Purification of a 107 kilodalton (kDa) casein kinase G substrate from thyroid cytosol

Summary A 107 kDa (pp107) casein kinase G (ck-G) substrate has been purified from mouse and beef thyroid cytosol; ck-G was purified from beef thyroid cytosol. Ck-G and pp107 were found to co-elute on DEAE cellulose chromatography at approximately 300 mM NaCl. Ck-G and pp107 were separated by spermine-agarose affinity chromatography; ppl07 is eluted with a stepped gradient at 250 mM NaCl and ck-G is eluted at 500 mM NaCl. Ck-G was subsequently purified by casein-agarose and GTP-agarose affinity chromatography. The 107 kDa protein was purified using heparin-agarose affinity chromatography. Phosphorylation of purified pp107 by ck-G was stimulated by spermine (ED₅₀ = 0.2 mM) and inhibited by low concentrations of heparin (0.1–5 µg/ml). The Km and Vmax for the reaction were 1.46 µM and 32.2 nmoles P transferred/20 min/mg protein, respectively; 1 mole pp107 incorporated 0.81 mole phosphorus. pp107 was found to be an acidic substrate with a pI of 3.87 and was absorbed to wheat-germ agglutinin-agarose. The specificity of pp107 phosphorylation was studied using diacylglycerol-activated calcium/phospholipid-dependent protein kinase C, calcium-activated calmodulin-dependent protein kinase, and the catalytic subunit of cAMP-dependent protein kinase A. Phosphorylation of pp107 by the other protein kinases tested never exceeded 4% of that of ck-G. Our data show that pp107 is an acidic glycoprotein which may serve as a high-affinity and specific substrate for ck-G.Abbreviations SDS-PAGE Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis - ck-G Casein Kinase G (casein kinase II) - PK-C Diacylglycerol-Activated Calcium/phospholipid-Dependent Protein Kinase - PK-A cAMP-Dependent Protein Kinase - CAMPK Calcium-Activated Calmodulin-Dependent Protein Kinase - EGTA Ethylene Glycol Bis (B-aminoethylether)-N,N,N,N-tetraacetic Acid - PMSF Phenylmethyl Sulfonyl Floride - TCA Trichloroacetic Acid     

Phosphorylation of the insulin receptor by casein kinase I

Insulin receptor was examined as a substrate for the multipotential protein kinase casein kinase I. Casein kinase I phosphorylated partially purified insulin receptor from human placenta as shown by immunoprecipitation of the complex with antiserum to the insulin receptor. Analysis of the phosphorylated complex by polyacrylamide gel electrophoresis under nonreducing conditions showed a major phosphorylated band at the position of the alpha 2 beta 2 complex. When the phosphorylated receptor was analyzed on polyacrylamide gels under reducing conditions, two phosphorylated bands, Mr 95,000 and Mr 135,000, were observed which corresponded to the alpha and beta subunits. The majority of the phosphate was associated with the beta subunit with minor phosphorylation of the alpha subunit. Phosphoamino acid analysis revealed that casein kinase I phosphorylated only seryl residues. The autophosphorylated alpha 2 beta 2 receptor purified by affinity chromatography on immobilized O-phosphotyrosyl binding antibody was also a substrate for casein kinase I. Reduction of the phosphorylated alpha 2 beta 2 receptor indicated that casein kinase I incorporated phosphate into seryl residues only in the beta subunit.     

An improved purification procedure and properties of casein kinase II from brain

A simple and short purification procedure applicable to casein kinase II has been developed, for fully characterizing the enzyme from calf cerebral cortex cytosol. The procedure consists of four chromatographic steps: DEAE-cellulose, phosphocellulose, phosvitin-Sepharose and ATP-agarose which yields 87% pure casein kinase II. The purified enzyme shows three major bands with apparent molecular masses of 42, 38, and 27 kDa by polyacrylamide gel electrophoresis in sodium dodecyl sulfate and is self-autophosphorylated on its 27 kDa polypeptide. The enzyme shows all the characteristics described for casein kinase II from other sources: it is independent of cyclic nucleotides, calcium/phospholipids, and double-stranded poly(I).poly(C); it can utilize both ATP and GTP as phosphoryl donors and can phosphorylate both casein and phosvitin but not histone. The kinetic studies establish that theK _m for ATP is 12.5 M and 25.1 M when using phosvitin and casein respectively as phosphoryl acceptors. TheK _m for phosvitin is 0.91 mg/ml and for casein 1.43 mg/ml, while theV _max is 315 nmol/min/per mg protein and 479 nmol/min/per mg protein for phosvitin and casein respectively. The activity of the kinase is highly stimulated by KCl or NaCl, and almost completely inhibited by heparin concentrations of 1 g/ml (92%). This inhibition is reduced to only 33% in the presence of optimal KCl concentrations (150 mM). Spermine stimulates enzyme activity, whilst hemin produces a slight inhibition.     

Assignment of the casein kinase II gene family to cattle chromosomes

Theileriosis, or East Coast fever, a parasitic disease in cattle, is associated with overexpression of casein kinase II. Casein kinase II is composed of two catalytic subunits (α or α') and two regulatory β subunits. The genes encoding these subunits of casein kinase II were mapped to bovine chromosomes by polymerase chain reaction analysis of a well-characterized bovine × rodent somatic hybrid cell panel. The α-subunit (CSNK2A1) was mapped to bovine chromosome 13, the α'-subunit (CSNK2A2) to chromosome 5 and the β-subunit (CSNK2B) to chromosome 23. Both CSNK2A1 and CSNK2B mapped to known regions of conserved synteny between human and cattle, while CSNK2A2 defined a new homology segment between the human and bovine genomes.     

Identification of a casein kinase II phosphorylation domain in NS1 protein of H5N1 influenza virus

Influenza virus causes febrile respiratory illness. The infection results in significant mortality, morbidity and economic disruption. In this bioinformatics study, we used the NS1 (the conserved nonstructural) protein of influenza A virus to demonstrate its role in infectivity. Our in silico study revealed a new Casein kinase II (CKII) phosphorylation domain at position 151-154. This domain was formed due to the mutation at position 151 (T151I). Moreover, considerable difference in the secondary structure of this protein due to mutation was also reported. It is also confirmed by contact residue analysis that the changes in secondary structure are due to mutations.     

Phosphorylation of membrane proteins by cytosolic casein kinases in human erythrocytes. Effect of monovalent ions, 2,3-bisphosphoglycerate and spermine

Summary Membrane proteins of human erythrocytes can be phosphorylated not only by membrane casein kinase (MS) but also by cytosolic casein kinases CS and CTS, resembling casein kinase I and II, respectively.Casein kinase CS, like membrane casein kinase MS, preferentially phosphorylates membrane proteins such as band 2 (spectrin, -subunit) and band 3, which are the major phosphate-acceptor proteins in the endogenous phosphorylation of isolated ghosts in the presence of [-³²P]ATP.By contrast, cytosolic casein kinase CTS phosphorylates, in addition to band 2, some membrane proteins, whose endogenous phosphorylation in isolated ghosts under the same conditions is negligible, if any.The CS- and CTS-catalyzed phosphorylations exhibit different response to increasing NaCl (or KCI) concentrations up to physiological levels (140 mM KCI, 20 mM NaCI); i.e. CS-and MS-catalyzed phosphorylations are strongly inhibited by 75–150 mM KCI (or NaCl), while CTS-catalyzed phosphorylation is practically unaffected.In the absence of added NaCl, CS- and MS-catalyzed phosphorylations are markedly inhibited by 1.5-3 mM 2,3-bisphosphoglycerate, whereas CTS-catalyzed phosphorylation appears to be practically unaffected.Finally, CS- and MS-catalyzed phosphorylations are slightly inhibited also by 1 mM spermine, while CTS-catalyzed phosphorylation is enhanced by this polycation concentration.     

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.     

Effect of Ethanol on Nuclear Casein Kinase II Activity in Brain

Casein kinase II (CK II) plays an important role in serine/threonine dependent protein phosphorylation. In brain it is associated with long term potentiation besides its involvement in DNA, RNA and protein metabolism. Ethanol has been shown to induce cognitive impairment and affects DNA, RNA and protein metabolism at various steps. Since CK II is central in all these events, which are specifically affected by ethanol, the role of nuclear CK II is investigated in the present study. Total nuclear casein kinase activity was unaffected while heparin sensitive nuclear casein kinase II activity showed a 30% decrease in the brain from chronic alcohol fed rats. Cytosolic CK II activity was also unaffected. Immunological detection by western analysis using CK II antibodies showed no alteration in the quantity of enzyme. The decrease in nuclear casein kinase II might be responsible for ethanol induced cognitive impairment in the brain.     

Effects of exogenous actin-binding casein kinase injected in oocytes and eggs ofXenopus laevis

The effects of microinjections of exogenous casein kinase 2 on the structural organization of the maturing oocytes and eggs were studied inXenopus laevis. Kinase inhibited the progesterone-stimulated oocyte maturation and induced dislocation of pigment granules. The morphological effect was shown to be dose-dependent. The results obtained are discussed in the light of the possible influence of casein kinase 2 on the organization of the cortical actin cytoskeleton through phosphorylation of actin-binding proteins.     

Involvement of protein kinase A and casein kinase II in thein vivo protein kinase activities in prophase arrested Xenopus oocytes

In vivo casein phosphorylation was analysed in Xenopus full-grown oocytes arrested in the prophase of the meiotic cell division. The phosphorylation was inhibited by the protein kinase inhibitor (PKI) and also by heparin (3 g/ml; final concentration). casein phosphorylation was increased by spermine (2 mM). Therefore, protein kinase A and casein kinase II are both activein vivo in full-grown oocytes and may be involved in the prophase arrest of meiotic cell division.     

A structural model for elongation factor 1 (EF-1) and phosphorylation by protein kinase CKII

EF-1a binds aminoacyl-tRNA to the ribosome with the hydrolysis of GTP; the complex facilitates the exchange of GDP for GTP to initiate another round of elongation. To examine the subunit structure of EF-1 and phosphorylation by protein kinase CKII, recombinant , , and subunits from rabbit were expressed in E. coli and the subunits were reconstituted into partial and complete complexes and analyzed by gel filtration. To determine the availability of the and subunits for phosphorylation by CKII, the subunits and the reconstituted complexes were examined as substrates for CKII. Formation of the nucleotide exchange complex increased the rate of phosphorylation of the subunit and reduced the Km, while addition of to or the complex inhibited phosphorylation by CKII. However, a had little effect on phosphorylation of . Thus, the and subunits in EF-1 were differentially phosphorylated by CKII, in that phosphorylation of was altered by association with other subunits, while the site on was always available for phosphorylation by CKII. From the availability of the subunits for phosphorylation by CKII and the composition of the reconstituted partial and complete complexes, a model for the subunit structure of EF-1 consisting of (22)2 is proposed and discussed.     

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.     

Proteins of theXenopus laevis zinc finger multigene family as targets for CK II phosphorylation

Zn finger proteins (ZFPs) of the C₂/H₂ type inXenopus laevis are encoded by a multigene family comprising several hundred members. Based upon conserved sequence features outside the Zn finger region, ZFPs can be subdivided into distinct subfamilies. Two of such subfamilies are characterized by conserved, N-terminal amino acid sequences termed the FAX and the FAR Domain. Here we present data suggesting that the zinc finger proteins of the FAR-ZFP subfamily are targets for CK II mediated phosphorylation. Expression of these proteins during oogenesis coincides with CK II activity in unfertilized eggs. Additionally, we have found that XIcOF 7.1, a member of the FAX-ZFP subfamily, is also phosphorylated by CK II. The target sites forin vitro phosphorylation are localized within the conserved N-terminal domains but not within the Zn finger regions. However, amino acid sequence comparison revealed that individual phosphoacceptor sites are not generally conserved among all members of the respective ZFP subfamilies. The relevance of a potential CK II phosphorylation for the regulation of ZFP activityin vivo is discussed.     

Casein kinase II activity in the brain of an insect,Acheta domesticus: Characterization and hormonal regulation

This study documented casein kinase II (CK II) activity in Acheta domesticus brain using specific antibodies and its regulation by polyamines. In control animals a transient decrease in CK II activity at day 3 after imaginal moult was observed in the brain but not in the fat body. If deprived of ecdysone by ovariectomy a different pattern was observed, with CK II activity being significantly higher on days 3 and 4 after emergence. After ecdysone injection in ovariectomized females, CK II activity decreased to levels similar to those in controls. The implications of ecdysone regulation of brain CK II activity are discussed. © 1997 Wiley-Liss, Inc.     

Identification of osteopontin phosphorylation sites involved in bone remodeling and inhibition of pathological calcification

Osteopontin is a noncollagenous, phosphorylated extracellular glycoprotein, expressed in mineralized and nonmineralized tissues, organs and body fluids. The protein contains an RGD tripeptide cell-binding motif, and is subjected to a variety of posttranslational modifications that play important roles in its multiple biological functions, such as bone remodeling and inhibition of pathological calcification. In this study, we have expressed bovine osteopontin in a prokaryotic system and identified the seven amino acid residues phosphorylated in vitro by CKII.     

Effect of spermine on membrane-associated and membrane-inserted forms of protein kinase C

Protein kinase C is reported to exist in two membrane-bound states: a reversible one which can be dissociated by calcium chelators (membrane-associated form) and an irreversible one which is chelator stable (membrane-inserted form).In the present work the effects of a naturally occurring polyamine (spermine) on the membrane-associated and membrane-inserted forms of protein kinase C were investigated using a reconstituted system consisting of partially purified protein kinase C from rat brain and phospholipid vesicles of defined composition. The active membrane-bound complex was conveniently determined by its ability to bind radioactive phorbol ester with an exact 1:1 stoichiometry.Our experimental data show that, in the absence of calcium ions, the amount of enzyme bound to phospholipids vesicles was dramatically reduced by the presence of spermine whereas the PDBu binding affinity was not significantly affected. The addition of the divalent cation increased the affinity of phorbol ester for the active complex but had no effect on N_max; spermine added in this experimental conditions was no longer able to decrease the total number of enzyme molecules bound to liposomes.Moreover gel filtration experiments of the protein kinase C-phospholipids complex formed in the presence of calcium, indicated that polyamine added during the association process was able to reduce the extent of enzyme insertion into liposomes. Since the increase in phospholipid concentration resulted in a higher level of non-dissociable protein kinase C-liposomes complex we propose that spermine, complexing to membrane binding sites both in the absence and in the presence of Ca⁺⁺, could promote binding conditions that oppose to the formation of the inserted form of the enzyme. As a consequence the distribution between the reversible and the irreversible membrane-bound forms of protein kinase C is affected.     

Casein kinase activity in etiolated Cucumis sativus cotyledons

Two calcium- and light-dependent protein kinases have been reported in etiolated Cucumis sativus cotyledons (Vidal et al. 2007). In the present work, we studied casein kinase (CK) activity in etiolated cucumber cotyledons of in-gel and in vitro kinase assays, using specific CK inhibitors, and ATP and GTP as phosphate donors. Two proteins with CK activity were detected in both casein gels and autophosphorylation assays. One of them, with a molecular mass of approximately 36 kDa, showed biochemical CK1 characteristics: it was inhibited by specific CK1 inhibitors and only used ATP as phosphate donor. The second, with a molecular mass of approximately 38 kDa, had CK2 characteristics; it used both ATP and GTP as phosphate donors, was inhibited by all specific CK2 inhibitors, and was recognized by a polyclonal antibody directed against the α catalytic subunit of a CK2 from tobacco. The kinase activity of the CK2 detected in etiolated cucumber cotyledons showed circadian rhythmicity in both in vitro and in-gel casein phosphorylation and in autophosphorylation assays. Thus, our results suggest that the CK2 of approximately 38 kDa could be related to the circadian oscillator of C. sativus cotyledons.     

Heat-shock inhibits protein synthesis and eIF-2 activity in cultured cortical neurons

Stress, such as heat-shock, hypoxia and hypoglycemia, inhibits the initiation of protein synthesis. The effects of heat-shock on protein synthesis, eucaryotic initiation factor 2 (eIF-2) activity, protein kinase C (PKC), and casein kinase II (CKII) activities were studied in primary cortical neuronal cultures. In neurons exposed to heat-shock at 44°C for 20 min, protein synthesis is inhibited by more than 80%, and is accompanied by a 60% decrease in eIF-2 activity. Steady state PKC and CK II activities were not affected by heat-shock. Vanadate (200 M), a protein phosphotyrosine phosphatase inhibitor, partially prevented the depression of eIF-2 activity during heat-shock, and increased CKII activity by 90%. In contrast, staurosporine (62nM), a protein kinase C inhibitor, did not affect eIF-2 activity. We conclude that heat-shock causes a change in the phosphorylation/ dephosphorylation of regulatory proteins leading to a depressed eIF-2 activity and protein synthesis in neurons.