Subunit structure of casein kinase II from bovine testis. Demonstration that the alpha and alpha' subunits are distinct polypeptides

The relationship between the alpha and alpha' subunits of casein kinase II was studied. For this study, a rapid scheme for the purification of the enzyme from bovine testis was developed. Using a combination of chromatography on DEAE-cellulose, phosphocellulose, hydroxylapatite, gel filtration on Sephacryl S-300 and heparin-agarose, the enzyme was purified approximately 7,000-fold. The purification scheme was completed within 48 h and resulted in the purification of milligram quantities of casein kinase II from 1 kg of fresh bovine testis. The purified enzyme had high specific activity (3,000-5,000 nmol of phosphate transferred per min/mg protein) when assayed at 30 degrees C with ATP and the synthetic peptide RRRDDDSDDD as substrates. The isolated enzyme was a phosphoprotein with an alkali-labile phosphate content exceeding 2 mol/mol protein. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis three polypeptides were apparent: alpha (Mr 45,000), alpha' (Mr 40,000), and beta (Mr 26,000). Several lines of evidence conclusively demonstrated that the alpha and alpha' subunits are distinct polypeptides. Two-dimensional maps of 125I-tryptic peptides derived from the two proteins were related, but distinct. An antipeptide antibody was raised in rabbits which reacted only with the alpha subunit on immunoblots and failed to react with either the alpha' or beta subunits. Direct comparison of peptide sequences obtained from the alpha and alpha' subunits revealed differences between the two polypeptides. The results of this study clearly demonstrate that the alpha and alpha' subunits of casein kinase II are not related by post-translational modification and are probably encoded by different genes.     

Effects of cationic polypeptides on the activity, substrate interaction, and autophosphorylation of casein kinase II: a study with calmodulin.

The effects of basic polypeptides on the ability of casein kinase II to phosphorylate an exogenous substrate (calmodulin) are correlated with steady-state autophosphorylation of the alpha- and beta-subunits of casein kinase II. Polylysine and polyarginine increase autophosphorylation of the alpha-subunit with a concomitant decrease in beta-subunit phosphorylation, while enhancing casein kinase II-stimulated phosphorylation of calmodulin over 100-fold. The highly basic carboxyl terminal segment of the endogenous p21c-Ki-ras has similar effects on the phosphorylation of calmodulin and the alpha- and beta-subunits of casein kinase II. Altering the concentration of cationic polypeptides produces a biphasic effect on the phosphorylation of both calmodulin and the alpha-subunit, which correlate positively with each other but do not correlate with beta-subunit phosphorylation. When the KCl concentration is changed, casein kinase II activity correlates positively only with alpha-subunit phosphorylation. In contrast, the biphasic response of calmodulin phosphorylation by casein kinase II at different Ca2+ concentrations correlates positively with both alpha- and beta-subunit phosphorylation. Therefore, in the presence of basic protein activators, the rate of phosphorylation of a substrate, calmodulin, correlates with steady-state phosphorylation of the alpha-subunit, but not with the beta-subunit under all conditions tested. Endogenous cationic factors may modulate the in vivo activity of casein kinase II and alter the interaction of the enzyme with specific intracellular substrates.     

Calmodulin and acidic compounds alter basic protein phosphorylation by the protein kinase from human platelets

A cyclic nucleotide-independent protein kinase of human platelets, which phosphorylated histones, myelin basic protein and protamine and did not catalyze the phosphorylation of acidic proteins such as casein, phosvitin and myosin light chain, has been purified approx. 1,500-fold from the crude extract by steps of DEAE-cellulose, Sephadex G-200, hydroxylapatite and phosphoryl cellulose column chromatography. The substrate phosphorylation by this kinase was markedly enhanced by calmodulin even in the absence of Ca²⁺, when mixed histone was used as a substrate. The interaction of the kinase with mixed histone resulted in an irreversible inactivation of the enzyme. Calmodulin prevented this inactivation, and this compound produced an apparent increase in histone phosphorylation by the kinase. It should be noted that acidic polypeptides such as troponin-C, phospholipids and nucleic acids have a similar ability. The addition of Ca²⁺ reduced the effect of calmodulin more than the effects of other acidic compounds.     

Identification of the substrates of the casein kinase II associated with non-polysomal messenger ribonucleoproteins of A. salina cryptobiotic embryos

The association of a protein kinase with cytoplasmic non-polysomal messenger ribonucleoproteins is demonstrated by chromatography on oligo(dT)-cellulose and sucrose gradient centrifugation. The cAMP-independent enzyme is inhibited by caffeine and poly(L)-glutamic acid and is classified as a casein kinase II. Among the exogenous proteins initiation factor eIF2 is the best substrate and is 7.8 times more efficiently phosphorylated than casein. Endogenous mRNP protein substrates have a Mr of 125000, 65000, 38000, 26000 and 23500. The main phosphate acceptor is the Mr38000 poly(A)-binding protein. Dephosphorylation of the poly(A)-binding protein by protein phosphatases decreases its RNA binding property. The effect of phosphorylation-dephosphorylation of mRNP proteins on the initiation of protein synthesis is discussed.Abbreviations PMSF phenylmethanesulfonyl fluoride - mRNP messenger ribonucleoprotein - iRNA small inhibitor RNA - eIF2 eukaryotic initiation factor 2     

Phosphorylation of DARPP-32, a dopamine- and cAMP-regulated phosphoprotein, by casein kinase II

DARPP-32 (dopamine- and cAMP-regulated phosphorprotein, Mr = 32,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) is an inhibitor of protein phosphatase-1 and is enriched in dopaminoceptive neurons possessing the D1 dopamine receptor. Purified bovine DARPP-32 was phosphorylated in vitro by casein kinase II to a stoichiometry greater than 2 mol of phosphate/mol of protein whereas two structurally and functionally related proteins, protein phosphatase inhibitor-1 and G-substrate, were poor substrates for this enzyme. Sequencing of chymotryptic and thermolytic phosphopeptides from bovine DARPP-32 phosphorylated by casein kinase II suggested that the main phosphorylated residues were Ser45 and Ser102. In the case of rat DARPP-32, the identification of these phosphorylation sites was confirmed by manual Edman degradation. The phosphorylated residues are located NH2-terminal to acidic amino acid residues, a characteristic of casein kinase II phosphorylation sites. Casein kinase II phosphorylated DARPP-32 with an apparent Km value of 3.4 microM and a kcat value of 0.32 s-1. The kcat value for phosphorylation of Ser102 was 5-6 times greater than that for Ser45. Studies employing synthetic peptides encompassing each phosphorylation site confirmed this difference between the kcat values for phosphorylation of the two sites. In slices of rat caudate-putamen prelabeled with [32P]phosphate, DARPP-32 was phosphorylated on seryl residues under basal conditions. Comparison of thermolytic phosphopeptide maps and determination of the phosphorylated residue by manual Edman degradation identified the main phosphorylation site in intact cells as Ser102. In vitro, DARPP-32 phosphorylated by casein kinase II was dephosphorylated by protein phosphatases-1 and -2A. Phosphorylation by casein kinase II did not affect the potency of DARPP-32 as an inhibitor of protein phosphatase-1, which depended only on phosphorylation of Thr34 by cAMP-dependent protein kinase. However, phosphorylation of DARPP-32 by casein kinase II facilitated phosphorylation of Thr34 by cAMP-dependent protein kinase with a 2.2-fold increase in the Vmax and a 1.4-fold increase in the apparent Km. Phosphorylation of DARPP-32 by casein kinase II in intact cells may therefore modulate its phosphorylation in response to increased levels of cAMP.     

The presence of protein kinase activity and acceptors of phosphate groups in nonpolysomal cytoplasmic messenger ribonucleoprotein complexes of embryonic chicken muscle

Nonpolysomal cytoplasmic (free) mRNA.protein (mRNP) complexes of embryonic chicken muscle were purified by a combination of oligo(dT)-cellulose chromatography and sucrose density gradient centrifugation. The protein moieties of the purified mRNP complex were analyzed by two-dimensional gel electrophoresis using separation according to charge in the first dimension and molecular weight in the second. Sixteen polypeptides of Mr = 27,000 to 75,000 were present in the mRNP complex. These mRNP polypeptides displayed different electrophoretic migration properties than those of ribosomal proteins. A protein kinase activity was found associated with the mRNP. This enenzyme was able to transfer phosphate group(s) from ATP to at least three acidic mRNP polypeptides of Mr = 27,000, 38,000, and 73,000 and one basic polypeptide of Mr = 75,000. Among these, the Mr = 38,000 acidic polypeptide was the best acceptor of phosphate groups.     

Phosphorylation of components of high molecular weight aminoacyl-tRNA-synthetase complex from the rabbit liver by associated casein kinase type I

The aminoacyl-tRNA synthetase complex from rabbit liver possesses an endogenous protein kinase activity. The associated protein kinase in the complex was defined as casein kinase I. Using FPLC, a fraction of the supramolecular complex with a high level of metabolic activity was isolated; this preparation was found to be enriched in the casein kinase activity. Incubation of this fraction with [gamma-32P] ATP leads to the intensive incorporation of labeled phosphate into 12 polypeptides of the complex, i.e., glutamyl-, isoleucyl-, leucyl-, methionyl-, lysyl-, arginyl- and aspartyl-tRNA synthetases. An addition of free homologous casein kinase I does not change the spectrum or level of phosphorylation of the complex substrates. The homologous casein kinase II phosphorylates polypeptides with Mr of 65, 43 and 20 kDa in the complex.     

Herpes simplex virus phosphoproteins. II. Characterization of the virion protein kinase and of the polypeptides phosphorylated in the virion.

The protein kinase associated with purified herpes simplex virus 1 and 2 virions partitioned with the capsid-tegument structures and was not solubilized by non-ionic detergents and low, non-inhibitory concentrations of urea. The enzyme required Mg2+ or Mn2+ and utilized ATP or GTP. The activity was enhanced by non-ionic detergents and by Na+ even in the presence of high concentrations of of Mg2+, but not by cyclic nucleotides. The enzyme associated with capsid-tegument structures phosphorylated virion polypeptides only; exogenously added substrates (acidic and basic histones, casein, phosphovitin, protamine, and bovine serum albumin) were not phosphorylated. The major phosphorylated species were virion polypeptides (VP) 1-2, 4, 11-12, 13-14, 18.7, 18.8 and 23. VP 18.7 and VP 18.8 have not been previously detected, but may be phosphorylated forms of polypeptides co-migrating with VP 19. Of the remainder, only VP 23 has been previously identified as a capsid protein; the others are constituents of the tegument or of the under surface of the virion envelope. The distribution of the phosphate bound to viral polypeptides varied depending on the Mg2+ concentration and pH. In the absence of dithiothreitol, in vitro phosphate exchange was demonstrable in VP 23 and to a lesser extent in two other polypeptides on sequential phosphorylation frist with saturating amounts off unlabeled ATP and then with [gamma-32P]ATP. Analysis of the virion polypeptides specified by herpes simplex virus 1 X herpes simplex virus 2 recombinants indicates that the genes specifying the polypeptides which serve as a substrate for the protein kinase map in the unique sequences near the left and right reinterated DNA sequences of the L component.     

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.     

A synthetic peptide substrate for selective assay of protein kinase C.

Among various phosphate acceptor proteins and peptides so far tested, a synthetic peptide having the sequence surrounding Ser(8) of myelin basic protein, Gln-Lys-Arg-Pro-Ser(8)-Gln-Arg-Ser-Lys-Tyr-Leu, (MBP4-14), is the most specific and convenient substrate which can be used for selective assay of protein kinase C. This peptide is not phosphorylated by cyclic AMP-dependent protein kinase, casein kinases I and II, Ca2+/calmodulin-dependent protein kinase II, or phosphorylase kinase, and can be routinely used for the assay of protein kinase C with low background in the crude tissue extracts. The Km value is considerably low (7 microM) with a Vmax value of twice as much as that for H1 histone.     

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.     

The ribosomal proteins phosphorylated in vitro by protein kinase activities from Krebs II ascites cells

Studies were performed to identify in cytoplasmic extracts of Krebs II ascites cells protein kinase activities that might be responsible for the phosphorylation of the ribosomal proteins previously identified as phosphoproteins in these cells in vivo. Column chromatography resolved a casein kinase activity that could use ATP or GTP as a phosphoryl donor to phosphorylate, in ribosomes, exclusively the acidic 60S phosphoprotein(s) phosphorylated in vivo. A second casein kinase fraction could use ATP, only, in a similar reaction, but also contained protein kinase activity with respect to other ribosomal proteins, including the basic ribosomal protein phosphorylated in vivo, ribosomal protein S6. This latter was also among several proteins phosphorylated by an activity in the cyclic AMP-independent histone kinase fraction.     

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.     

Cyclic AMP-stimulated protein kinases at brain synaptic junctions.

We have examined endogenous cyclic AMP-stimulated phosphorylation of subcellular fractions of rat brain enriched in synaptic plasma membranes (SPM), purified synaptic junctions (SJ), and postsynaptic densities (PSD). The analyses of these fractions are essential to provide direct evidence for cyclic AMP-dependent endogenous phosphorylation at discrete synaptic junctional loci. Protein kinase activity was measured in subcellular fractions using both endogenous and exogenous (histones) proteins as substrates. The SJ fraction possessed the highest kinase activity toward endogenous protein substrates, 5-fold greater than SPM and approximately 120-fold greater than PSD fractions. Although the kinase activity as measured with histones as substrates was only slightly higher in SJ than SPM fractions, there was a marked preference of kinase activity toward endogenous compared to exogenous substrates in SJ fractions but in SPM fractions. Although overall phosphorylation in SJ fractions was increased only 36% by 5 micron cyclic AMP, there were discrete proteins of Mr = 85,000, 82,000, 78,000, and 55,000 which incorporated 2- to 3-fold more radioactive phosphate in the presence of cyclic AMP. Most, if not all, of the cyclic AMP-independent kinase activity is probably catalyzed by catalytic subunit derived from cyclic AMP-dependent kinase, since the phosphorylation of both exogenous and endogenous proteins was greatly decreased in the presence of a heat-stable inhibitor protein prepared from the soluble fraction of rat brain. The specific retention of SJ protein kinase(s) activity during purification and their resistance to detergent solubilization was achieved by chemical treatments which produce interprotein cross-linking via disulfide bridges. Two SJ polypeptides of Mr = 55,000 and 49,000 were photoaffinity-labeled with [32P]8-N3-cyclic AMP and probably represent the regulatory subunits of the type I and II cyclic AMP-dependent protein kinases. The protein of Mr = 55,000 was phosphorylated in a cyclic AMP-stimulated manner suggesting autophosphorylation as previously observed in other systems.     

Polyamine-sensitive protein kinase from chick intestinal mucosa

Summary A cyclic nucleotide-independent protein kinase which phoshorylates preferentially acidic proteins such as casein or phosvitin was isolated from cytosol of chick duodenal mucosa. The enzyme was purified more than 633 fold to apparent homogeneity by ammonium sulfate fractionation, column chromatography on DEAE-cellulose, phosphocellulose, hydroxylapatite and by sucrose density gradient centrifugation. The native enzyme has a molecular weight of 131000 as measured by gel filtration. The enzyme is a complex protein containing three polypeptides of molecular weight of 39 000, 36 000 and 27 000. It behaves as a complex throughout its purification and gel filtration but its components are readily separated by electrophoresis in denaturing buffer. The 27 000 molecular weight band was selectively autophosphorylated when the enzyme was incubated in the presence of [-³²P]ATP.When casein was used as substrate, physiological concentrations of naturally occurring polyamines such as spermine and spermidine markedly stimulated enzyme activity. However with phosvitin as substrate polyamines were strong inhibitors of the enzyme activity. This contrasting effect on intestinal kinase activity was also apparent using cytoplasmic proteins as endogenous phosphate acceptors. A characterization of this differential effect is presented and some possible physiological implications are discussed.     

Protein kinase activity associated with Fc gamma 2a receptor of a murine macrophage like cell line, P388D1

The properties of protein kinase activity associated with Fc receptor specific for IgG2a (Fc gamma 2aR) of a murine macrophage like cell line, P388D1, were investigated. IgG2a-binding protein isolated from the detergent lysate of P388D1 cells by affinity chromatography on IgG-Sepharose was found to contain four distinct proteins of Mr 50,000, 43,000, 37,000, and 17,000, which could be autophosphorylated upon incubation with [gamma-32P]ATP. The autophosphorylation of Fc gamma 2a receptor complex ceased when exogenous phosphate acceptors (casein or histone) were added in the reaction mixture. Casein was found to be a much better phosphate acceptor than histone in this system, as casein incorporated about 32-fold more 32P than histone did. Phosphorylation of casein catalyzed by Fc gamma 2a receptor complex was dependent on casein concentration (maximum phosphate incorporation being at 0.5 mg/mL), increased with time or temperature, was dependent on the concentration of ATP and Mg2+, and was maximum at pH near 8. Casein phosphorylation was significantly inhibited by a high concentration of Mn2+ (greater than 25 mM) or KCl (greater than 100 mM) or by a small amount of heparin (greater than 10 units/mL) and was enhanced about 2-fold by protamine. Casein kinase activity associated with Fc gamma 2a receptor used ATP as substrate with an apparent Km of 2 microM as well as GTP with an apparent Km of 10 microM. Prior heating (60 degrees C for 15 min) or treatment with protease (trypsin or Pronase) of Fc gamma 2a receptor complex almost totally abolished casein kinase activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and properties of a nuclear protein kinase from rat mammary gland

A nuclear protein kinase that shows a high degree of substrate specificity for the phosphorylation of the acidic proteins casein, phosvitin and non-histone chromatin proteins, rather than the basic proteins histones and protamine, was partially purified from lactatingrat mammary gland. The enzyme is associated with the acidic protein fraction of chromatin. Nuclear kinase requires Co²⁺ for activity, and other bivalent cations such as Mg²⁺ and Mn²⁺ can substitute partially for Co²⁺. The kinase is further activates (2–3-fold) by various salts, their concentration for maximum stimulation being: NaCl, 150mm; KCl, 200mm; sodium acetate, 300mm. The sedimentation coefficient of the nuclear kinase is 8.9S and its mol.wt. is approx. 300000 by gel-exclusion chromatography. The enzyme is not activated by cyclic AMP or cyclic GMP and is inhibited neither by the regulatory subunit of mammary cyclic AMP-dependent protein kinase nor by the heat-stable protein kinase inhibitor from ox heart. Analysis of ³²P-labelled protein products reveals that the kinase transfers the terminal phosphate of ATP to serine and threonine residues of proteins. The enzyme, however, has specificity for the phosphorylation of threonine in casein and serine in phosvitin. Molecular size and enzymic characteristics of the nuclear protein kinase are clearly different from those of the cytosol enzyme previously characterized.     

The protein phosphatases involved in cellular regulation. 1. Modulation of protein phosphatases-1 and 2A by histone H1, protamine, polylysine and heparin

The phosphorylase phosphatases in rat and rabbit liver cytosol that are markedly stimulated by histone H1, protamine and polylysine were identified as protein phosphatases-2A0, 2A1 and 2A2 by anion-exchange chromatography, gel-filtration and immunotitration experiments. Histone H1 and protamine also stimulated the dephosphorylation of phosphorylase kinase, glycogen synthase, fructose-1,6-bisphosphatase, pyruvate kinase, acetyl-CoA carboxylase and phenylalanine hydroxylase by phosphatases-2A1 and 2A2, and with several of these substrates activation was even more striking (20-100-fold) than that observed with phosphorylase (approximately 5-fold). Activation by basic polypeptides did not involve dissociation of these phosphatases to the free catalytic subunit. The dephosphorylation of phosphorylase by protein phosphatase-1 was suppressed by basic polypeptides, protamine and polylysine being the most potent inhibitors. However, the dephosphorylation of glycogen synthase, pyruvate kinase and acetyl-CoA carboxylase were markedly stimulated by histone H1 and protamine (2-13-fold). Consequently, with the appropriate substrates, protein phosphatase-1 can also be regarded as a basic-polypeptide-activated protein phosphatase. Heparin stimulated (1.5-2-fold) the dephosphorylation of phosphorylase by phosphatases-2A0 and 2A1, provided that Mn2+ was present, but phosphatase-2A2 and the free catalytic subunit of phosphatase-2A were unaffected. Heparin, in conjunction with Mn2+, also stimulated (1.5-fold) the dephosphorylation of glycogen synthase (labelled in sites 3 abc), phosphorylase kinase and phenylalanine hydroxylase by phosphatase-2A1, but not by phosphatase-2A2. By contrast, the dephosphorylation of phosphorylase and phosphorylase kinase by protein phosphatase-1 was inhibited by heparin. However, dephosphorylation of glycogen synthase and pyruvate kinase by phosphatase-1 was stimulated by this mucopolysaccharide. The studies demonstrate that basic proteins can be used to distinguish protein phosphatase-1 from protein phosphatase-2A, but only if phosphorylase is employed as substrate. Optimal differentiation of the two phosphatases is observed at 30 micrograms/ml protamine or at heparin concentrations greater than 150 microM.     

Properties of neurofilament protein kinase.

Neurofilament (NF) protein kinase, partially purified from NF preparations [Toru-Delbauffe & Pierre (1983) FEBS Lett. 162, 230-234], was found to be distinct from both the casein kinase present in NFs and the cyclic AMP-dependent protein kinase which is able to phosphorylate NFs. NF-kinase phosphorylated the three NF protein components. The amount of phosphate incorporated per molecule was higher for NF 200 than for NF 145 and NF 68. Other proteins present in the NF preparations were also used as NF-kinase substrates. Two of them might correspond to the myelin basic proteins with Mr values of 18,000 and 21,000. Four other substrates in the NF preparation were not identified (respective Mr values 53,000, 55,000, 65,000 and greater than 300,000). NF kinase also phosphorylated two additional brain-cell cytoskeletal elements: GFAp and vimentin. Casein, histones and phosvitin, currently used as substrates for protein kinase assays, were very poor phosphate acceptors. Half-maximal NF-kinase activity was obtained at an NF protein concentration of about 0.25 mg/ml in heated, salt-washed, NF preparations. The specific activity was about 5 pmol of 32P incorporated/min per microgram of NF kinase preparation protein. ATP was a phospho-group donor (Km 8 X 10(-5) M), but GTP was not. NF-kinase activity remained stable at 65 degrees C for more than 1 h. The enzyme was not degraded by storage at -20 degrees C for several months in a buffer containing 50% (w/v) sucrose. Maximal activity was obtained with 5 mM-Mg2+ (Mg2+ could be replaced by Co2+); Zn2+ and Cu2+ inhibited the reaction. NF-kinase was not dependent on cyclic AMP, cyclic GMP, Ca2+ or Ca2+ plus dioleoylglycerol and phosphatidylserine.     

Protein kinases of the chick oviduct: a study of the cytoplasmic and nuclear enzymes.

Subcellular fractionation of oviduct tissue from estrogen-treated chicks indicated that the bulk of the protein kinase activity of this tissue is located in the cytoplasmic and nuclear fractions, DEAE-cellulose chromatography of cytosol revealed a major peak of cAMP stimulatable activity eluting at 0.2 M KCl. This peak was further characterized and found to exhibit properties consistent with cytoplasmic cAMP dependent protein kinases isolated from other tissues; it had a Km for ATP of 2 X 10(-5) M, preferred basic proteins such as histones, as substrate, and had a M of 165 000. Addition of 10(-6) M cAMP caused the holoenzyme to dissociate into cAMP binding regulatory subunit and a protein kinase catalytic subunit. Extraction of purified oviduct nuclei with 0.3 M KCl released greater than 80% of the kinase activity in this fraction. Upon elution from phospho-cellulose, the nuclear extract was resolved into two equal peaks of kinase activity (designated I and II). Peak I had a sedimentation coefficient of 3S and a Km for ATP of 13 muM. while peak II had a sedimentation coefficient of 6S and a Km for ATP of 9 muM. Both enzymes preferred alpha-casein as a substrate over phosvitin or whole histone, although they exhibited different salt-activity profiles. The cytoplasmic and nuclear enzymes were well separated on phospho-cellulose and this resin was used to quantitate the amount of cAMP dependent histone kinase activity in the nucleus and the amount of casein kinase activity in the cytosol. Protein kinase activity in nuclei from estrogen-stimulated chicks was found to be 40% greater than hormone-withdrawn animals. This increase in activity was not due to translocation of the cytoplasmic protein kinase in response to hormone, but to an increase in nuclear (casein) kinase activity. During the course of this work, we observed small but significant amounts of cAMP binding activity very tightly bound to the nuclear fraction. Solubilization of the binding activity by sonication in high salt allowed comparison studies to be performed which indicated that the nuclear binding protein is identical with the cytoplasmic cAMP binding regulatory subunit. The possible role of the nuclear binding activity is discussed.