Protein phosphokinase activity of rat liver nuclear membrane

The presence of protein phosphokinase activity in a purified nuclear-membrane preparation from adult rat liver was demonstrated by measuring the incorporation of ³²P from γ-³²P-ATP into endogenous nuclear-membrane proteins as well as into the exogenous protein substrates, dephosphophosvitin (DPV) and lysine-rich histone (LRH). The activity of this enzyme toward DPV was 60 times greater than that toward LRH. cAMP and cGMP did not appear to affect the phosphorylation of endogenous-membrane proteins.     

Nuclear envelope of the seminal-vesicle epithelium.

The nuclear envelope of seminal-vesicle epithelium was isolated by a procedure involving enzymic digestion with deoxyribonuclease I, sonication in the presence of 0.34 M-sodium citrate, and centrifugation through sucrose density gradients. The mass of envelope DNA was only 0.8% of that of envelope protein, and by transmission electron microscopy the envelope was 98-99% pure. We showed that the envelope possess a protein kinase activity which is uninfluenced by cyclic nucleotides. Both lysine-rich histone and dephosphophosvitin as substrates gave a greater specific activity than did envelope protein itself. Optimum requirements with respect to Na+, Mg2+, pH and ATP were established for each substrate, and the influence of other factors on enzyme activity was investigated. Data, obtained mainly with the use of lysine-rich histone, are presented which indicate that nuclear envelope from intact and 96 h-castrated guinea pigs may have equal protein kinase activities and, in separate experiments, equal phosphoprotein phosphatase activities. Clarification of these initial observations must await identification of the natural substrates or the envelope's phosphorylation-dephosphorylation reactions.     

Effects of polyamines on prostatic chromatin- and non-histone-protein-associated protein kinase reactions.

Studies are presented on the influence of polyamines on prostatic chromatin- and non-histone-protein-associated protein kinase reactions involving both exogenous and endogenous substrates. The activities toward the model acidic protein substrate, dephosphophosvitin, were maximal at 160--200mM-NaCl (or -KCl or -NH4Cl). Under these conditions, spermidine and spermine added in concentrations up to 2mM were essentially without effect. However, without addition of NaCl to the medium, marked stimulation of these reactions was elicited by these polyamines at 1--2mM concentrations. The stimulatory effects were not due to non-specific changes in the ionic strength or to substitution of spermine for Mg2+, as maximal stimulation by 1 mM-spermine was observed only at optimal (2--4mM) Mg2+ concentrations. Qualitatively similar effects of polyamines were observed with enzyme preparations from the prostates of castrated rats, and with chromatin and non-histone-protein preparations from other tissues besides ventral prostate. When phosphorylation of endogenous non-histone proteins of the chromatin was measured, spermine stimulated both the initial rates and the final extent of transphosphorylation, even in the presence of optimal concentration of NaCl. By contrast, spermine or spermidine had no effect on the chromatin- and non-histone-protein-associated protein kinase reactions determined with lysine-rich histones as substrates. Chemically NN-dimethylated dephosphophosvitin was a less active substrate for the chromatin-associated protein kinase, but its phosphorylation was more markedly stimulated by spermine in comparison with unmodified dephosphophosvitin. These observations hint that the polyamine stimulations of the various protein kinase reactions may be due to effects on the conformations of the non-histone protein substrates rather than on the kinases themselves.     

Protein kinases and their substrates in brown adipose tissue from newborn rats.

The 10000 X g supernatant fraction of brown fat from newborn rats catalyzed the cyclic AMP-dependent phosphorylation of both histone and a preparation of proteins from the same subcellular fraction (endogenous proteins). The apparent affinity for ATP was lower for the phosphorylation of the endogenous proteins than for the phosphorylation of histone. In order to discover whether the phosphorylation of histone and the endogenous proteins were catalyzed by different enzymes, the 100000 X g supernatant was fractionated by ion-exchange and adsorption chromatography. Three different cyclic AMP-dependent protein kinases and one cyclic AMP-independent protein kinase were separated and partially purified. Each of these enzymes catalyzed the phosphorylation of both substrates, and the difference in apparent Km for ATP remained. Neither affinity chromatography on histone-Sepharose, nor electrophoresis on polyacrylamide gels resulted in the separation of the phosphorylation of histone from that of the endogenous proteins of any of the partially purified kinases. Moreover, experiments in which the phosphorylated substrates were separated by differential precipitation with trichloroacetic acid showed that the endogenous proteins competitively inhibited the phosphorylation of lysine-rich histone. It is concluded that each of the partially purified kinase preparations contains protein kinase, which catalyzes the phosphorylation of both substrates. The difference in apparent Km for ATP was found to be due to the presence in the endogenous protein preparation of a low molecular weight compound which competes with ATP. This was not ATP nor the modulator protein. The ratio of the phosphorylation of endogenous proteins to that of histone was much higher for the cyclic AMP-independent kinase preparation than for the other enzymes. Electrophoresis of the endogenous substrates in the presence of sodium dodecyl sulphate showed that the enzyme phosphorylated a greater number of proteins than did the cyclic AMP-dependent kinases. The phosphorylation of endogenous proteins relative to that of histone was significantly lower for one of the cyclic AMP-dependent kinases than for the other two. This difference was not reflected in a different pattern of phosphorylation of the individual proteins of the endogenous mixture.     

Chromatin-associated protein phosphokinases of rat ventral prostate. Characteristics and effects of androgenic status.

Protein phosphokinase activity endogenous to rat ventral prostate chromatin was assayed by using edphosphophosvitin as an exogenous substrate. For maximal activity of the kinase reaction, the presence of 200 mM NaCl, 5 mM MgCl2, and 1 mM dithiothreitol was essential. Two apparent pH optima were observed, a broad one between pH 7 and 7.4, and one at pH 7.89. At pH 7.4 the apparent Km for 31% dephosphophosvitin was 0.3 mg per ml. With respect to ATP, two apparent Km values (0.04 and 0.41 mM) were found. The kinase activity was minimal toward exogenous histones when used as substrates (3% for lysine-rich and 0.3% for arginine-rich (f3) histones, compared with dephosphophosvitin controls). The protein phosphokinases were not significantly stimulated by cyclic adenosine 3':5'-monophosphate (cyclic AMP) when histones used as substrate. With dephosphophosvitin as substrate, cyclic AMP produced a small inhibition (5 to 15%). Orchiectomy of adult rats resulted in a rapid decline in the chromatin-associated protein phosphokinase activity assayed using optimal experimental condition described above. At 9 hours postorchiectomy, a 30% decline in the activity was observed; this was further reduced to about 50% of the control by 18 hours. This decrease in the kinase activity (e.g. at 9 hours postorchiectomy) appears to precede measurable changes in the protein and RNA complements of chromatin. Testosterone replacement following orchiectomy abolished this decline in the chromatin-associated activity. The chromatin-associated protein phosphokinase activity toward lysine-rich and arginine-rich histones was also sensitive to androgenic status of the animals and declined rapidly postorchiectomy. The results suggest the presence of multiple and androgen-sensitive protien phosphokinases associated with rat ventral prostate chromatin, which may modulate the phosphorylation of nuclear nonhistone phosphoproteins with changing gene action mediated by testosterone in this target tissue.     

PHOSPHORYLATION AND METHYLATION OF CHROMATIN PROTEINS FROM MOUSE BRAIN NUCLEI

Abstract— Chromatin protein kinase and histone methyltransferase are present in two nuclear populations, neuronal and oligodendroglial. At least 30% of the enzymes are tightly bound to chromatin. The specific activities of both enzymes are higher in neuronal populations than in oligodendroglial. Protein kinase from these nuclear populations phosphorylates endogenous protein; however, the methyltransferase requires exogenous histone as substrate. The methyltransferase from both nuclear populations preferentially methylates lysine-rich histone.     

Membrane-associated protein kinase activities in seminal plasma from vasectomized men

Differential centrifugation was used to prepare heavy and light membrane fractions from the seminal plasma of vasectomized men. The two membrane fractions combined contained half of the phosvitin and histone kinase activities but only 7% of the total protein content in vasectomy semen. These two kinase activities as well as phosphorylation of endogenous membrane proteins were optimally stimulated by Mg2+; Mn2+ could effectively substitute for Mg2+ only in endogenous phosphorylation reactions. Neither the phosvitin nor histone kinase responded to cAMP or cGMP, but the histone kinase was strongly inhibited by the heat-stable cAMP-dependent protein kinase inhibitor. The phosvitin kinase was not affected by this inhibitor. The phosphorylation of endogenous proteins in the heavy membrane fraction was not affected by the protein kinase inhibitor but protein phosphorylation in the light membrane fraction was partly (45%) inhibited. The differential effects of increased ionic strength, sulphydryl protecting agents, and the protein kinase inhibitor on protein kinase activity towards lysine-rich histones, phosvitin and endogenous proteins, as well as differential extractability and binding to an anion exchange column of histone kinase and phosvitin kinase activities, indicate that more than one kinase activity is present in these membrane subfractions. Electron microscopic examination showed that there are several kinds of membrane-limited components in vasectomy seminal fluid that vary in size, density, and ultrastructure. The association of type(s) of protein kinase to individual membrane components remains to be established.     

Phosphoprotein phosphatase activity of rat liver nuclear membrane.

Nuclear membranes from rat liver contain a phosphoprotein phosphatase activity capable of dephosphorylating endogenous nuclear membrane phosphoproteins. This activity was also expressed towards the ³²P-labeled exogenous phosphoprotein substrates phosvitin and lysine-rich histone. Differential effects of altered ionic strength, EDTA, pyrophosphate, and 2-mercaptoethanol on the phosphatase activity towards the two exogenous substrates suggest the presence of multiple phosphatases in the nuclear membrane. ATP, ADP, and sodium fluoride inhibited activity towards both exogenous substrates, while cyclic AMP or cyclic GMP at 10^?6M had no apparent effect.     

Purification of nuclear cAMP-independent protein kinases from rat ventral prostate

Two nuclear cAMP-independent protein kinases (designated PK-N1 and PK-N2) were purified from rat ventral-prostate and liver. The yield of enzyme units was 4-5% and 7-9% for each enzyme from the prostatic nuclei and liver nuclei, respectively. The average fold purification for prostatic nuclear protein kinase N1 and N2 was 1360 and 1833, respectively. The respective average specific activity of the two enzymes towards casein was 81,585 and 110,000 nmol 32P incorporated/hr/mg of enzyme. Protein kinase N1 comprised one polypeptide of Mr 35,000 which underwent phosphorylation in the presence of Mg2+ + ATP. Protein kinase N2 comprised two polypeptides Mr 40,000 and 30,000 of which only the Mr 30,000 polypeptide was autophosphorylated. Both enzymes were active towards casein, phosvitin, dephosphophosvitin, spermine-binding protein, and non-histone proteins in vitro. Little activity was detected towards histones. Both enzymes were stimulated by 150-200 mM NaCl. MgCl2 requirement varied with the protein substrate but was between 2-4 mM for both enzymes. With dephosphophosvitin as substrate, the apparent Km for ATP for N1 protein kinase was 0.01 mM. GTP did not replace ATP in this reaction. Protein kinase N2 was active in the presence of ATP or GTP. The apparent Km was 0.01 mM for ATP, but 0.1 mM for GTP.     

Hydroxylation of lysyl residues in lysine-rich and arginine-rich histones by lysyl hydroxylase in vitro.

Lysine-rich and arginine-rich histones were examined as substrates for lysyl hydroxylase. Both proteins are known to be rich in lysyl residues, and lysine-rich histone also contains -X-Lys-Gly-sequences, whereas no such sequences are found in the arginine-rich histone. Both histones were found to be hydroxylated by lysyl hydroxylase, and the time courses of the hydroxylation reactions with these substrates were linear for at least 60 min. The Km values observed where 3 - 10(-6)M for heat-denatured lysine-rich histone and 6 - 10(-6)M for heat-denatured arginine-rich histone. Heat-denatured lysine-rich histone was hydroxylated at a higher rate than non-denatured both at 37 and 25 degrees C. No such phenomenon was found, however, when arginine-rich histone was examined as a substrate. Furthermore, at 37 degrees C lysine-rich histone was a better substrate for lysyl hydroxylase then arginine-rich histone, but this relationship was reversed at 25 degrees C. The synthesis of hydroxylysine observed with arginine-rich histone indicates that the lysyl hydroxylase preparation used in these experiments catalyzes the synthesis of hydroxylysine not only in the sequence -X-Lys-Gly-, but also in some other sequences. Certain collagen polypeptide chains are known to contain one hydroxlysyl residue in a sequence other than -X-Lys-Gly-, and the present results may explain this finding.     

The identification and properties of phosphatases in skeletal muscle with activity towards the inhibitory subunit of troponin, and their relationship to other phosphoprotein phosphatases.

1. Phosphoprotein phosphatases with activity towards the inhibitory subunit of troponin (troponin I), phosphorylase a and lysine-rich histone (fraction F1) have been fractionated from rat skeletal muscle by chromatography on Sephadex G-200 and polylysine-Sepharose. Six separate fractions were identified on the basis of substrate specificity and behaviour during chromatography. 2. All fractions showed similar Km values for any given protein substrate. The Km for troponin I (5 muM) was significantly lower than that previously reported. 3. Phosphatase activities towards troponin I and hosphorylase a did not show a requirement for bivalent-metal ions. Two of the fractions with only minor activity towards histone were activated by Mn2+. 4. Discontinuous polyacrylamide-gel-electrophoresis studies indicated that several of the fractions contained more than one phosphatase activity, and additionally showed that several of the activities could exist in different aggregation states. On the basis of these studies at least two phosphatases with activity only towards troponin I were identified. In addition, phosphorylase phosphatase (which has considerable activity towards troponin I) and a general phosphatase with activity towards all three substrates were found. 5. A fraction with mol.wt. of 150000 could be activated by freezing with 2-mercaptoethanol or by heating to 55 degrees C. This activation was accompanied by a decrease in mol.wt. to 25000. 6. The total amount of phosphatase with activity towards troponin I which was extracted would be sufficient to dephosphorylate all the troponin I present in skeletal muscle in approximately 10s.     

Inhibition by gossypol of phospholipid-sensitive Ca2+-dependent protein kinase from pig testis

Gossypol, a polyphenolic binaphthalene-dialdehyde extracted from cotton plants which possesses male antifertility action in mammals, is a potent inhibitor of phospholipid-sensitive Ca2+-dependent protein kinase from pig testis. Gossypol inhibited Ca2+-dependent activity of the enzyme without affecting its basal activity. The IC50 value (concentration causing 50% inhibition) was 31 microM when lysine-rich histone was used as substrate. Kinetic analysis indicated that the compound inhibited the enzyme non-competitively with respect to ATP (Ki = 31 microM) or lysine-rich histone (Ki = 30 microM), and competitively with respect to phosphatidylserine (Ki = 2.1 microM). With Ca2+, irrespective of the presence or absence of 1,3-diolein, the compound lowered Vmax and increased the apparent Ka for Ca2+. The compound also inhibited phosphorylation by the enzyme of high-mobility-group 1 protein (one of the endogenous substrates in the testis for the enzyme located in nucleosome), with an IC50 value of 88 microM. These results suggested that a phospholipid-sensitive Ca2+-dependent protein phosphorylation system in the testis is involved in the regulation of spermatogenesis.     

Immunological identification of protein kinase C-alpha and protein kinase C-delta in cultured rat mesangial cells: differential sensitivity of the two isoforms towards the protein kinase inhibitor H7.

Rat mesangial cells contain both calcium-dependent protein kinase C (PKC) activity, which phosphorylates histone H1 and endogenous proteins, and calcium-independent, phospholipid-dependent PKC activity, which phosphorylates only endogenous proteins. The calcium-dependent PKC was identified as PKC alpha by immunoblot analysis and hydroxyapatite chromatography (HPLC). The calcium-insensitive, phospholipid-dependent isoform was identified as PKC delta using similar techniques. The inhibition of these two PKC isoforms by the protein kinase inhibitor H7 [1-(iso-quinolinyl sulphonyl)-2-methyl piperazine] was examined using both histone H1 and endogenous proteins as substrates. Phosphorylations catalyzed by the calcium-dependent PKC isoform alpha were almost 90% inhibited when histone H1 was used, and only 55% when endogenous proteins were the substrate. In contrast, the phosphorylation of endogenous proteins catalysed by the calcium-insensitive, phospholipid-dependent PKC delta was not significantly affected by the inhibitor.     

Purification of cytosolic cAMP-independent protein kinases from rat ventral prostate

Two cAMP-independent protein kinases were purified from rat ventral-prostate and liver cytosol, and were designated PK-C1 and PK-C2 to distinguish them from the nuclear protein kinases described in the preceding paper. The yield of the prostate enzymes was about 5% each, and about 10% each for the liver enzymes. The average fold purification of the prostatic enzymes was 1892 and 3176 for protein kinase C1 and C2, respectively. Their average respective specific activity towards casein was 40,111 and 67,340 nmol 32P incorporated/hr per mg of enzyme protein. protein kinase C1 comprised one polypeptide of Mr 39,000 which underwent phosphorylation in the presence of Mg2+ + ATP. Protein kinase C2 comprised three polypeptides of Mr 41,000; 38,000; 26,000. Of these only the Mr 26,000 polypeptide was autophosphorylated. The Mg2+ requirement for protein kinase C1 and C2 was between 1 and 4 mM depending on the nature of the protein substrate. Both enzymes were stimulated by 100-200 mM NaCl. Km for ATP for C1 and C2 kinases was 0.01 mM; GTP could be used only by protein kinase C2 but with a markedly lower affinity. The enzymes were active towards casein, phosvitin, dephosphophosvitin, and spermine-binding protein in vitro, but demonstrated little activity towards histones. Despite several similarities in these general properties of cytosolic protein kinases C1 and C2 with those of nuclear protein kinases N1 and N2, a number of differences are also noted.     

Cyclic nucleotide-independent protein kinases bound to cytoplasmic and nuclear polyribosomes in non-infected and adenovirus-infected HeLa cells

Cyclic nucleotide-independent protein kinase activity bound to cytoplasmic and nuclear polyribsomes from non-infected and adenovirus-infected HeLa cells was compared. The enzymes catalysed the incorporation of phosphate from gamma-(32)P-labelled ATP or GTP into acid-precipitable material in the absence of exogenous substrates. Their activity was not affected by cyclic AMP or cyclic GMP and was not inhibited by a cyclic nucleotide-dependent protein kinase-inhibitor protein. The kinases are tightly bound to polyribosomes of either origin from infected and non-infected cells, since treatment with 0.5m-NaCl did not dissociate the activity. The enzymes and the enzyme-associated endogenous substrates of cytoplasmic polyribosomes are significantly different from those of the nucleus, and adenovirus infection of the cells did not alter the nature of the enzymes or the substrates at 18-20h after infection. Nuclear kinases catalysed 3-4-fold more phosphate incorporation than did the cytoplasmic kinases. They did not phosphorylate endogenous substrates in the cytoplasmic preparations, and vice versa, which suggests that such substrates for cytoplasmic and nuclear kinases are specific. Polyacrylamide-gel electrophoresis of the phosphorylated proteins revealed the presence of a higher number of endogenous substrates in the nuclear preparation. The nuclear kinases phosphorylated all histones from HeLa cells, but the cytoplasmic ones phosphorylated predominantly the histone of mol.wt. 12000. Bovine heart kinase phosphorylated several low-molecular-weight cytoplasmic proteins and no nuclear proteins. With a DEAE-cellulose column either enzyme activity could be resolved into a number of peaks. The substrate specificities of these peaks indicate that there are at least two different forms of the enzyme in each preparation of polyribosomes.     

Protein kinases and their protein substrates associated with chromatin and ribosomes in SV40-transformed rat cells.

The level of endogenous protein phosphorylation in non-histone chromosomal and ribosomal wash proteins is 7--10 times greater in SV40-transformed rat cells than in untransformed parental cells. Protein kinase activity in these proteins was fractionated by either phosphocellulose or DEAE-cellulose chromatography. One major and one minor component were detected in non-histone proteins and only one component in ribosomal wash proteins when the activity in each fraction was measured with an exogenous substrate, casein. These enzymes prefer casein to whole histone as substrate and are cyclic AMP-independent. The enzyme activity in a major peak of non-histone proteins and in ribosomal wash proteins measured with casein as substrate is 3 times greater in transformed cells than in untransformed cells, whereas pH optimum, cation requirements and apparent Km values for casein and ATP are identical or very similar in the two cell types. No significant phosphatase was detected in non-histone and ribosomal wash proteins from the two types of cell. The patterns of endogenous protein phosphorylation in these protein fractions analysed by gel electrophoresis are significantly different between these cells. These results suggest that the high level of endogenous protein phosphorylation in non-histone and ribosomal wash proteins from SV40-transformed cells is caused mainly by the increased activity of protein kinase and the nature of protein substrates.     

Histones H3 and H4 inhibit protein kinase C specifically

The lysine-rich histone H1 is a preferred substrate for the Ca2+-phospholipid-dependent protein kinase (protein kinase C). Histones H3 and H4 are poor substrates but potent inhibitors of the enzyme. The inhibitory effect of H3 and H4 seems to result mainly from a decreased sensitivity of protein kinase C to stimulation by phosphatidylserine (PS). These observations suggest that site-specific phosphorylation of one histone type can be regulated by other histones.     

A lysine-rich protein functions as an H1 histone in Dictyostelium discoideum chromatin.

Mononucleosomes released from Dictyostelium discoideum chromatin by micrococcal nuclease contained two distinctive DNA sizes (166-180 and 146 bp). Two dimensional gel electrophoresis suggested a lysine-rich protein protected the larger mononucleosomes from nuclease digestion. This was confirmed by stripping the protein from chromatin with Dowex resin. Subsequently, only the 146 bp mononucleosome was produced by nuclease digestion. Reconstitution of the stripped chromatin with the purified lysine-rich protein resulted in the reappearance of the larger mononucleosomes. Two-dimensional gel electrophoresis showed the protein was associated with mononucleosomes. Hence, the protein functions as an H1 histone in bringing the two DNA strands together at their exit point from the nucleosome. Trypsin digestion of the lysine-rich protein in nuclei resulted in a limiting peptide of approx. 10 kilodaltons. Trypsin concentrations which degraded the protein to peptides of 12-14 kilodaltons and partially degraded the core histones did not change the DNA digestion patterns obtained with micrococcal nuclease. Thus, the trypsin-resistant domain of the lysine-rich protein is able to maintain chromatosome structure.     

A cyclic adenosine 3',5'-monophosphate-dependent histone kinase from pig brain. Purification and some properties of the enzyme.

A cyclic adenosine 3',5'-monophosphate-dependent histone kinase (ATP: protein phosphotransferase, EC 2.7.1.37) was isolated from pig brain. The enzyme has been purified 1140-fold; it is homogeneous on polyacrylamide gel electrophoresis and gel filtration. The estimated molecular weight of the enzyme is 120 000. Histone kinase dissociates into a catalytic subunit and a regulatory one (molecular weights 40 000 and 90 000, respectively). The catalytic subunit has been obtained in homogeneous state as evidenced by sodium dodecylsulphate-polyacrylamide gel electrophoresis. At all purification steps, enzymatic activity is stimulated 5-fold by cyclic AMP. An apparent Km value for cyclic AMP is about 3.3 - 10- minus 7 M. In the presence of cyclic AMP(5 - 10- minus 6 M), the Km value for ATP and F1 histone were 1.2 - 10- minus five and 3 - 10- minus 5 M, respectively. Optimum pH value for histone kinase is 6.5, its isoelectric point is situated at pH 4.6. The purified enzyme displays high specificity for the lysine-rich and moderately lysine-rich histones F1, F2a2 and F2b. Arginine-rich histones and other known protein substrates for cyclic AMP-dependent protein kinases (casein, Escherichia coli RNA polymerase, etc.) are extremely poor substrates for this enzyme.