out the possibility that an acyl-phosphate linkage or phosphomannosylglycopeptide was present in the reaction products. The protein kinase(s) and the reaction products were located on the cell surface of the cells, as shown by the fact that the products were removed by mild trypsinization of cells carefully controlled so that the cells remained in an intact state. Phosphorylation of exogenous proteins (phosvitin and casein) by intact cells further supported the location of the enzyme. The phosphorylated proteins of the cells were found to be metabolically stable and remained on the cell surface even at 120 min after the phosphorylation reaction. Possible roles of ecto-protein kinase activity in macrophage functions and macrophage-activation are also discussed.     

Inhibitors of casein kinase 1 block the growth of Leishmania major promastigotes in vitro

Casein kinase 1 (CK1) is a family of multifunctional Ser/Thr protein kinases that are ubiquitous in eukaryotic cells. Recent studies have demonstrated the existence of, and role for, CK1 in protozoan parasites such as Leishmania, Plasmodium and Trypanosoma. The value of protein kinases as potential drug targets in protozoa is evidenced by the successful exploitation of cyclic guanosine monophosphate-dependent protein kinase (PKG) with selective tri-substituted pyrrole and imidazopyridine inhibitors. These compounds exhibit in vivo efficacy against Eimeria tenella in chickens and Toxoplasma gondii in mice. We now report that both of these protein kinase inhibitor classes inhibit the growth of Leishmania major promastigotes and Trypanosoma brucei bloodstream forms in vitro. Genome informatics predicts that neither of these trypanosomatids codes for a PKG orthologue. Biochemical studies have led to the unexpected discovery that an isoform of CK1 represents the primary target of the pyrrole and imidazopyridine kinase inhibitors in these organisms. CK1 from extracts of L. major promastigotes co-fractionated with [(3)H]imidazopyridine binding activity. Further purification of CK1 activity from L. major and characterization via liquid chromatography coupled tandem mass spectrometry identified CK1 isoform 2 as the specific parasite protein inhibited by imidazopyridines. L. major CK1 isoform 2 expressed as a recombinant protein in Escherichia coli displayed biochemical and inhibition characteristics similar to those of the purified native enzyme. The results described here warrant further evaluation of the activity of these kinase inhibitors against mammalian stage Leishmania parasites in vitro and in animal models of infection, as well as studies to genetically validate CK1 as a therapeutic target in trypanosomatid parasites.