Inactivation of bovine kidney cytosolic protamine kinase by the catalytic subunit of protein phosphatase 2A

Incubation of highly purified preparations of the bovine kidney cytosolic protamine kinase in the presence of near homogeneous preparations of the catalytic subunit of protein phosphatase 2A (PrP2Ac) from bovine kidney resulted in time-dependent inactivation of the protamine kinase. By contrast, incubation of bovine kidney cytosolic casein kinase II with PrP2Ac had no effect on the activity of this casein kinase II. In the presence of 10 mM sodium fluoride, 10 mM inorganic orthophosphate, 1 mM pyrophosphate or 0.1 mM ATP, the inactivation of the protamine kinase by PrP2Ac was completely inhibited. Half-maximal inhibition by ATP occurred at about 20 microM. The rate of inactivation of the protamine kinase by PrP2Ac was unaffected by Mg2+, Mn2+, Ca2+, EDTA or EGTA at 1 mM. The results strongly indicate that the activity of the cytosolic protamine kinase is regulated by phosphorylation/dephosphorylation.     

Enhanced casein kinase II activity in COS-1 cells upon overexpression of either its catalytic or noncatalytic subunit.

Casein kinase II consists of catalytic (alpha) and regulatory (beta) subunits complexed into a heterotetrameric alpha 2 beta 2 structure. Full-length cDNAs encoding the alpha and beta subunits of human casein kinase II were subcloned into an expression vector containing the cytomegalovirus promotor, yielding the expression constructs pCMV-alpha and pCMV-beta. Northern analyses of total cellular RNA prepared from COS-1 fibroblasts 65 h after transfection with pCMV-alpha or pCMV-beta or with both expression constructs showed marked specific increases in corresponding alpha and beta subunit RNAs. Immunoblot analysis utilizing anti-casein kinase II antiserum of cytosolic extracts prepared from COS-1 cells co-transfected with pCMV-alpha and pCMV-beta showed 2- and 4-fold increases in immunoreactive alpha and beta subunit protein, respectively, relative to vector-transfected cells. These same cytosolic fractions exhibited an average 5-fold increase in casein kinase II catalytic activity. COS-1 cells transfected with pCMV-alpha alone exhibited a 3-fold increase in immunoreactive alpha subunit protein and a nearly 2-fold increase in cytosolic casein kinase II catalytic activity. Transfection with the cDNA coding for the noncatalytic beta subunit alone also caused a near doubling of cytosolic casein kinase II catalytic activity. No increase in immunoreactive alpha subunit protein was observed in pCMV-beta-transfected cells, and no increase in immunoreactive beta subunit protein was observed in pCMV-alpha-transfected cells. These results indicate that a portion of the endogenous cellular casein kinase II protein is not fully active and that raising the concentration of the alpha or beta subunit stimulates this latent activity.     

cAMP-independent casein kinase mediates phosphorylation of many T3-dependent phosphoproteins in rat liver cytosol

Administration of T3 (20 micrograms/100 g BW) for 3 days increases phosphorylation of several proteins in rat liver cytosol in vitro. To help elucidate the mechanism of T3-induced phosphorylation, we studied which protein kinase(s) mediate phosphorylation of endogenous cytosolic proteins. Five different protein kinases were obtained by DEAE+ cellulose column chromatographic fractionation of liver cytosol. When their ability to phosphorylate heat-inactivated cytosol was investigated, casein kinase, a cAMP independent protein kinase, showed the strongest effect. Casein kinase, purified by phosphocellulose chromatography, phosphorylated more than 10 cytosolic proteins. Several T3-dependent (and cAMP independent) phosphoproteins were included among these. One protein with Mr 39 X 10(3), of which phosphorylation is stimulated by T3 within five hours after injection, was the most active substrate for casein kinase. The results suggest that casein kinase is the enzyme responsible for phosphorylation of many rat liver cytosolic proteins and that several phosphoproteins, apparently under T3-regulation, might be phosphorylated by this enzyme.     

Polyamine-like effects of aminoglycosides on various messenger-independent protein kinase reactions

Gentamicin and several other aminoglycoside antibiotics in millimolar concentrations directly stimulate the phosphorylation of casein by purified preparations of cAMP- and Ca2+-independent protein kinases PK-C2 (equivalent to cytosolic casein kinase II) and its nuclear counterpart PK-N2 from rat liver and ventral prostate. These stimulatory effects of aminoglycoside antibiotics were similar to those exerted by the aliphatic polyamine spermine. Phosphorylation of casein by purified preparations of messenger-independent protein kinases PK-C1 (equivalent to cytosolic casein kinase I) and its nuclear counterpart PK-N1 was much less enhanced by spermine and the aminoglycoside antibiotics tested. Stimulations of PK-N2 reactions evoked by gentamicin or spermine (at 0.5 and 1.0 mM) were not additive. Several amino sugars tested were without effect on these protein kinases. Methylglyoxal bis(guanylhydrazone) which is known to block the stimulatory effects of polyamines on certain other enzymes did not alter spermine-stimulated phosphorylation of casein catalyzed by PK-N2 preparations.     

Differential effects of polyamines on rat thyroid protein kinase activities

Ornithine decarboxylase, the rate-limiting enzyme in polyamine biosynthesis, has been shown to be regulated in thyroid by thyrotropin both in vivo and in vitro. Little, however, is known of the role of polyamines in thyroid cell function. Since studies in other tissues suggest that polyamines may influence protein phosphorylation, we studied the effect of the polyamines on various protein kinase activities in rat thyroid. Putrescine, spermidine, and spermine inhibit cyclic-AMP-dependent histone H1 kinase activity when measured in the cytosol fraction of rat thyroid; this effect is largely reproduced by NaCl concentrations of equivalent ionic strength. Both spermidine and spermine effect a 1.6-2.4-fold increase in cytosolic cyclic-AMP-independent (messenger-independent) casein kinase activity; stimulation by both polyamines is maximal at 5mM. A similar profile of stimulation is observed for messenger-independent casein kinase activity in crude nuclear preparations. Sodium chloride fails to stimulate both cytosolic and nuclear messenger-independent casein kinase activities at ionic strength equivalent to the spermine concentrations used. Spermine, but not putrescine, spermidine, or sodium chloride, inhibits calcium/phospholipid-dependent protein kinase C activity in cytosol extracts partially purified by DEAE chromatography. These findings suggest that regulation of protein kinase(s) by polyamines may represent a proximal locus (i) of action of thyrotropin-regulated ornithine decarboxylase activity in thyroid.     

Insulin stimulates a novel Mn2+-dependent cytosolic serine kinase in rat adipocytes

The cytosolic fraction of insulin-treated adipocytes exhibits a 2-fold increase in protein kinase activity when Kemptide is used as a substrate. The detection of insulin-stimulated kinase activity is critically dependent on the presence of phosphatase inhibitors such as fluoride and vanadate in the cell homogenization buffer. The cytosolic protein kinase activity exhibits high sensitivity (ED50 = 2 X 10(-10) M) and a rapid response (maximal after 2 min) to insulin. Kinetic analyses of the cytosolic kinase indicate that insulin increases the Vmax of Kemptide phosphorylation and ATP utilization without affecting the affinities of this enzyme toward the substrate or nucleotide. Upon chromatography on anion-exchange and gel filtration columns, the insulin-stimulated cytosolic kinase activity is resolved from the cAMP-dependent protein kinase and migrates as a single peak with an apparent Mr = 50,000-60,000. The partially purified kinase preferentially utilizes histones, Kemptide, multifunctional calmodulin-dependent protein kinase substrate peptide, ATP citrate-lyase, and acetyl-coenzyme A carboxylase as substrates but does not catalyze phosphorylation of ribosomal protein S6, casein, phosvitin, phosphorylase b, glycogen synthase, inhibitor II, and substrate peptides for casein kinase II, protein kinase C, and cGMP-dependent protein kinase. Phosphoamino acid analyses of the 32P-labeled substrates reveal that the insulin-stimulated cytosolic kinase is primarily serine-specific. The insulin-activated cytosolic kinase prefers Mn2+ to Mg2+ and is independent of Ca2+. Unlike ribosomal protein S6 kinase and protease-activated kinase II, the insulin-sensitive cytosolic kinase is fluoride-insensitive. Taken together, these results indicate that a novel cytosolic protein kinase activity is activated by insulin.     

Casein kinase I is regulated by phosphatidylinositol 4,5-bisphosphate in native membranes.

Casein kinase I activity is present in cells as a cytosolic and a membrane-bound enzyme. Previously, the erythroid membrane-bound casein kinase I was shown to associate with purified integral membrane proteins; this association and protein kinase activity was regulated by phosphatidylinositol 4,5-bisphosphate (PIP2) (Bazenet, C.E., Brockman, J.L., Lewis, D., Chan, C., and Anderson, R.A. (1990) J. Biol. Chem. 265, 7369-7376). Here we show that both the membrane-bound and the cytosolic casein kinase interact with native membranes and that this interaction is regulated by the membrane content of PIP2. On native membranes, casein kinase I activity is potently inhibited by small increases (10-20%) in the membrane content of either exogenously added or intrinsic PIP2. However, the majority of the intrinsic content of PIP2 in isolated membranes does not inhibit casein kinase, suggesting that this PIP2 is not accessible. Regulation of the casein kinases on membranes is sensitive to detergents and to chymotrypsin treatment of membranes.     

Erythroid membrane-bound protein kinase binds to a membrane component and is regulated by phosphatidylinositol 4,5-bisphosphate

In the erythrocyte, a membrane-bound serine/threonine protein kinase (a casein kinase) has been shown to phosphorylate a number of membrane proteins, modulating their function. Here we report that the membrane-bound protein kinase binds to membranes by an association with a minor membrane component contained in preparations of glycophorin (possibly a minor glycophorin). The binding of the kinase to glycophorins does not significantly modify kinase activity. However, upon binding, the kinase activity is potently inhibited by phosphatidylinositol 4,5-bisphosphate, and the affinity of the kinase for the glycophorins is increased. Other phospholipids or polyanions such as inositol 1,4,5-trisphosphate or 2,3-diphosphoglycerate do not affect protein kinase activity when the kinase is bound to membranes but do inhibit the solubilized membrane-bound kinase. In the erythrocyte, there is a cytosolic form of the casein kinase which is very similar, having the same molecular weight and substrate specificity as the membrane-bound casein kinase. The cytosolic casein kinase is inhibited by 2,3-diphosphoglycerate but much less so by glycophorin preparations containing phosphoinositol 4,5-bisphosphate. When the sequences of both casein kinases were compared by two-dimensional peptide mapping, it was found that the two kinases were very similar but not identical.     

Regulation of casein kinase II activity by epidermal growth factor in human A-431 carcinoma cells

In order to characterize more fully the mechanism by which casein kinase II is regulated in mammalian cells, the effect of epidermal growth factor (EGF) on the activity of the kinase in human A-431 carcinoma cells was examined. Treatment of cells with EGF prior to lysis consistently resulted in a transient 4-fold increase in the activity of cytosolic casein kinase II. Activity rose sharply between 20 and 30 min, peaked at approximately 50 min, and returned to basal levels by approximately 120 min. Similar results were obtained using the casein kinase II specific peptide substrate, Arg-Arg-Arg-Glu-Glu-Glu-Thr-Glu-Glu-Glu, or DNA topoisomerase II (which is specifically modified by the kinase in vivo and serves as a high affinity substrate in vitro) as the phosphate acceptor in assays. Identification of casein kinase II as the stimulated activity was confirmed by partial proteolytic mapping and phosphoamino acid analysis of modified topoisomerase II, by inhibition at nanomolar levels of heparin or micromolar levels of nonradioactive GTP, and by the ability to employ radioactive GTP as a direct phosphate donor. The EGF stimulation of casein kinase II was dependent on the availability of intracellular (but not extracellular) calcium. In addition, hormonal action was modulated by calcium/phospholipid-dependent protein kinase (protein kinase C). Casein kinase II stimulation did not require an increase in the concentration of the kinase, protein synthesis, the continual presence of a small effector molecule, or a direct interaction with the EGF receptor/tyrosine kinase. In contrast, hormonal activation of the kinase was dependent on the phosphorylation of casein kinase II or a terminal stimulatory factor.     

Phosphorylation of the medium chain subunit of the AP-2 adaptor complex does not influence its interaction with the tyrosine based internalisation motif of TGN38

Tyrosine based motifs conforming to the consensus YXXphi (where phi represents a bulky hydrophobic residue) have been shown to interact with the medium chain subunit of clathrin adaptor complexes. These medium chains are targets for phosphorylation by a kinase activity associated with clathrin coated vesicles. We have used the clathrin coated vesicle associated kinase activity to specifically phosphorylate a soluble recombinant fusion protein of mu2, the medium chain subunit of the plasma membrane associated adaptor protein complex AP-2. We have tested whether this phosphorylation has any effect on the interaction of mu2 with the tyrosine based motif containing protein, TGN38, that has previously been shown to interact with mu2. Phosphorylation of mu2 was shown to have no significant effect on the in vitro interaction of mu2 with the cytosolic domain of TGN38, indicating that reversible phosphorylation of mu2 does not play a role in regulating its direct interaction with tyrosine based internalisation motifs. In addition, although a casein kinase II-like activity has been shown to be associated with clathrin coated vesicles, we show that mu2 is not phosphorylated by casein kinase II implying that another kinase activity is present in clathrin coated vesicles. Furthermore the kinase activity associated with clathrin coated vesicles was shown to be capable of phosphorylating dynamin 1. Phosphorylation of dynamin 1 has previously been shown to regulate its interaction with other proteins involved in clathrin mediated endocytosis.     

Casein Kinase II-Type Protein Kinase from Pea Cytoplasm and Its Inactivation by Alkaline Phosphatase in Vitro

A casein kinase II-type protein kinase has been purified from the cytosolic fraction of etiolated pea (Pisum sativum L.) plumules to about 90% purity as judged from Coomassie blue stained sodium dodecyl sulfate-polyacrylamide gels. This kinase has a tetrameric [alpha][alpha]'[beta]2 structure with a native molecular mass of 150 kD, and subunit molecular masses of 41 and 40 kD for the two catalytic subunits ([alpha] and [alpha]') and 35 kD for the putative regulatory subunit ([beta]).Casein and phosvitin can be used as artificial substrates for this kinase. Both serine and threonine residues were phosphorylated when mixed casein, [beta]-casein, or phosvitin were used as the substrate, whereas only serine was phosphorylated if [alpha]-casein or histone III-S was the substrate. The kinase activity was stimulated 130% by 0.5 mM spermine (the concentration required for 50% of maximal enzyme activity [A50] = 0.1 mM) and 80% by 2.5 mM spermidine (A50 = 0.4 mM), whereas putrescine and cadaverine had no effect. The kinase was very sensitive to inhibition by heparin (concentration for 50% inhibition [I50] = 0.025 [mu]g/mL). In contrast to most other casein kinase II-type protein kinases, this preparation was inhibited by K+ and Na+, with I50 values of 75 and 65 mM, respectively. Pretreatment of the purified kinase preparation in vitro with alkaline phosphatase caused a 5-fold decrease in its activity. Additionally, this kinase also lost its activity when its [beta] subunit was autophosphorylated in the absence of substrate. These results suggest that the activity of this casein kinase II protein kinase may be regulated by the phosphorylation state of two different sites in its multimeric structure.     

Phosphorylation of cytosolic proteins by casein kinases in human erythrocytes. Response to ionic strength and to 2,3-bisphosphoglycerate

The endogenous phosphorylation of human erythrocyte cytosolic proteins is markedly increased when the crude cytosol, prior to incubation in the presence of [y-³²P] ATP, is submitted to DEAE-cellulose chromatography. Some proteins, including 22 and 23 kDa proteins, are preferentially phosphorylated by cytosolic casein kinase CS, whereas other proteins, including 42 kDa protein, are preferentially phosphorylated by casein kinase CTS. The CS-catalyzed phosphorylation is strongly inhibited by physiological ionic strength (150 mM KCl or NaCl) and by physiological levels (3 mM) of 2,3-bisphosphoglycerate, while CTS-catalyzed phosphorylation is unaffected. The very poor endogenous phosphorylation of these proteins in the crude cytosol may be due to the presence of other cytosolic inhibitors which are removed by DEAE-cellulose chromatography.     

Casein kinase II activity and polyamine-stimulated protein phosphorylation of cytosolic and plasma membrane proteins in bovine sperm

A highly purified preparation of sperm cytosolic protein kinase was obtained by repeated chromatography with phosphocellulose. The preferred substrate of the enzyme was casein and the activity was not stimulated by added Ca2+, calmodulin, or cAMP. With casein as substrate, both ATP and GTP served as phosphate donors and the activity was inhibited by low micromolar heparin and stimulated by low millimolar spermine and spermidine. These properties are characteristic of casein kinase II from other cells. Endogenous protein substrates of the enzyme in sperm cytosolic fractions and in plasma membranes were demonstrated by incubating the preparations with [gamma-32P]GTP, under conditions unfavorable to other protein kinases, and analyzing the products by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Spermine greatly enhanced the phosphorylation of three (55, 92, and 106 kDa) proteins in both cytosolic and plasma membrane preparations. Our results indicate that polyamines play a role in modulating the phosphorylation state of proteins in sperm and may further regulate sperm function through this mechanism.     

Inhibition of casein kinase II by heparin

Casein kinase II, a cyclic nucleotide-independent protein kinase from rabbit reticulocytes, was shown to be inhibited by heparin. Heparin specifically inhibited the enzyme and had no effect on other protein kinases, including casein kinase I, the type I and II cAMP-dependent protein kinases, protease-activated kinase I, and the hemin-controlled repressor. Heparan sulfate was found to be 40-fold less effective than heparin towards casein kinase II; other acid mucopolysaccharides had little or no effect on the enzymatic activity. Steady state studies revealed that heparin acted as a competitive inhibitor with respect to the substrate, casein. A value of 20 ng/ml or about 1.4 nM was obtained for the apparent Ki. The inhibition was not reversed by ATP and varying the ATP and heparin concentrations in the assay only altered the maximum velocity.     

Major vault protein is a substrate of endogenous protein kinases in CHO and PC12 cells.

Major vault protein (MVP) is the predominant member of a large cytosolic ribonucleoprotein particle, termed vault. We have previously shown that MVP derived from electric ray electric organ becomes phosphorylated by protein kinase C in vitro and by tyrosine kinase in vivo. Here we show that MVP from two mammalian cell lines (CHO and PC12 cell) becomes highly phosphorylated by endogenous protein kinases in cell-free systems. The susceptibility to protein kinases differs substantially from those observed in MVP derived from electric organ. Phosphorylation of MVP depends on the presence of Mg2+ and can be inhibited by the chelating agent EDTA. Inhibitors of casein kinase II attenuate the phosphorylation of MVP. In contrast to CHO cells, addition of recombinant casein kinase II enhances the phosphorylation of MVP in PC12 cells. Endogenous kinase activity is of particulate nature and copurifies with vault particles. Immuno-affinity purified vaults containing recombinant tagged MVP expressed in CHO cells reveal no autophosphorylation, suggesting that protein kinase activity is not an intrinsic property of vaults. Our results suggest that cell-specific phosphorylation of MVP may play a critical role in vault function.     

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.     

A high-beef diet alters protein kinase C isozyme expression in rat colonic mucosa

We recently reported that a red meat (beef) diet relative to a casein-based diet increases protein kinase C (PKC) activity in rat colonic mucosa. The purpose of this study was to further elucidate the effects of a high-beef diet on colonic intracellular signal transduction by analyzing steady-state protein levels of different PKC isozymes as well as activities of the three types of sphingomyelinases. Male Wistar rats (n = 12/group) were fed AIN93G-based diets either high in beef or casein for 4 weeks. Rats fed the beef diet had significantly (P < 0.05) higher cytosolic PKC alpha and lower membrane PKC delta protein levels than rats fed the casein diet. The beef-fed rats also had alterations in subfractions of PKC zeta/lambda so that they had a significantly (P = 0.001) lower level of membrane 70 & 75 kDa fraction and a higher (P = 0.001) level of cytosolic 40 & 43 kDa fraction than rats fed the casein diet. Because protein levels analyzed with a PKC zeta-specific antibody were similar, these differences in PKC zeta/lambda were probably due to changes in PKC lambda expression. PKC beta2 levels did not differ between the dietary groups. Diet had no significant effect on the activity of acid, neutral, or alkaline sphingomyelinase. This study demonstrated that consumption of a high-beef diet is capable of modulating PKC isozyme levels in rat colon, which might be one of the mechanisms whereby red meat affects colon carcinogenesis.     

Isolation and study of cAMP-independent chromatin protein kinases from brain cells]

Two cAMP-independent protein kinases were purified from rat brain neuron chromatin by using extraction with ammonium sulfate with subsequent chromatography on DEAE-Sephadex A-25 and Sephadex G-150. These enzymes were identified as casein kinases NI and NII, respectively. The molecular masses of the proteins as determined by gel filtration are 4500 and 130 Da. Casein kinase NII utilizes ATP (Km = 7.5 mM) and GTP (Km = 8.5 mM) as substrates, while casein kinase NI utilizes only ATP (Km = 6 mM). The activities of the both enzymes are inhibited by Mn2+ and Ca2+, while heparin (1 microgram/ml) inhibits only casein kinase NII. The memory stimulator ethymizol (ethylnorantipheine) increases the activity of casein kinase NII only when brain proteins extracted by 0.35 M NaCl or rat liver HMG-proteins are used as reaction substrates. This substance has no effect on the phosphorylation of casein and histone HI. The role of casein kinase NII of neuronal chromatin in the realization of stimulatory effects of physiologically active substances on RNA synthesis is discussed.     

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.     

Spermine Stimulation of a Nuclear NII Kinase from Pea Plumules and Its Role in the Phosphorylation of a Nuclear Polypeptide

We have previously demonstrated that spermine stimulates the phosphorylation of a 47 kilodalton nuclear polypeptide from pea plumules (N Datta, LK Hardison, SJ Roux 1986 Plant Physiol 82: 681-684) In this paper we report that spermine stimulates the activity of a cyclic AMP independent casein kinase, partially purified from a chromatin fraction of pea plumule nuclei. This effect of spermine was substrate specific; i.e. with casein as substrate, spermine stimulated the kinase activity, and with phosvitin as substrate, spermine completely inhibited the activity. The stimulation by spermine of the casein kinase was, in part, due to the lowering of the Mg²⁺ requirement of the kinase. Heparin could partially inhibit this casein kinase activity and spermine completely overcame this inhibition. By further purification of the casein kinase extract on high performance liquid chromatography, we fractionated it into an NI and an NII kinase. Spermine stimulated the NII kinase by 5- to 6-fold but had no effect on the NI kinase. Using [γ-³²P]GTP, we have shown that spermine promotes the phosphorylation of the 47 kilodalton polypeptide(s) in isolated nuclei, at least in part by stimulating an NII kinase.