Purification and characterization of a casein-kinase-II-type enzyme from Xenopus laevis ovary. Biological effects on the meiotic cell division of full-grown oocyte

A casein kinase of type II has been highly purified from Xenopus laevis ovary. A new experimental protocol has been developed for the purification, consisting in four chromatographic steps: hydrophobic on tyrosine-agarose, ion exchange on DEAE-Sepharose, affinity on heparin-Sepharose and fast protein liquid on Mono Q. The purification was greater than 20,000, taking into account an inhibitor present in the starting material which masked the activity in the crude fraction. The overall yield was greater than 20%. Full-grown Xenopus oocytes contain 64 milliunits per oocyte corresponding to an intracellular concentration in the nanomolar range. The enzyme shares the following features with the mammalian casein kinase II: (a) comparable subunit composition (42-kDa doublet, 38 kDa and 26 kDa), (b) autophosphorylation of the 26-kDa subunit, (c) ability to use GTP as well as ATP as phosphate donor, (d) inability to use Mn2+ instead of Mg2+ to support the activity, (e) phosphorylation of both threonine and serine residues of casein, (f) inhibition by low doses of heparin. Biological effects of the highly purified enzyme have been investigated upon microinjection into Xenopus full-grown oocytes. At nanomolar concentrations (approximately 3 nM) the enzyme inhibited progesterone induction of meiotic cell division whereas it facilitates meiotic maturation at the level of maturation-promoting factor. These results suggest a role for the kinase in the phosphorylation cascade involved during the prophase/metaphase transition of meiotic cell division, both in the mechanism of the meiotic prophase arrest and in the activity of the cytoplasmic factor maturation-promoting factor. When microinjected into oocytes above 45 nM, the kinase provoked complex changes in the profile of the in ovo 32P-labelled proteins indicating that its targets could be other kinase/phosphatase regulatory proteins.     

Progesterone-induced activation of RNA-binding casein kinase type II during maturation of frog oocytes

During progesterone-induced maturation of the Rana temporaria oocytes phosphorylation of RNA(heparin)-binding proteins changes drastically due to the alterations in the activity of RNA-binding casein kinase II. Its activity increases 7 hr later administration of progesterone and correlates with the level of translation in oocytes. Cycloheximide almost completely inhibits the protein biosynthesis but has no effect on the activity of RNA-binding casein kinase and the set of phosphorylated polypeptides. The possibility is discussed that this enzyme participates in the translation control mechanisms.     

In vivo progesterone regulation of protein phosphatase activity in Xenopus oocytes

Exogenous beta casein, previously phosphorylated in vitro by protein kinase A and casein kinase II, was microinjected into Xenopus oocytes to monitor in vivo protein phosphatase activities. Phosphatase activities were 1.6 and 3.4 fmol/min/oocyte, respectively, for beta casein phosphorylated by casein kinase II and beta casein phosphorylated by protein kinase A. Progesterone induced an early decrease (35% after 10 min) in phosphatase activity restricted to the protein kinase A sites of beta casein.     

Protein phosphorylation during meiotic maturation of Xenopus oocytes: cdc2 protein kinase targets

M-Phase specific protein kinase or cdc2 protein kinase is a component of MPF (M-Phase promoting factor). During meiotic maturation of Xenopus oocytes, cdc2 protein kinase is activated in correlation with MPF activity. A protein phosphorylation cascade takes place involving several protein kinases, among which casein kinase II, and different changes associated with meiosis occur such as germinal vesicle breakdown, chromosome condensation, cytoskeletal reorganization and increase in protein synthesis. Our results provide a biochemical link between cdc2 protein kinase and protein synthesis since they show that the kinase phosphorylates in vitro a p47 protein identified as elongation factor EF1 (gamma subunit) and that the in vitro site of p47 corresponds to the site phosphorylated in vivo. Immunofluorescence showed that the elongation factor (EF1-beta gamma) is localized in the oocyte cortex. Furthermore, they show that cdc2 kinase phosphorylates and activates casein kinase II in vitro, strongly supporting the view that casein kinase II is involved in the phosphorylation cascade originated by cdc2 kinase.     

Polyamine levels during Xenopus laevis oogenesis: a role in oocyte competence to meiotic resumption

The results presented here show that a decrease in the concentration of total polyamines, due to a decrease in putrescine and spermine, occurs during oogenesis in Xenopus laevis. The microinjection of spermine or spermidine decreases the hormonal responsiveness (maturation) of the fully-grown oocytes. This effect is synergistic with that already described for the microinjection of casein kinase II (Mulner-Lorillon, O. et al. (1987) Eur. J. Biochem. 171, 107-117), a polyamine dependent enzyme. Therefore a decrease in polyamine concentration, via its effect on endogeneous casein kinase II, could constitute one of the molecular changes required for the acquisition of competence to mature.     

Isolation and characterization of casein kinases I and II from rabbit liver. Analysis of their effects on protein biosynthesis in the cell

Two cAMP-independent protein kinases isolated from rabbit liver extracts phosphorylate casein far more effectively than histones. The first protein kinase consists of one polypeptide chain (Mr = 37,000), utilizes exclusively ATP and is not inhibited in the presence of low heparin and RNA concentrations. The second protein kinase consists of three subunits (Mr = 42,000, 40,000 and 25,000 Da), utilizes both ATP and GTP and is inhibited by low heparin and RNA concentrations. The latter enzyme has Mr approximately 140,000 Da and possesses a polyanion-binding activity. These characteristics allow to relate the above enzymes to casein kinases I and II, respectively. Injection of casein kinase I into frog oocytes results in the inhibition of the rate of amino acid incorporations into the soluble and detergent extractable proteins. Casein kinase II has no effect on the amino acid incorporation into the recipient oocytes.     

Phosphorylation pattern of the p90rsk and mitogen-activated protein kinase (MAPK) molecule: comparison of in vitro and in vivo matured porcine oocytes

The overall objective was to elucidate the phosphorylation pattern and activity of the kinase p90rsk, a substrate of mitogen-activated protein kinase (MAPK), during in vitro and in vivo maturation of pig oocytes. Cumulus-oocyte complexes were collected from slaughtered pigs and matured in vitro (0, 22, 26, 30, 34, 46 h) with and without the MEK inhibitor U0126. For in vivo maturation, gilts were stimulated with equine chorionic gonadotrophin (eCG) (600-800 IU). Maturation was induced 72 h later with hCG (500 IU). Oocytes were obtained surgically (0, 22, 30 h). The samples were submitted to electrophoresis and protein blotting analysis. Enhanced chemiluminescence was used for visualization. In vitro matured oocytes were further submitted to a commercially available radioactive kinase assay to determine kinase activity. It was shown that oocytes, as well as cumulus cells, already possess a partially phosphorylated p90rsk at the time of removal from follicles, with a further phosphorylation of the molecule occurring between 22-24 h after the initiation of culture, and in vivo maturation. The phosphorylation of p90rsk coincides with the phosphorylation of MAPK and can be prevented by U0126, indicating a MAPK-dependent phosphorylation of p90rsk. Phosphorylation of the in vivo matured oocytes occurred shown as a band of less than 200 kDa. This is presumably a molecule complex, with MAPK not being a component. Therefore, the p90rsk molecule in vivo exists as a dimer. Determination of kinase activity demonstrated decreasing enzyme activities. This led to the conclusion that the assay is not specific for p90rsk, instead measuring p70S6 kinase activities.     

Identification and isolation of casein kinase type II from RNA-binding proteins of amphibian oocytes

Protein kinase previously detected in RNA-binding proteins of amphibian oocytes phosphorylates casein far more efficiently than histones to form phosphoserine and phosphothreonine and utilizes both ATP and GTP. Heparin in concentrations below 1 microgram/ml inhibits protein kinase. This allows to relate the enzyme to casein kinases II. Protein kinase was extensively purified (more than 15000-fold) with respect to proteins of ribosome-free extract. The homogeneous enzyme consists of three polypeptide chains (Mr 43,000, 41,000, and 29,000). The 125I-labelled enzyme possessing casein kinase and RNA-binding activities when injected into amphibian oocytes was detected in the particles identical to free cytoplasmic informosomes in terms of their sedimentation properties.     

Protein phosphorylation during Rhodnius prolixus embryogenesis: protein kinase casein kinase II activity.

Protein kinase casein kinase II (CK II) activity was assayed during Rhodnius prolixus embryogenesis. Vitellin (VT) is the main endogenous substrate during the whole development. It is maximally phosphorylated at the third day of embryogenesis by CK II and then its phosphorylation decreases to a basal level by the time of first instar eclosion. When dephosphorylated casein was used as an exogenous substrate a different profile of enzyme activity was obtained. CK II activity increases on day 1 after fertilization and reaches a plateau on day 7 and its activity remains elevated until eclosion. Extracts obtained from oocytes or from 3-day old eggs were fractionate through gel filtration chromatography. CK II activity was assayed in each fraction and the enzyme obtained from the 3-day old eggs was shown to be three times more active than that obtained from oocytes, although the amount of enzyme present in the fractions was the same. These enriched CK II fractions were assayed against different effectors, such as: cAMP, H-8, H-89, calphostin C, sphingosine, polylysine and heparin. Heparin was the most effective one. When CK II activity was assayed in non-fertilized eggs, no activation of the enzyme was observed when compared to fertilized eggs. These data indicate that CK II is activated in a fertilization dependent process. The decrease in CK II activity against VT coincides with the beginning of VT proteolysis processing suggesting a possible relationship between protein phosphorylation and yolk degradation.     

Modulation of casein kinase II activity by the polar head group of an insulin-sensitive glycosyl-phosphatidylinositol

A phospho-oligosaccharide, whose production is stimulated by insulin, modulated the activity of partially purified casein kinase II. Whereas at 2 microM the phospho-oligosaccharide stimulated casein kinase II 1.3-fold, higher concentrations of this molecule were inhibitory. 50% inhibition of the enzyme was obtained at 15 microM phospho-oligosaccharide. This biphasic effect of the phospho-oligosaccharide on casein kinase II activity was observed using as substrate both casein or the specific peptide for casein kinase II, Arg-Arg-Arg-Glu-Glu-Glu-Thr-Glu-Glu-Glu. The effect of the phospho-oligosaccharide on casein kinase II was still observed after gel filtration. Deamination of the phospho-oligosaccharide with nitrous acid abolished both the activation and the inhibition of casein kinase II. The glycophospholipid precursor of the phospho-oligosaccharide did not affect casein kinase II activity. Moreover, modulation of casein kinase II activity was not observed with other compounds structurally related to the phospho-oligosaccharide, when used in the micro-molar range. In conclusion, the present results indicate that the phospho-oligosaccharide that mimics and might mediate some of the actions of insulin modulates casein kinase II activity in vitro.     

Induction of a substrate for casein kinase II during lymphocyte mitogenesis

Particulate fractions prepared from concanavalin A-activated murine T lymphocytes contain an endogenous protein kinase that phosphorylates an endogenous protein substrate of Mr 112 000. The phosphorylation of 112 kDa protein is greatly reduced or absent in unstimulated T cells. Phosphoamino acid analysis indicates that 112 kDa protein is labeled on a serine. Add-back experiments using purified protein kinases indicate that 112 kDa protein serves as a substrate for casein kinase II. Phosphorylation of 112 kDa protein by the endogenous kinase is inhibited by heparin, a known casein kinase II inhibitor. The site or sites modified by the endogenous kinase and exogenous casein kinase II appear identical by peptide-mapping experiments. A time-course of the appearance of phosphorylated 112 kDa protein following stimulation with concanavalin A, measured in the presence or absence of added casein kinase II, suggests that 112 kDa protein is induced in activated T cells. Subcellular localization studies suggest that 112 kDa protein is a nuclear protein. Silver-binding and purification studies suggest that 112 kDa protein is of the nucleolar organizing region.     

Clathrin-coated vesicles contain two protein kinase activities. Phosphorylation of clathrin beta-light chain by casein kinase II

Incubation of clathrin-coated vesicles with Mg2+-[gamma-32P]ATP results in the autophosphorylation of a 50-kDa polypeptide (pp50) (Pauloin, A., Bernier, I., and Jollès, P. (1982) Nature 298, 574-576). We describe here a second protein kinase that is associated with calf brain and liver coated vesicles. This kinase, which phosphorylates casein and phosvitin but not histone and protamine using either ATP or GTP, co-fractionates with coated vesicles as assayed by gel filtration, electrophoresis, and sedimentation. The enzyme can be extracted with 0.5 M Tris-HCl or 1 M NaCl, and can be separated from the pp50 kinase as well as the other major coat proteins. We identified this enzyme as casein kinase II based on physical and catalytic properties and by comparative studies with casein kinase II isolated from brain cytosol. It has a Stokes radius of 4.5 nm, a catalytic moiety of approximately 45 kDa, and labels a polypeptide of 26 kDa when the pure enzyme is assayed for autophosphorylation. Its activity is inhibited by heparin and not affected by cAMP, phospholipids, or calmodulin. This protein kinase preferentially phosphorylates clathrin beta-light chain. The phosphorylation is markedly stimulated by polylysine and inhibited by heparin. Isolated beta-light chain as well as beta-light chain in triskelions or in intact coated vesicles is phosphorylated. All of the phosphate (0.86 mol of Pi/mol of clathrin beta-light chain) is incorporated into phosphoserine.     

Antibodies to Xenopus egg S6 kinase II recognize S6 kinase from progesterone- and insulin-stimulated Xenopus oocytes and from proliferating chicken embryo fibroblasts.

Ribosomal protein S6 becomes highly phosphorylated during progesterone- or insulin-induced maturation of Xenopus laevis oocytes. We have previously purified an Mr 92,000 protein as one of the major S6 kinases from Xenopus unfertilized eggs. In this paper we confirm by renaturation of activity from a sodium dodecyl sulfate-polyacrylamide gel that this protein is an S6 kinase. This enzyme, termed S6 kinase II (S6 K II), was used for the preparation of polyclonal antiserum. Immunocomplexes formed with the antiserum and purified S6 K II were able to express kinase activity with the same substrate specificity as that of the purified enzyme, including autophosphorylation of S6 K II itself. The antiserum did not react with S6 kinase I, another major S6 kinase present in Xenopus eggs, which is chromatographically distinct from S6 K II. The administration of progesterone to oocytes resulted in a 20- to 25-fold increase in S6 kinase activity in extracts of these cells. Immunocomplex kinase assays done on extracts revealed that anti-S6 K II serum reacted with S6 kinase from progesterone-treated oocytes. This antiserum also reacted with the activated S6 kinase from insulin-stimulated oocytes. In addition, anti-S6 K II serum reacted with activated S6 kinase from chicken embryo fibroblasts stimulated with serum or transformed by Rous sarcoma virus. These results indicate that S6 K II or an antigenically related S6 kinase(s) is subject to regulation by mitogenic stimuli in various cell types.     

pH-dependent changes in structure and RNA-binding activity of casein kinase 2 from Rana temporaria oocytes

It is demonstrated by filter-binding assay that casein kinase 2 from Rana temporaria oocytes binds rRNA in vitro with high affinity. Ligand-blotting shows that rRNA-binding activity is inherent to alpha and alpha' subunits of the enzyme. Increase of pH from 6.5 to 7.5 has little effect on casein kinase but completely suppresses rRNA-binding activity of the enzyme. Sedimentation coefficient of casein kinase 2 also depends on pH: at pH 7.5 it is mainly 10 S, and at pH 6.5-18 S. At pH 6.95 the amounts of both forms are equal. The heavy form of casein kinase 2 practically lacks rRNA-binding activity.     

Multiple phosphorylation sites of rat liver glycogen synthase

Rat liver glycogen synthase was purified to homogeneity by an improved procedure that yielded enzyme almost exclusively as a polypeptide of Mr 85,000. The phosphorylation of this enzyme by eight protein kinases was analyzed by cleavage of the enzyme subunit followed by mapping of the phosphopeptides using polyacrylamide gel electrophoresis in the presence of SDS, reverse-phase high-performance liquid chromatography and thin-layer electrophoresis. Cyclic AMP-dependent protein kinase, phosphorylase kinase, protein kinase C and the calmodulin-dependent protein kinase all phosphorylated the same small peptide (approx. 20 amino acids) located in a 14 kDa CNBr-fragment (CB-1). Calmodulin-dependent protein kinase and protein kinase C also modified second sites in CB-1. A larger CNBr-fragment (CB-2) of approx. 28 kDa was the dominant site of action for casein kinases I and II, FA/GSK-3 and the heparin-activated protein kinase. The sites modified were all localized in a 14 kDa species generated by trypsin digestion. Further proteolysis with V8 proteinase indicated that FA/GSK-3 and the heparin-activated enzyme recognized the same smaller peptide within CB-2, which may also be phosphorylated by casein kinase 1. Casein kinase 1 also modified a distinct peptide, as did casein kinase II. The results lead us to suggest homology to the muscle enzyme with regard to CB-1 phosphorylation and the region recognized by FA/GSK-3, which in rabbit muscle is characterized by a high density of proline and serine residues. A striking difference with the muscle isozyme is the apparent lack of phosphorylations corresponding to the muscle sites 1a and 1b. These results provide further evidence for the presence of liver- and muscle-specific glycogen synthase isozymes in the rat. That the isozymes differ subtly as to phosphorylation sites may provide a clue to the functional differences between the isozymes.     

Casein kinase II accumulation in the nucleolus and its role in nucleolar phosphorylation

A rabbit antiserum against highly purified casein kinase II from mouse tumor cells was used for immunolocalization of the enzyme in fixed, permeabilized mouse cells. Casein kinase II was highly accumulated in nucleoli compared to the extra-nucleolar space of the nucleus or to the cytoplasma. Casein kinase II samples highly purified from the cytoplasma, from the extra-nucleolar fraction of the nucleus or from nucleoli exhibited no differences with respect to structure and function. All samples originally had an alpha 2 beta 2 structure (alpha, 42 kDa; beta, 24 kDa) showing formation of the alpha'-chain (36 kDa) only in the late steps of purification. The isoelectric point of the alpha-chain of all three samples was pH 7.7 and that of the beta-chain was pH 6.4-6.6. Using ATP or GTP, all three casein kinase II samples gave the same results of maximum phosphorylation of purified nucleolar marker phosphoproteins pp105/C23, pp135 and B23, yielding pp135 as one of the most highly phosphorylated proteins with an incorporation of about 75 phosphate groups per molecule pp135. Studies on optimum conditions of phosphorylation of nucleolar phosphoproteins by casein kinase II revealed that each of the protein substrates individually responded to alterations of assay parameters such as pH, magnesium ion and sodium chloride concentrations indicating that predominantly individual structural criteria were responsible for optimum phosphorylation. The determination of the apparent Km of casein kinase II for purified nucleolar phosphoproteins yielded values of 0.15 microM (pp105/C23), 0.1 microM (pp135) and 1.0 microM (B23) identifying them as high-affinity substrates of casein kinase II.     

Regulation of Xenopus laevis DNA topoisomerase I activity by phosphorylation in vitro

DNA topoisomerase I has been purified to electrophoretic homogeneity from ovaries of the frog Xenopus laevis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the most purified fraction revealed a single major band at 110 kDa and less abundant minor bands centered at 62 kDa. Incubation of the most purified fraction with immobilized calf intestinal alkaline phosphatase abolished all DNA topoisomerase enzymatic activity in a time-dependent reaction. Treatment of the dephosphorylated X. laevis DNA topoisomerase I with a X. laevis casein kinase type II activity and ATP restored DNA topoisomerase activity to a level higher than that observed in the most purified fraction. In vitro labeling experiments which employed the most purified DNA topoisomerase I fraction, [gamma-32P]ATP, and the casein kinase type II enzyme showed that both the 110- and 62-kDa bands became phosphorylated in approximately molar proportions. Phosphoamino acid analysis showed that only serine residues became phosphorylated. Phosphorylation was accompanied by an increase in DNA topoisomerase activity in vitro. Dephosphorylation of DNA topoisomerase I appears to block formation of the initial enzyme-substrate complex on the basis of the failure of the dephosphorylated enzyme to nick DNA in the presence of camptothecin. We conclude that X. laevis DNA topoisomerase I is partially phosphorylated as isolated and that this phosphorylation is essential for expression of enzymatic activity in vitro. On the basis of the ability of the casein kinase type II activity to reactivate dephosphorylated DNA topoisomerase I, we speculate that this kinase may contribute to the physiological regulation of DNA topoisomerase I activity.     

Kinetics of meiotic progression, M-phase promoting factor (MPF) and mitogen-activated protein kinase (MAP kinase) activities during in vitro maturation of porcine and bovine oocytes: species specific differences in the length of the meiotic stages

The kinetics of nuclear maturation, M-phase promoting factor (MPF) and mitogen-activated protein kinase (MAP kinase) activities during in vitro maturation of porcine and bovine oocytes were examined. A further objective was to determine the duration of the meiotic stages during the maturation process. Porcine and bovine cumulus-oocyte complexes (COCs) were incubated in TCM 199 supplemented with 20% (v/v) heat inactivated fetal calf serum (FCS), 0.05microg/ml gentamycin, 0.02mg/ml insulin, 2.5microg/ml FSH and 5microg/ml LH. COCs were removed from the culture media in hourly intervals starting immediately after recovery from the follicle until 24 (bovine) or 48h (porcine) of culture. Oocytes were either fixed to evaluate the maturation status or the activity of MPF, assessed by its histone H1 kinase activity, and MAP kinase were determined by a radioactive assay simultaneously. In oocytes of both species, the MPF activity oscillated during the culture period with two maxima corresponding with the two metaphases: between 27-32 and after 46h (porcine) and between 6-9 and after 22h (bovine). There was a temporary decline in activity after 33-38 (porcine) and after 19h (bovine), which corresponded with anaphase I and telophase I. MAP kinase activity increased during the whole culture period and reached maximum levels after 47 (porcine) and after 22h (bovine). In porcine oocytes, the MAP kinase was activated before GVBD and MPF activation. In bovine oocytes, MPF and MAP kinase were activated at approximately the same time as the GVBD (8-9h of incubation). In average porcine, oocytes remain 23.4h in the germinal vesicle (GV) stage (13h in GV I, 5.7h in GV II, 3.2h in GV III and 1.5h in GV IV), 0.9h in diakinese, 9.6h in the metaphase I, 2.8h in anaphase I and 1.9h in telophase I of the first meiotic division. In bovine oocytes, the temporal distribution of the meiotic stages were 8.5h for the GV stage, 1.2h for diakinese, 8.3h for metaphase I, 1.6h for anaphase I and 1.9h for telophase I. These results indicate that the duration of the meiotic stages differs between the species and that MAP kinase is activated before MPF and GVBD in porcine oocytes.     

Roscovitine, a specific inhibitor of cyclin-dependent protein kinases, reversibly inhibits chromatin condensation during in vitro maturation of porcine oocytes.

In the present study the effects of roscovitine on the in vitro nuclear maturation of porcine oocytes were investigated. Roscovitine, a specific inhibitor of cyclin-dependent protein kinases, prevented chromatin condensation in a concentration-dependent manner. This inhibition was reversible and was accompanied by non-activation of p34cdc2/histone H1 kinase. It also decreased enzyme activity of MAP kinase, suggesting a correlation between histone H1 kinase activation and the onset of chromatin condensation. The addition of roscovitine (50 microM) to extracts of metaphase II oocytes revealed that the MAP kinase activity was not directly affected by roscovitine, which indicates a possible link between histone H1 and MAP kinase. Chromatin condensation occurred between 20 and 28 h of culture of cumulus-oocyte complexes (COCs) in inhibitor-free medium (germinal vesicle stage I, GV1: 74.6% and 13.7%, respectively). Nearly the same proportion of chromatin condensation was detected in COCs incubated initially in inhibitor-free medium for 20-28 h and subsequently in roscovitine-supplemented medium (50 microM) for a further 2-10 h (GV I: 76.2% and 18.8%, respectively). This observation indicates that roscovitine prevents chromatin condensation even after an initial inhibitor-free cultivation for 20 h. Extending this initial incubation period to > or = 22 h led to an activation of histone H1 and MAP kinase and increasing proportions of oocytes exhibiting chromatin condensation in the presence of roscovitine. It is concluded that histone H1 kinase is involved in the induction of chromatin condensation during in vitro maturation of porcine oocytes.     

Protein phosphorylation in Rhodnius prolixus oocytes: identification of a type II casein kinase

A protein kinase activity in chorionated oocytes of Rhodnius prolixus phosphorylates in vitro vitellin (VT), the major yolk protein. Phosphatase inhibitors including NaF, sodium vanadate, beta-glycerophosphate and okadaic acid did not alter the protein phosphorylation profile to a visible extent. Among the exogenous protein substrates tested, casein was readily phosphorylated, but histones were not. Several different protein kinase activators, including cAMP, Ca2+ plus calmodulin, Ca2+ plus diolein and phosphatidylserine, were added to the reaction media but spermidine was the only effective one, inducing a 2-fold increase in the phosphorylation of VT. A strong inhibition was obtained with nanomolar levels of heparin. The enzyme could also accept GTP as the phosphate donor instead of ATP. These properties identify the major protein kinase activity as a type II casein kinase (CK II). The pH dependence and the effects of mono- and divalent cations on VT phosphorylation were also studied. Gel filtration revealed only one peak of protein kinase activity, with a molecular mass of 170 K, similar to values previously reported in the literature for CK IIs from other organisms.