Phosphorylation Of Non-histone Proteins Research Articles

Casein kinase II activity of buffalo sperm chromatin.

Two cyclic AMP-independent protein kinase activities have been found associated with buffalo sperm chromatin: a histone kinase highly specific for arginine-rich histone was reported recently (Mudgal et al., 1997: Arch Andrology 38:191-199) and a casein kinase II is described here. Casein kinase activity was solubilized with 0.35 M NaCl, which extracted 90% of the initial enzyme activity associated with buffalo sperm chromatin. Of the two acidic proteins tested, casein was preferred substrate over phosvitin. Among the casein fractions, the order of preference for casein kinase was beta-casein > alpha-casein > casein. Cyclic AMP at concentrations up to 50 microM had no effect on the phosphorylation of casein. Phosphoamino acid analysis using casein as the substrate showed threonine to be the acceptor amino acid for phosphoester link. Phosphorylation specificity was determined by phosphorylating buffalo beta-casein followed by the preparation of tryptic peptides and identification of amino acid residue phosphorylated. Threonine residue at position 41 having clusters of acidic amino acid residues (Thr. Glu. Asp. Glu) C-terminal to it was phosphorylated, a phosphorylation specificity akin to CKII. It is thought that phosphorylation of histones decreases their association with DNA and probably makes the DNA more available for replication, while phosphorylation of nonhistone proteins modifies their interaction with histones, allowing control of template activity. Two protein kinases found in buffalo sperm chromatin may perform a similar function.

Nuclear casein kinase 2 (CK-2) activity in human normal, benign hyperplastic, and cancerous prostate.

In previous work, we had observed that chromatin-associated nonhistone protein phosphorylation, catalyzed by intrinsic protein kinase reaction in chromatin preparations from human benign prostatic hyperplasia (BPH) prostate samples was markedly elevated, compared with the normal prostate chromatin samples [Rayan et al: Cancer Res 45:2277-2282, 1985]. The properties of this protein kinase reaction were suggestive of the involvement of casein kinase(s). By employing the specific synthetic substrate for casein kinase 2 (CK-2) for assays in cellular fractions, we have shown that this protein kinase is present in human prostate chromatin. Its activity is increased in BPH chromatin by about 25-fold, as compared with its activity in the normal prostate chromatin. This suggests that CK-2 is a possible mediator of the enhanced phosphorylation of chromosomal proteins in BPH chromatin. By comparison, CK-2 activity in chromatin preparations from prostatic carcinoma samples was markedly less elevated than that of the BPH chromatin. Immunohistochemical analysis of the enzyme in human frozen sections of prostate tissue samples showed that the enzyme immunostaining was diffuse in the cytoplasm, but more intense in the nucleus, especially in the nucleoli. In general, the staining corresponded with the enzymic data. However, sections from prostatic carcinoma samples appeared to show differential staining, depending on the Gleason's grade of the sample. The samples with higher Gleason's grade showed less intense immunostain in the nucleus, compared with samples of lower Gleason's grade. Further, regions of sections in samples with higher Gleason's grade did not show any immunostaining. These differences in the characteristics of CK-2 expression in prostatic carcinoma samples may be potentially significant, but need to be evaluated further for their significance to the pathobiology of prostatic neoplasia.

Nature of the intrinsic protein kinases involved in phosphorylation of non-histone proteins in intact prostatic nuclei: further identification of androgen-sensitive protein kinase reactions

Nuclei isolated from rat ventral prostate contain a number of messenger-dependent and -independent protein kinases. Studies were undertaken to determine the relative contribution of these protein kinases in phosphorylation of non-histone proteins (NHPs) in isolated nuclei. The data suggest that messenger-dependent protein kinases such as those dependent on cAMP or Ca2+/calmodulin or Ca2+/phospholipid may be present in very small amounts in intact isolated nuclei, and thus appear not to be significantly involved in phosphorylation of endogenous NHPs. Messenger-independent nuclear associated protein kinases PK-N1 and PK-N2 are known to catalyze the phosphorylation of NHPs in vitro (Goueli Sa, et al., Eur J Biochem 113: 45-51, 1980). Of these, the intrinsic heparin-sensitive PK-N2 as compared with heparin-insensitive PK-N1 appeared to be the predominant protein kinase engaged in phosphorylation of NHPs in intact nuclei. About 78-88% of NHP phosphorylation in intact nuclei was inhibited by heparin suggesting that the remaining 12-22% phosphorylation of NHPs was catalyzed via the heparin-insensitive protein kinase(s). Further, the data provide additional evidence that heparin-sensitive PK-N2 is the one that is most responsive to androgenic status in the animal.

ラット唾液腺核蛋白質のりん酸化の加齢に伴う動態

Development, growth, maturation and aging processes of secretory cells of rat salivary glands progress mainly after birth. Nuclear non-histone proteins, phosphorylated actively and reversively, have an important role as regulatory molecules of gene activity and have a possibility to bring about specific changes in these cellular processes. We examined in the present study the age-dependent changes in the phosphorylation of non-histone proteins of rat salivary glands. Nuclei purified from submandibular and parotid glands of 8-week-old rats rapidly incorporated 32P from gamma-32P-ATP into the nuclear phosphoproteins and reached equilibrium within 9 min. A preponderant amount of the 32P was present in non-histone proteins. The levels of phosphorylation of non-histone proteins in salivary gland nuclei increased rapidly after birth, reaching a maximum in both gland nuclei of 4-week-old rats and then decreasing to the levels observed in submandibular and parotid gland nuclei from 20 and 16-week-old rats, respectively. These levels were still maintained in nuclei from aged rats. Moreover, age-dependent changes in the protein kinase activity of submandibular and parotid gland nuclei were linked up with the changes in the phosphorylation of non-histone proteins. However, changes were not observed in the phosphorylation of histone proteins after birth. These results suggest that protein kinase activity in salivary gland nuclei may have an important role on age-dependent changes in cell function, mediated through the control of the phosphorylation of non-histone proteins.

Nuclear protein synthesis in thymocytes of X-irradiated rats.

The biosynthesis and phosphorylation of nuclear proteins of thymocytes were investigated in rats after 4.0 Gy whole-body X-irradiation during the period which precedes DNA degradation in lymphoid cells. Proteins were separated by two-dimensional gel-electrophoresis, detected by Coomassie blue staining. Incorporation of [35S]methionine into proteins and phosphorylation of proteins with [32P]inorganic phosphate were determined by scanning densitometry of autofluorograms and autoradiograms, respectively. No change in the quantity of proteins was observed 1 h after irradiation. Decrease in specific activity of [35S]methionine-labelled proteins was seen in most protein fractions. Significant enhancement of phosphorylation of three proteins was established, characterized by molecular weight and pH: MW 20 kD, pH 6.8; MW 35 kD, pH 5.8 and MW 48 kD, pH 5.8. These results suggest that immediately after X-irradiation a short-term increase of chromatin-bound non-histone protein phosphorylation occurs. This finding, along with the previously described enhancement of RNA polymerase II in thymocytes (Zhivotovsky et al. 1982) suggests a temporary gene activation shortly after X-irradiation of the rat.

Changes in phosphorylation of nonhistone proteins during differentiation of a lower eukaryote Physarum polycephalum

During starvation-induced differentiation of a slime mold Physarum polycephalum several changes in the phosphorylation of nuclear proteins occur. The overall content of serine- and threonine-bound phosphate drops by 50% and de novo phosphorylation of a number of nonhistone proteins is drastically altered. On the contrary, no selective dephosphorylation of nuclear proteins phosphorylated under normal growth accompanies differentiation.

Molecular mechanisms of the chromosome condensation and decondensation cycle in mammalian cells.

AbstractThe chromosomes undergo a condensation‐decondensation cycle within the life cycle of mammalian cells. Chromosome condensation is a complex and critical event that is necessary for the equal distribution of genetic material between the two daughter cells. Although chromosome condensation‐decondensation and segregation is mechanistically complex, it proceeds with high fidelity during the eukaryotic cell division cycle. Cell fusion studies have indicated the presence of chromosome condensation factors in mammalian cells during mitosis. If extracts from mitotic cells are injected into immature oocytes of Xenopus laevis, they induce meiotic maturation (i.e. germinal vesicle breakdown and chromosome condensation) within 2–3 hours. Recently, we showed that the maturation‐promoting activity of the mitotic cell extracts is inactivated by certain protein factors present in cells during the G1 period. The activity of the G1 factors coincides with the process of chromosome decondensation that begins at telophase and continues throughout the G1 period. These studies have revealed that the mitotic factors and the G1 factors play a pivotal role in the regulation of condensation and decondensation of chromosomes. Furthermore, our studies strongly suggest that nonhistone protein phosphorylation and dephosphorylation may mediate chromosome condensation and decondensation, respectively.

Differential localization and androgen sensitivity of prostatic nuclear protein kinases in euchromatin and heterochromatin fractions.

We have examined the distribution and androgenic regulation of protein kinases and phosphoproteins in euchromatin and heterochromatin fractions of rat ventral prostate chromatin. Available procedures to prepare euchromatin and heterochromatin fractions were found to result in the loss of various chromatin-associated protein kinases even though there was no gross change in the gel electrophoretic profile of proteins in these fractions. This loss was prevented by the addition of 0.5 mM phenylmethylsulfonyl fluoride throughout the preparative procedures, which indicates that the protein kinases associated with the chromatin may be particularly susceptible to proteolytic degradation during further subfractionation. By utilizing an improved method for fractionation of chromatin, we have demonstrated a marked enrichment of protein kinase activity (towards phosvitin and endogenous chromosomal proteins) in the euchromatin fraction as compared with heterochromatin. Both of these fractions were also examined for the incorporation of 32P into two main classes of nonhistone proteins (namely, H2SO4-soluble and -insoluble nonhistones). The amount of 32P incorporated into heterochromatin-associated proteins of both classes was markedly less than that in the euchromatin-associated proteins. Protein kinase activities (especially those active towards phosvitin and nonhistone proteins) in the euchromatin fraction as compared with the heterochromatin were significantly reduced within 24 h after androgenic deprivation in the animal. The decreased phosphorylation of nonhistone proteins could be attributed to the loss of endogenous protein kinase activity. The results indicate that not only are chromatin-associated protein phosphokinases preferentially localized in euchromatin fractions but also that these euchromatin-associated protein kinases display the greatest sensitivity to androgenic status of the animal.

Posttranslational phosphorylation of specific chromosomal proteins and transcription of hnRNA genes in isolated nuclei: retention of in vivo sensitivity to 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB).

The rapidly turning over phosphorylation of specific nuclear nonhistone proteins, especially 42-, 33-, and 30-kDa polypeptides, and its relation to the transcriptional activity of hnRNA genes was investigated in isolated nuclei from salivary gland cells of Chironomus tentans. Incubation conditions promoting the phosphorylation of nonhistone proteins as well as the transcriptional activity of RNA polymerase II were established. The pattern of 32P incorporation into the nonhistone proteins found in isolated nuclei resembled that obtained in experiments with intact cells, and the endogenous RNA polymerase II retained its ability to reinitiate the transcription under in vitro assay conditions. In addition, the in vivo sensitivity of the phosphorylation of 42-, 33-, and 30-kDa polypeptides, like the sensitivity of the initiation of hnRNA transcription to 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), were preserved in the nuclear preparation. The experimental data taken together provide further support for the idea that the activation of hnRNA genes is causally related to the phosphorylation of specific nonhistone proteins.

Phosphorylation of nonhistone proteins during premature chromosome condensation in a temperature-sensitive mutant, tsBN2.

In tsBN2 cells, a temperature-sensitive (ts) mutant of the BHK21 cell line, with a ts-defect in its regulatory system for chromosome condensation, antigens that react with mitotic specific mouse monoclonal antibody MPM-2 were produced when premature chromosome condensation (PCC) was induced by a temperature shift. The polypeptides of antigens recognized by MPM-2 in tsBN2 cells with PCC were identical to those of antigens in mitotic cells. These antigens appeared concomitantly with chromosome condensation, which suggests that these mitotic-specific antigens may be related to chromosome condensation. As the production of mitotic-specific antigens was inhibited by W-7, a specific and potent antagonist of calmodulin, calmodulin may function in the mitotic phosphorylation of nonhistone protein.

Phosphorylation of non-histone proteins associated with mitosis in HeLa cells

Our previous studies indicated that certain non-histone proteins (NHP) extractable with 0.2 M NaCl from mitotic HeLa cells induce germinal vesicle breakdown and chromosome condensation in Xenopus laevis oocytes. Since the maturation-promoting activity of the mitotic proteins is stabilized by phosphatase inhibitors, we decided to examine whether phosphorylation of NHP plays a role in the condensation of chromosomes during mitosis. HeLa cells, synchronized in S phase, were labeled with 32P at the end of S phase, and the cells subsequently collected while they were in G2, mitosis, or G1. Cytoplasmic, nuclear, or chromosomal proteins were extracted and separated by gel electrophoresis. The labeled protein bands were detected by radioautography. The results indicated an 8–10-fold increase in the phosphorylation of NHP from mid-G2 to mitosis, followed by a similar-size decrease as the cells divided and entered G1. The NHP phosphorylation rate increased progressively during G2 traverse and reached a peak in mitosis. Radioautography of the separated NHP revealed eight prominent, extensively phosphorylated protein bands with molecular masses ranging from 27.5 to 100 kD. These NHP were rapidly dephosphorylated during M-G1 transition. Phosphorylation—dephosphorylation of NHP appeared to be a dynamic process, with the equilibrium shifting to phosphorylation during G2-M and dephosphorylation during M-G1 transitions. These results suggest that besides histone H1 phosphorylation, phosphorylation of this subset of NHP may also play a part in mitosis.

Phosphorylation of nonhistone proteins during the HeLa cell cycle. Relationship to DNA synthesis and mitotic chromosome condensation.

Cell cycle variations in the phosphorylation of chromatin-associated nonhistones were determined. Cells were radiolabeled with [32P]orthophosphate and chromatin was obtained by mild digestion of nuclei with micrococcal nuclease. The experiments were performed in the presence of a substrate inhibitor of alkaline phosphatase, beta-glycerophosphate. The results show that, while similar molecular weight species of phosphorylated nonhistones are associated with interphase chromatin through the HeLa cell cycle, the incorporation (32P cpm/micrograms of protein) profiles of selected major phosphononhistones show substantial changes. The most prominent peaks of specific radioactivity occur in the DNA synthesis phase (S phase). The phosphorylation states of the proteins of isolated metaphase chromosomes were also determined. Nonhistone proteins of isolated metaphase chromosomes are strikingly dephosphorylated, especially in comparison to histone H1. The phosphorylation of the major phosphononhistone of chromatin, which has a molecular weight of 55,000, was further characterized by techniques that included one-dimensional peptide mapping in sodium dodecyl sulfate-polyacrylamide gels and nonequilibrium pH gradient slab gel electrophoresis. Phosphoproteins are also components of the nuclear scaffold, and cell cycle variations in these proteins were investigated. The primary phosphorylated species has a molecular weight of 119,000. As with chromatin-associated nonhistones, this nuclear scaffold protein shows substantial incorporation of 32P in S phase, and a high level of incorporation also occurs close to mitosis.

Differential effects of polyamines on the phosphorylation of chromatin-associated proteins.

Studies are presented on the nature of chromatin-associated phosphoproteins whose phosphorylation is influenced by polyamines. After labelling with 32P, chromatin-associated proteins were separated into four fractions. Fraction I comprised neutral and basic non-histone phosphoproteins, including high-mobility-group non-histones; fraction II consisted mostly of histones; fraction III consisted of a class of (salt-soluble) acidic non-histone phosphoproteins; and fraction IV consisted of residual (salt-insoluble) acidic non-histone phosphoproteins. The average relative distribution of protein in the four fractions (I-IV) was about 1:4:2:1 for both liver and prostate. However, tissue-dependent differences were observed in the incorporation of 32P in various protein fractions. In the presence of polyamines (e.g. 1 mM-spermine or 2 mM-spermidine) maximal stimulation of phosphorylation was observed in non-histone proteins of fraction I (160-180%), followed by that in non-histone proteins of fraction III (80-110%). The phosphorylation of residual non-histone proteins in fraction IV, and the small extent of phosphorylation of histones in fraction II, remained unaltered in the presence of polyamines. Thus polyamines do not stimulate the phosphorylation of all non-histone proteins; their stimulative effect is most prominent in the phosphorylation of neutral and basic non-histone proteins and a class of salt-soluble acidic non-histone proteins. In accord with our hypothesis, these differential effects of polyamines on phosphorylation of endogenous non-histone proteins may relate to the conformation of these substrates rather than to endogenous kinases.

Effect of calcium and calmodulin on RNA synthesis in isolated nuclei from rat liver cells

Calcium regulates a great variety of intracellular processes [l-4]. In muscular contraction [5] or the activation of adenylate [6,7], calcium, acting as a coupling or second messenger 141, binds to specific proteins like troponin [5] or calmodulin 16-81 to exert its action. In RNA synthesis, activation by calcium is not well understood. Calcium stimulates RNA synthesis in slices of ventricular tissue [9], GH3 cells [lo] and cultures of chicken pectoralis muscle [ 111. But only indirect evidence is available about the molecular mechanism of such activation. Calciumdependent protein kinases in the nuclei of rat liver cells [ 121, the participation of calcium in the phosphorylation of non-histone proteins [ 13,141 and the presence of calmodulin in the nuclei of some cells [15], suggests that calcium could stimulate RNA synthesis through interaction of calcium with calmodulin, activation of calcium-dependent protein kinases by this calcium-calmodulin complex, phosphorylation of specific non-histone proteins and, finally, derepession of the genome. To test this hypothesis, we have investigated the effects of calcium, calmodulin and the calmodulin inhibitor, chlorpromazine, on RNA synthesis in isolated nuclei from rat liver cells. The results on the stimulation of RNA synthesis by calcium were not confirmed by our study, which has shown instead that calcium inhibits RNA synthesis and that this effect (again unlike the previous hypothesis) is not dependent on calmodulin. 2. MATERIALS AND METHODS

Modifications of chromatin structure in control of gene expression

Repetitive sequences in intron and spacer DNA could be sites for binding of chromosomal proteins which maintain chromatin structure and control gene activity. Methylation of DNA guides the binding of acidic nonhistone proteins and maintains the differentiation state during DNA replication. Differentiation inducers modify repressor proteins permiting unfolding of chromatin. Histone H 1 must be removed for gene activity. Phosphorylation of nonhistone proteins probably induces allosteric modifications which permit unfolding of chromatin. Acetylation of nucleosomal histones is necessary to permit passage of RNA polymerase. Deacetylation quickly returns the gene to a normal histone repressed state. Chromosomal RNA attached to nonhistone proteins aids the binding of RNA polymerase to the DNA template. Carcinogens can disrupt normal gene control leading to circumvention of normal cell cycle controls.

DNA replication, chromatin structure, and histone phosphorylation altered by theophylline in synchronized HeLa S3 cells.

The onset of DNA replication normally is coincident with an increase in histone 1 phosphorylation and a relaxation in chromatin structure. In this paper we show that 5 mM theophylline, added 2 h after selective detachment to synchronized HeLa-S-3 cells, delays the onset and reduces the rate of DNA synthesis while theophylline treatment beginning at 8 h has no effect on subsequent DNA synthesis. These actions of theophylline are accompanied by an inhibition of histone 1 phosphorylation and a prevention of the normal relaxation in chromatin structure between G1 and S phases as revealed by image analysis of Feulgen-stained nuclei. The time courses of intracellular cyclic AMP levels, nonhistone protein phosphorylation, and [3H]lysine incorporation are also compared in the same treated and untreated synchronized HeLa cells. Comparison with experiments using 1-beta-D-arabinofuranosylcytosine (Ara-C) shows that the above phenomena are not a direct result of inhibition of DNA synthesis. We interpret our results as evidence that the associations between histone 1 phosphorylation, chromatin relaxation, and the onset of DNA synthesis are temporally and causally related.

Effects of polyamines and histone on the phosphorylation of non-histone proteins in isolated rat liver nuclei

Addition of polyamines to isolated nuclei increases the rate and extent of phosphate incorporation from ATP into non-histone proteins several-fold. Similar results are obtained when histones are added to phosphorylating nuclei or when nuclei are incubated with DNAase prior to the addition of ATP. Electrophoretic analysis of the reaction products in SDS polyacrylamide gels reveals that specific non-histone proteins are preferentially phosphorylated in the presence of polyamines, some of which appear to be the same as in the presence of histones or DNAase. Removal of protein-bound phosphate during prolonged incubation of nuclei occurs with the same kinetics in the presence or absence of polyamines. Our results suggest that polyamines and histones stimulate nuclear protein phosphorylation by rendering additional phosphate acceptors accessible to the kinases.

Biochemical studies on the nuclei of rat salivary glands stimulated with isoproterenol: Phosphorylation of chromatin proteins

1. The phosphorylation in vitro of chromatin proteins was determined in nuclei isolated from submaxillary and parotid glands of rats after injection of isoproterenol. 2. A greater incorporation of radioactivity was observed in the non-histone proteins than in the histones. 3. The phosphorylation of non-histone proteins was diminished during the first 14 hr after isoproterenol injection with a return to control values by 48 hr. 4. A reduction in the non-histone protein/DNA ratio was observed during the period of 24 hr following isoproterenol injection and was particularly marked in the case of the parotid.

Induction and properties of cytoplasmic factor(s) which enhance nuclear nonhistone protein phosphorylation in lymphocytes stimulated by anti-Ig.

An increased in vitro phosphorylation of nonhistone nuclear proteins (NHP) was observed in the nuclei isolated from rabbit lymphocytes which had been stimulated with anti-Ig for 4 h. No concomitant increase of phosphorylation in histones or 0.14 M NaCl-soluble proteins was observed. The increase of in vitro phosphorylation of NHP was also observed in the nuclei isolated from nonstimulated cells when these nuclei were preincubated for 2 h with cell-free extracts from anti-Ig-stimulated cells. The active substance in cell-free extracts was maximally induced when lymphocytes were stimulated with anti-Ig for 2 h. The induction of an increased phosphorylation of NHP in nonstimulated nuclei with the cell-free extracts was not due to decrease of the adenosine triphosphate pool in the extracts from anti-Ig-stimulated cells. The active substance in cell-free extracts was not NHP-protein kinase itself, but it probably activated NHP-protein kinase in quiescent nuclei. The active substance was nondialyzable and probably protein. It was resistant against heating at 56 degrees C for 30 min, but the activity was completely destroyed by heating at 90 degrees C for 30 min. The active substance may be responsible for the transduction of the membrane-mediated signals given through Ig receptors to nuclei.

Apparent protein kinase activity in oligodendroglial chromatin after chronic morphine treatment

The oligodendroglial nuclei were purified from mouse brain with more than 97 per cent homogeneity. Cyclic AMP-independent phosphorylation of non-histone protein in oligodendroglial chromatin was studied. Morphine sulfate, in vitro, had no effect on phosphorylation. However, chronic morphine treatment resulted in an increase of phosphorylation in high molecular weight regions of sodium dodecylsulfate (SDS) electrophoresis gel. The increase was not the result of a decrease in phosphoprotein phosphatase activity.