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.     

Casein kinase II is a major protein phosphorylating activity in the nuclei of Xenopus laevis oocytes

The nuclei of Xenopus laevis oocytes contain kinases capable of phosphorylating endogenous and exogenous proteins using either ATP or GTP as phosphoryl donors. These enzymes are much more active with casein and phosvitin as substrates than with histones or protamines. The protein phosphorylating activity of oocyte nuclear extracts is not regulated by cyclic nucleotides, phorbol esters, calmodulin and calcium, or phospholipids. However, the casein phosphorylating activity can be greatly enhanced by the polyamines spermine or spermidine and drastically inhibited by heparin. Fractionation of the nuclear casein kinase activities by DEAE-Sephadex chromatography and glycerol gradient centrifugation indicate that the nuclei contain enzymes with the properties of casein kinases I and II as characterized in other species. Oocyte casein kinase I (Mr 37,000) is specific for ATP as phosphoryl donor, is only slightly inhibited by 10 micrograms/ml heparin, and is not significantly stimulated by polyamines. Casein kinase II (Mr 135,000) can use both ATP and GTP as substrates, and is very sensitive to heparin inhibition and polyamine stimulation. The fact that low concentrations of heparin (10 micrograms/ml) can inhibit a large percentage of the endogenous phosphorylation of nuclear extracts or of whole nuclei indicates that casein kinase II is probably the major protein phosphorylating activity of these oocyte organelles.     

Phosphorylation of varicella-zoster virus glycoprotein gpI by mammalian casein kinase II and casein kinase I.

Varicella-zoster virus (VZV) glycoprotein gpI is the predominant viral glycoprotein within the plasma membranes of infected cells. This viral glycoprotein is phosphorylated on its polypeptide backbone during biosynthesis. In this report, we investigated the protein kinases which participate in the phosphorylation events. Under in vivo conditions, VZV gpI was phosphorylated on its serine and threonine residues by protein kinases present within lysates of either VZV-infected or uninfected cells. Because this activity was diminished by heparin, a known inhibitor of casein kinase II, isolated gpI was incubated with purified casein kinase II and shown to be phosphorylated in an in vitro assay containing [gamma-32P]ATP. The same glycoprotein was phosphorylated when [32P]GTP was substituted for [32P]ATP in the protein kinase assay. We also tested whether VZV gpI was phosphorylated by two other ubiquitous mammalian protein kinases--casein kinase I and cyclic AMP-dependent kinase--and found that only casein kinase I modified gpI. When the predicted 623-amino-acid sequence of gpI was examined, two phosphorylation sites known to be optimal for casein kinase II were observed. Immediately upstream from each of the casein kinase II sites was a potential casein kinase I phosphorylation site. In summary, this study showed that VZV gpI was phosphorylated by each of two mammalian protein kinases (casein kinase I and casein kinase II) and that potential serine-threonine phosphorylation sites for each of these two kinases were present in the viral glycoprotein.     

Purification of a yeast protein kinase sharing properties with type I and type II casein kinases

Cyclic nucleotide independent protein kinases preferring casein as in vitro substrates were resolved into four distinct species. Only one of the enzymes (CKII) was retained by DEAE-cellulose, whereas the three other enzymes (CKI-1, CKI-2, and CKI-3) were absorbed to CM-Sephadex, eluted with 250 and 600 mM NaCl, and fractionated by heparin-Sepharose chromatography. The casein kinase CKI-3 eluting at the highest NaCl concentration (550 mM) was purified to electrophoretic homogeneity by fast protein liquid chromatography. CKI-1 and CKI-2 correspond to mammalian type I casein kinase, because they bind to CM-Sephadex, they are monomeric enzymes of molecular weights below 50,000, they accept ATP exclusively (CKI-1) or predominantly (CKI-2) as phosphate donor, and they are either completely or relatively heparin insensitive. CKII corresponds to type II casein kinase due to its chromatographic properties, complex quaternary structure, nucleotide specificity (both ATP and GTP are phosphate donors), and heparin sensitivity. CKI-3 shares the following properties with type I casein kinases: it is retained by CM-Sephadex but not by DEAE-cellulose, and it consists of a monomeric protein having a molecular weight of 38,000. On the other hand, CKI-3 accepts both ATP and GTP with equal efficiency, and it is heparin sensitive (50% inhibition at 0.3 microgram/mL) like type II casein kinases. CKI-3 differs from the other three yeast casein kinases in requiring a low pH (5.5) and a high MgCl2 concentration (50 mM) for optimal activity. All four casein kinases phosphorylate their own catalytic protein at serine and threonine residues.     

Nuclear protein phosphorylation in isolated nuclei from HeLa cells. Evidence that 32P incorporation from [gamma-32P]GTP is catalyzed by nuclear kinase II

A nuclear system for studying nuclear protein phosphorylation is characterized, using as phosphate donor either low levels of [gamma-32P]GTP, low levels of [gamma-32P]ATP, or low levels of labeled ATP plus excess unlabeled GTP. Since nuclear casein kinase II is the only described nuclear protein kinase to use GTP with high affinity, low levels of GTP should specifically assay this enzyme. ATP should measure all kinases, and ATP plus unlabeled GTP should measure all kinases except nuclear casein kinase II (ATP-specific kinases). The results are consistent with these predictions. In contrast with the ATP-specific activity, endogenous phosphorylation with GTP was enhanced by 100 mM NaCl, inhibited by heparin and quercetin, stimulated by polyamines, and did not use exogenous histone as substrate. The GTP- and ATP-specific kinases phosphorylated different subsets of about 20 endogenous polypeptides each. Addition of purified casein kinase II enhanced the GTP-supported phosphorylation of the identical proteins that were phosphorylated by endogenous kinase. These results support the hypothesis that activity measured with GTP is catalyzed by nuclear casein kinase II, though other minor kinases which can use GTP are not ruled out. Preliminary observations with this system suggest that the major nuclear kinases exist in an inhibited state in nuclei, and that the effects of polyamines on nuclear casein kinase II activity are substrate specific. This nuclear system is used to determine if the C-proteins of hnRNP particles, previously shown to be substrates for nuclear casein kinase II in isolated particles, is phosphorylated by GTP in intact nuclei. The results demonstrate that the C-proteins are effectively phosphorylated by GTP, but in addition they are phosphorylated by ATP-specific kinase activity.     

Characterization of a cyclic nucleotide- and calcium-independent neurofilament protein kinase activity in axoplasm from the squid giant axon

The phosphorylation activity associated with a neurofilament-enriched cytoskeletal preparation isolated from the squid giant axon has been studied and compared to the phosphorylation activities in intact squid axoplasm. The high molecular weight (greater than 300 kDa) and 220-kDa neurofilament proteins are the major endogenous substrates for the kinases in the axoplasm and the neurofilament preparation, whereas 95- and less than 60-kDa proteins are the major phosphoproteins in the ganglion cell preparation. The squid axon neurofilament (SANF) protein kinase activity appeared to be both cAMP and Ca2+ independent and could phosphorylate both casein (Km = 40 microM) and histone (Km = 180 microM). The SANF protein kinase could utilize either ATP or GTP in the phosphotransferase reaction, with a Km for ATP of 58 microM and 129.4 microM for GTP when casein was used as the exogenous substrate; and 25 and 98.1 microM for ATP and GTP, respectively, when the endogenous neurofilament proteins were used as substrates. The SANF protein kinase activity was only slightly inhibited by 2,3-diphosphoglycerate and various polyamines at high concentrations and was poorly inhibited by heparin (34% inhibition at 100 micrograms/ml). The failures of heparin to significantly inhibit and the polyamines to stimulate the SANF protein kinase indicate that it is not a casein type II kinase. The relative efficacy of GTP as a phosphate donor indicates that SANF protein kinase differs from known casein type I kinases. Phosphorylated (32P-labeled) neurofilament proteins were only slightly dephosphorylated in the presence of axoplasm or stellate ganglion cell supernatants, and the neurofilament-enriched preparation did not dephosphorylate 32P-labeled neurofilament proteins. The axoplasm and neurofilament preparations had no detectable protein kinase inhibitor activity, but a strong inhibitor activity, which was not dialyzable but was heat inactivatable, was found in ganglion cells. This inhibitor activity may account for the low phosphorylation activity found in the stellate ganglion cells and may indicate inhibitory regulation of SANF protein kinase activity in the ganglion cell bodies.     

Nuclear protein phosphorylation in isolated nuclei from HeLa cells. Evidence that 32P incorporation from [γ-32P]GTP is catalyzed by nuclear kinase II

A nuclear system for studying nuclear protein phosphorylation is characterized, using as phosphate donor either low levels of [γ-³²P]GTP, low levels of [γ-³²P]ATP, or low levels of labeled ATP plus excess unlabeled GTP. Since nuclear casein kinase II is the only described nuclear protein kinase to use GTP with high affinity, low levels of GTP should specifically assay this enzyme. ATP should measure all kinases, and ATP plus unlabeled GTP should measure all kinases except nuclear casein kinase II (ATP-specific kinases). The results are consistent with these predictions. In contrast with the ATP-specific activity, endogenous phosphorylation with GTP was enhanced by 100 mM NaCl, inhibited by heparin and quercetin, stimulated by polyamines, and did not use exogenous histone as substrate. The GTP- and ATP-specific kinases phosphorylated different subsets of about 20 endogenous polypeptides each. Addition of purified casein kinase II enhanced the GTP-supported phosphorylation of the identical proteins that were phosphorylated by endogenous kinase. These results support the hypothesis that activity measured with GTP is catalyzed by nuclear casein kinase II, though other minor kinases which can use GTP are not ruled out. Preliminary observations with this system suggest that the major nuclear kinases exist in an inhibited state in nuclei, and that the effects of polyamines on nuclear casein kinase II activity are substrate specific. This nuclear system is used to determine if the C-proteins of hnRNP particles, previously shown to be substrates for nuclear casein kinase II in isolated particles, is phosphorylated by GTP in intact nuclei. The results demonstrate that the C-proteins are effectively phosphorylated by GTP, but in addition they are phosphorylated by ATP-specific kinase activity.     

Properties of several protein kinases that copurify with rat spinal cord neurofilaments

Several protein kinases that copurify with neurofilaments (NF) were identified and each kinase was assessed for its ability to phosphorylate NF proteins. NFs were isolated using an axonal flotation procedure and the kinases were extracted from NFs with 0.8 M KCl. NF kinases were incubated with peptide substrates for selected protein kinases, [32P]ATP and protein kinase cofactors and inhibitors to characterize the kinases. Using peptide substrates, three types of kinase were identified, and a fourth was identified using NF protein as substrate. The first three kinases were the catalytic subunit of cAMP-dependent protein kinase, calcium-calmodulin dependent protein kinase II and a cofactor-independent kinase that phosphorylated prepro VIP sequence 156-170 and was inhibited by heparin. Using NF proteins as substrate, a fourth kinase was identified which was cofactor-independent and was not inhibited by heparin. Neither cofactor-independent kinase was casein kinase II. NF proteins were phosphorylated in vitro on serine and threonine, primarily by the two cofactor-independent kinases. Using [alpha-32P]8-N3ATP for affinity labeling, one kinase of 43,800 Da was identified. Thus, in addition to cAMP-dependent protein kinase and calcium-calmodulin dependent protein kinase II, two kinases have been found which are primarily responsible for NF phosphorylation in vitro and are cofactor-independent.     

A newly synthesized selective casein kinase I inhibitor, N-(2-aminoethyl)-5-chloroisoquinoline-8-sulfonamide, and affinity purification of casein kinase I from bovine testis

When screening various isoquinolinesulfonamide compounds which we synthesized, CKI-7, N-(2-amino-ethyl)-5-chloroisoquinoline-8-sulfonamide, was found to have a potent inhibitory action against casein kinase I and a much weaker effect on casein kinase II and other protein kinases. Kinetic analysis indicated that CKI-7 inhibited casein kinase I competitively with respect to ATP and that the Ki values were 8.5 microM for casein kinase I and 70 microM for casein kinase II. An affinity chromatography absorbent was synthesized by coupling CKI-8 (1-(5-chloroisoquinoline-8-sulfonyl], a derivative of CKI-7, to cyanogen bromide-activated Sepharose 4B. Partially purified casein kinase I from bovine testis was subjected to affinity chromatography. Analysis of the purified casein kinase I by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed a single band with molecular weight 37,000. These newly synthesized compounds, CKI-7 and CKI-8, should serve as useful tools for elucidating the biological significance of casein kinase I-mediated reactions.     

Characterization of insulin-like growth factor binding protein-1 kinases from human hepatoma cells

The phosphorylation of insulin-like growth factor binding protein-I (IGFBP-1) alters its binding affinity for insulin-like growth factor I (IGF-I) and thus regulates the bioavailability of IGF-I for binding to the IGF-I receptor. The kinase(s) responsible for the phosphorylation of IGFBP-1 has not been identified. This study was designed to characterize the IGFBP-1 kinase activity in HepG2 human hepatoma cells, a cell line that secretes IGFBP-1 primarily as phosphorylated isoforms. IGFBP-1 kinase activity was partially purified from detergent extracts of the cells by phosphocellulose chromatography and gel filtration. Two kinases of approximate M_r 150,000 (peak I kinase) and M_r 50,000 (peak II kinase) were identified. Each kinase phosphorylated IGFBP-1 at serine residues that were phosphorylated by intact HepG2 cells. The kinases were distinct based on their differential sensitivity to inhibition by heparin (IC₅₀ = 2.5 and 16.5 μg/ml, peak I and II kinase, respectively) and inhibition by the isoquinoline sulfonamide CKI-7 (IC₅₀ = 50 μM and 100 μM, peak I and II kinase, respectively). In addition, a tenfold molar excess of nonradioactive GTP relative to [gamma-³²P]ATP lowered the incorporation of ³²P into IGFBP-1 by 80% when the reaction was catalyzed by the peak I kinase, whereas GTP had no effect on the reaction catalyzed by the peak II kinase. In the presence of polylysine, IGFBP-1 was radiolabeled by the partially purified kinase activity when [gamma-³²P]GTP served as the phosphate donor indicating the presence of casein kinase II activity. Furthermore, IGFBP-1 was phosphorylated by purified casein kinase I and casein kinase II at sites phosphorylated by the peak I and peak II kinases. Our data suggest that at least two kinases could be responsible for the phosphorylation of IGFBP-1 in intact HepG2 cells and that the kinases are related to the casein kinase family of protein kinases. © 1996 Wiley-Liss, Inc.     

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.     

Differential kinase systems are involved in the rapidly turning over phosphorylation of prominent nuclear proteins

The activity of endogenous nuclear protein kinases has been probed in an vitro assay system of isolated nuclei from Chironomus salivary gland cells. The phosphorylation of a set of seven prominent rapidly phosphorylated non-histone proteins and of histones H3, H2A and H4 was analyzed using ATP or GTP as phosphoryl donor and heparin as protein kinase effector. The core histones H2A and H3 both incorporate 32P from [gamma-32P]ATP as well as from [gamma-32P]GTP but their phosphorylation is differentially affected by heparin. The phosphorylation of H2A is blocked by heparin while that of H3 is even stimulated in the presence of heparin when ATP is used as phosphate donor. H4 is unable to incorporate phosphate groups from GTP but its ATP-based phosphorylation is heparin sensitive. Of the non-histone protein kinase substrates, we could only detect two, the 44-kDa and 115-kDa proteins, which are heparin sensitive with either ATP or GTP and, thus, strictly meet the criteria for casein kinase type II-specific phosphorylation. The investigated histones and non-histone proteins can be grouped into three broad categories on the basis of their phosphorylation properties. (A) Proteins very likely affected by casein kinase NII. (B) Proteins phosphorylated by strictly ATP-specific protein kinases. (C) Proteins phosphorylated by ATP as well as GTP utilizing protein kinase(s) other than casein NII. Category B proteins can be subdivided into proteins phosphorylated in a heparin-resistant (B1) and heparin-sensitive (B2) manner. The phosphorylation of category C proteins may be heparin sensitive with ATP only (C1), heparin sensitive with GTP only (C2), heparin insensitive with both ATP and GTP (C3) or stimulated by heparin (C4).     

Casein kinases I and II bound to pig brain microtubules

1. Microtubules prepared from pig brain by two cycles of assembly-disassembly comprise cyclic nucleotide-independent protein kinase activity with phosvitin and troponin T as substrates. 2. Phosphocellulose chromatography resolved two phosvitin kinase activity peaks, one of which coincided with the troponin T kinase peak. 3. The activity peak corresponding to troponin T kinase was inhibited by heparin (I50 = 0.06 micrograms/ml), whereas the other phosvitin kinase peak was unaffected. 4. Both kinase fractions phosphorylated tubulin and microtubule-associated protein (MAP-2). 5. It is concluded that pig brain microtubules contain bound casein kinases I and II. The association may target the action of these kinases toward microtubular proteins in vivo.     

Epidermal growth factor, but not insulin, stimulates tyrosine phosphorylation of an endogenous protein of Mr 95,000 in triton extracts of human placental syncytiotrophoblast membranes.

1. Triton extracts of syncytiotrophoblast membranes were incubated with [gamma-32P]ATP, MgCl2 and MnCl2. Addition of epidermal growth factor (EGF) resulted in increased phosphorylation not only of the EGF receptor and a Mr-35,000 protein as previously described, but also a protein of Mr 95,000 on both tyrosine and serine residues. In addition, a small increase in the phosphorylation of a protein of Mr 105,000 was observed. Spermine had a similar effect on the phosphorylation of the Mr-95,000 protein, without affecting the phosphorylation of the other proteins. In the absence of MnCl2, the effect of spermine on the phosphorylation of Mr-95,000 protein was still evident, whereas that of EGF was greatly diminished. 2. The Mr-95,000 protein bound poorly to wheat-germ-lectin-Sepharose and was not precipitated by antisera specific for insulin and EGF receptors. The protein continued to exhibit serine and tyrosine phosphorylation on addition of [gamma-32P]ATP, MgCl2 and MnCl2 to a glycoprotein-depleted fraction prepared by chromatography on wheat-germ-lectin-Sepharose. The extent of phosphorylation was no longer increased by spermine or EGF, but was inhibited by heparin. 3. It is suggested that the Mr-95,000 protein not only is a possible direct substrate for the EGF-receptor (but not the insulin receptor) tyrosine kinase but is a substrate for other endogenous kinases, including a protein tyrosine kinase which is probably not a glycoprotein, and a protein serine kinase with properties similar to those of casein kinase II.     

Phosphorylation of DNA topoisomerase II in vivo and in total homogenates of Drosophila Kc cells. The role of casein kinase II

The phosphorylation of DNA topoisomerase II in Drosophila Kc tissue culture cells was characterized by in vivo labeling studies and in vitro studies that examined the modification of exogenous enzyme in total homogenates of these embryonic cells. Several lines of evidence identified casein kinase II as the kinase primarily responsible for phosphorylating DNA topoisomerase II. First, the only amino acyl residue modified in the enzyme was serine. Second, partial proteolytic maps of topoisomerase II which had been labeled with [32P]phosphate by Drosophila cells in vivo, by cell homogenates in vitro, or by purified casein kinase II were indistinguishable from one another. Third, phosphorylation in cell homogenates was inhibited by micrograms/ml concentrations of heparin, micromolar concentrations of nonradioactive GTP, or anti-Drosophila casein kinase II antiserum. Fourth, cell homogenates were able to employ [gamma-32P]GTP as a phosphate donor nearly as well as [gamma-32P]ATP. Although topoisomerase II was phosphorylated in homogenates under conditions that specifically stimulate protein kinase C, calcium/calmodulin-dependent protein kinase, or cAMP-dependent protein kinase, modification was always sensitive to anti-casein kinase II antiserum or heparin. Thus, under a variety of conditions, topoisomerase II appears to be phosphorylated primarily by casein kinase II in the Drosophila embryonic Kc cell system.     

Characterization of two tyrosine-specific protein kinases from pig spleen. Substrate-specific effect of autophosphorylation.

Spontaneously active tyrosine-specific protein kinases I and II (designated TyrK I and TyrK II) have been purified to electrophoretic homogeneity from a particulate fraction of porcine spleen based on an assay that used poly(4Tyr, Glu) as a substrate. SDS/polyacrylamide gels revealed a doublet of bands of about Mr 51,000 for TyrK I and two protein bands of Mr 55,000 and 54,000 for TyrK II. After incubation in the presence of [gamma-32P]ATP, the bands corresponding to both protein kinases contained phosphotyrosine. The two tyrosine protein kinases showed high activities with poly(Tyr, 4Glu) and poly(Tyr, 3Ala, 6Glu) as substrates and lower activity with angiotensin II. Neither histone, phosvitin, casein nor bovine serum albumin were phosphorylated. Both protein tyrosine kinases were activated by millimolar concentrations of Mg2+ whereas Mn2+ was less effective. The effects of various polyanionic and polycationic substances depended on the nature of the peptide substrate. With poly(Tyr, 4Glu) as a substrate, the substances either inhibited the activities of TyrK I and TyrK II or had no effect. However, activation was observed with angiotensin II as substrate in the presence of polylysine, polyornithine, protamine sulfate, and heparin as effectors. When angiotensin II was used as substrate, activation also occurred by autophosphorylation, in parallel to the phosphate incorporation into the protein kinases. Activation by autophosphorylation was not observed with the synthetic peptide substrates, poly(Tyr, 4Glu) and poly(Tyr, 3Ala, 6Glu).     

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.     

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.     

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.