Studies on chromatin-associated protein phosphokinase of submandibular gland from isoproterenol-treated rats

Water-soluble chromatin from rat submandibular gland nuclei was isolated, and had a DNA: RNA:protein ratio of 8:1:20. The spectral properties of this preparation were similar to those described for chromatins from other tissues. The rat submandibular gland chromatin possessed protein phosphokinase activity. It was able to incorporate ³²P from [γ-³²P]-ATP into chromatin proteins, and into dephospho-phosvitin. The chromatin-associated protein phosphokinase activity (measured with dephospho-phosvitin as substrate) required Mg²⁺, Na⁺ or K⁺ and dithiothreitol for optimal activity. A single injection of isoproterenol influenced the activity of this enzyme system, so that it was decreased at 2 h, showed a transient increase at 4 h, and a large increase at 10–16 h after the injection. This event appears to precede the increase in ribosomal RNA induced by Ipr [13]. By 48 h the chromatin-associated protein kinase returned to the normal control levels. These changes appeared to be commensurate with the corresponding alterations in the non-histone acidic protein complement of these chromatins. Actinomycin D or cycloheximide, when administered 30 min prior to isoproterenol, blocked the increase in chromatin-associated protein kinase at 4 as well as 10 h after the injection of isoproterenol. Injection of pilocarpine did not influence the chromatin-associated protein phosphokinase activity. Dichloroisoproterenol appeared to be antagonistic to the influence of isoproterenol in mediating changes in chromatin-associated protein kinase. The results suggest that the isoproterenol-induced increase in chromatin-bound protein phosphokinase which precedes the increase in RNA synthesis is related to the eventual onset of DNA synthesis in rat submandibular gland stimulated by isoproterenol. The chromatin-bound protein phosphokinase activity (or activities) may have a regulatory role on gene action, mediated through the control of phosphorylation of nuclear non-histone acidic proteins [26].     

Co-ordinated changes in the cyclic AMP signalling system and the phosphorylation of two nuclear proteins of Mr 130,000 and 110,000 during proliferative stimulation of the rat parotid gland by isoprenaline. Possible identity of the two proteins with pp135 and nucleolin.

Parotid glands were stimulated to growth by repeated injection of the beta-agonist isoprenaline into rats. Incubation of intact parotid-gland lobules with [32P]Pi and subsequent analysis of nuclear proteins revealed in the stimulated glands an increased 32P incorporation into two acid-soluble non-histone proteins with apparent Mr values of 110,000 and 130,000 (p110 and p130). After a single injection of isoprenaline, leading to a biphasic increase in DNA synthesis (maximum at 24 h), the same two proteins showed a transiently increased 32P incorporation at 17 h after injection. At this time point at the onset of DNA synthesis the total activity of soluble cyclic AMP-dependent protein kinase decreased. No change in p110/p130 phosphorylation was observed at 0.3 h after stimulation, a time of maximal stimulation of secretion. Administration of the beta-antagonist propranolol 8 h after the injection of isoprenaline suppressed the increase in DNA synthesis, the preceding changes in the concentration of cyclic AMP and in the activity of cyclic AMP-dependent protein kinase, as well as the increased phosphorylation of p110 and p130. Cross-reactivity of p110 and p130 with specific antisera against two nucleolar phosphoproteins of similar molecular mass (nucleolin and pp135), as well as their localization in a nucleolar cell fraction, indicated a possible identity of p110 and p130 with these two proteins. Our results suggest that nucleolin and pp135 are nuclear target proteins of cyclic AMP in the cyclic AMP-influenced regulation of the transition of cells from the G1 to the S phase.     

Synthesis and phosphorylation of chromatin-associated proteins in cAMP-induced "differentiated" neuroblastoma cells in culture.

Prostaglandin E₁ and a cAMP phosphodiesterase inhibitor 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone, RO20-1724, were used to induce differentiation in mouse neuroblastoma cells in culture. The incorporation of amino acids and phosphate into nuclear proteins of control and drug-treated cells (1 h and 3 days after treatment) was examined using double radioisotopic techniques. A marked decrease in histone synthesis and H1-histone phosphorylation were observed in ‘differentiated’ neuroblastoma cells after 3 days of prostaglandin E₁ and RO20-1724 treatment, but only small differences were noted in the synthesis and phosphorylation of non-histone chromatin associated proteins after 3 days of drug treatment. Minimal changes were observed in the labeling of histone and non-histone nuclear proteins if the cells were treated for 1 h with prostaglandin E₁ and RO20-1724.     

Nuclear protein synthesis and phosphorylation in Friend erythroleukemia cells stimulated with DMSO

Patterns of nuclear protein synthesis and phosphorylation have been investigated in Friend erythroleukemia cells. The rate of incorporation of [³H]leucine and [³²P]phosphate remains relatively constant during the first 48 h of dimethylsulfoxide (DMSO) stimulation, when more than 90% of the cells commit to erythroid differentiation, but falls to 20% by 120 h. Histone H2A phosphorylation is greatly increased during DMSO treatment, but no significant changes were found in the non-histone phosphoprotein patterns as determined by gel electrophoresis. There is also a small, but reproducible, change in the relative amounts of the two sub-fractions of histone H2A. There are no striking changes in the electrophoretic patterns of [¹⁴C]leucine-labelled nuclear proteins during the first 48 h, but the amount and the synthesis of two proteins of 46 000 and 280 000 D are increased somewhat during this period. Another protein, of molecular weight 65 000, appears to be induced in low amounts.     

Phosphorylation and acetylation of chromatin conjugate protein A24.

An analysis was made of the phosphorylation and acetylation of chromatin protein A24, a conjugate of histone 2A and ubiquitin. ³²P-orthophosphate was incorporated into phosphoserine of histone 2A and protein A24 in Novikoff hepatoma ascites cells in culture. The ratio of ³²P incorporation and the pattern of tryptic digestion of ³²P-labeled protein A24 indicated that the histone 2A component was phosphorylated and the ubiquitin component was not. Analysis of ε-N-acetyl lysine in protein A24, histone 2A and ubiquitin showed that while protein A24 and histone 2A were acetylated, ubiquitin was not. Apparently, even though it is conjugated with ubiquitin, the histone 2A portion of protein A24 has the same modifications as free histone 2A. The lack of modification of ubiquitin differs from that of high mobility group (HMG) non-histone chromatin proteins with which it is co-extracted from chromatin.     

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.     

Action of Miracil D and related compounds on histone synthesis and phosphorylation in regenerating liver

The effect of Miracil D and hycanthone on ³H-amino acid incorporation into histones was studied under conditions known to cause a greater than 90% inhibition of thymidine incorporation into DNA of regenerating rat liver. A dose level of 50 mg of either drug per kg body weight administered 8 h after partial hepatectomy caused an approximate 50% inhibition of amino acid incorporation into fl, f2b and combined f2a plus f3 histone in 24-h regenerating liver. There was little or no effect on amino acid nitrogen concentration or incorporation of ³H-amino acid into the acid-soluble fraction, cytoplasmic proteins or acid-insoluble nuclear proteins. Under the same conditions, Miracil D caused a 65% inhibition of ³²P incorporation into lysirierich f1 histone whereas a structurally related compound, GE-99, did not have a significant inhibitory effect on this parameter nor on [³H]thymidine incorporation into DNA. Temporal studies with hycanthone revealed a suppression of the increased phosphorylation of fl histone in regenerating rat liver without influencing the phosphorylation of other histones. The data support the concept of coordinated control of DNA synthesis and phosphorylation of fl histone.     

Associations of benzo[a]pyrene with rat-liver chromatin and the chromatin protein

Rat-liver nuclei were prepared in the course of time after the i.p. injection of [G-3H]benzo[a]pyrene ([3H]BP). The nuclei were lysed in the hypotonic buffer and centrifuged at 4000 X g. The recovery of the radioactivity of resulting supernatant (chromatin) was thus 91% at 24 h, 68% at 48 h and 74% at 168 h after the i.p. injection. The incorporation into nucleosome-oligomer fraction was always much more than into those of monomer and DNA-rich fractions. The preferential incorporation was found in the fraction which was enriched in non-histone chromatin proteins (NHCPs) of 49 000-55 000 daltons. This fraction steadily raised the incorporation level until at 168 h after the i.p. injection. In contrast, the levels of histone and DNA fractions were always very low. The significant incorporation was observed in the fraction which was composed of five classes of histones and low molecular-weight NHCPs (less than 30 000 daltons), despite the very low incorporation into the histone fraction. The fluorographic analysis revealed the predominant incorporations at the positions of molecular weight of 65 000, 52 000 and 44 000 daltons. In addition, the incorporations were clearly observed at the positions of 59 000, 49 000, 45 000, H1 histone, A24 protein and another one. On the other hand, these fractions were, at the final preparation steps, subjected to either dialysis of SDS-phenol treatment and/or acetone precipitation. The total recovery of radioactivity was thus 21% at 24 h, 32% at 48 h and 52% at 168 h after the i.p. injection. These results suggest that the chromatin contains considerable amounts of water-soluble, phenol and/or acetone-soluble BP-conjugates in the early period after the i.p. injection.     

Chromosomal protein synthesis during erythropoiesis in the mouse spleen

Induced erythropoiesis in the mouse spleen was employed to study chromosomal protein synthesis during erythroid cell development. Splenic erythropoiesis occurring after phenylhydrazine induced hemolysis can be divided into an early phase during which nuclear RNA polymerase activity and RNA production are maximal and a late phase in which hemoglobin synthesis and DNA accumulation are maximal. Chromatin was isolated from splenic tissue during both the early and late phases of erythropoiesis as well as from non-anemic animals. The total protein content of chromatin from the early erythroid phase was greater than that of chromatin from the late erythroid phase or from non-anemic controls. The increase was due to a coordinate increase in the concentration of both histone and nonhistone proteins. During late erythropoiesis, the concentration of each returned to pre-anemic levels. Total histone synthesis increased 2.6-fold during early erythropoiesis as compared with the pre-anemic state and remained elevated in late erythropoiesis. The increase in histone synthesis was due to an increase in the synthesis of all five major histone proteins. Nonhistone protein synthesis was more active than that of histones in the pre-anemic spleen and rose only slightly during early erythropoiesis, returning to preanemic levels during late erythropoiesis. Fractionation of nonhistone proteins on SDS-urea polyacrylamide gels revealed complex patterns with significant differences between the pattern of erythroid spleen non-histone proteins and that of the pre-anemic spleen. Analysis of the incorporation of ³H-valine into the non-histone proteins indicated that during early erythropoiesis there was a generalized increase in nonhistone protein synthesis. During the late erythroid phase, the decline in non-histone protein synthesis was most marked for the higher molecular weight proteins.     

Protein phosphorylation: Localization in regenerating optic axons

A number of axonal proteins display changes in phosphorylation during goldfish optic nerve regeneration (Larrivee and Grafstein, 1989). (1) To determine whether the phosphorylation of these proteins was closely linked to their synthesis in the retinal ganglion cell body, cycloheximide was injected intraocularly into goldfish whose optic nerves had been regenerating for 3 weeks. Cycloheximide reduced the incorporation of [³H]proline and³²P orthophosphate into total nerve protein by 84% and 46%, respectively. Of the 20 individual proteins examined, 17 contained less than 15% of the [³H]proline label measured in corresponding controls, whereas 18 proteins contained 50% or more of the³²P label, suggesting that phosphorylation was largely independent of synthesis. (2) To deterine whether the proteins were phosphorylated in the ganglion cell axons, axonal transport of proteins was blocked by intraocular injection of vincristine. Vincristine reduced [³H]proline labeling of total protein by 88% and³²P labeling by 49%. Among the individual proteins [³H]proline labeling was reduced by 90% or more in 18 cases but³²P labeling was reduced only by 50% or less. (3) When³²P was injected into the cranial cavity near the ends of the optic axons, all of the phosphoproteins were labeled more intensely in the optic tract than in the optic nerve. These results suggest that most of the major phosphoproteins that undergo changes in phosphorylation in the course of regeneration are phosphorylated in the optic axons.Abbreviations SDS sodium lauryl sulfate - GAP growth associated protein - TCA trichloracetic acid - kD kilodalton     

Synthesis and phosphorylation of histone H1 and high mobility group proteins in the regenerating rat liver after X irradiation

A rat was irradiated to the upper abdomen including the liver and then partially hepatectomized. The subsequent synthesis and phosphorylation of histone H1 and nonhistone chromosomal high mobility group (HMG) proteins were investigated. Incorporation of [3H]lysine into histone H1 was increased and reached its peak at 27 h after hepatectomy, and 14 Gy of X rays inhibited the increase. Increase in the incorporation of [3H]lysine into HMG (1 + 2), 14, and 17 which occurred around 27 h after hepatectomy was not inhibited by 14 Gy irradiation. Phosphorylation of histone H1, measured with 32Pi incorporation in vivo, was maximal between 21 and 24 h, and it was inhibited by 4.8 Gy of X rays and delayed with 1.9 Gy. Phosphorylation of HMG 14, which was the only HMG protein phosphorylated under present conditions, was not affected by X irradiation. The [3H]thymidine incorporation into nuclear DNA started increasing at 21 h and reached its maximum at 27 h after hepatectomy. X irradiation with 4.8 Gy inhibited the incorporation, and 1.9 Gy lowered it.     

Non-histone chromatin proteins of B lymphocytes stimulated by lipopolysaccharide. II. Phosphorylation.

Stimulation of mouse lymphocytes with the B lymphocyte specific mitogen lipopolysaccharide results in an increased rate of phosphorylation of non-histone chromatin proteins. An initial small increase in phosphorylation occurs during the first 2 h and a much larger increase after 24 h of culture with mitogen. The phosphorylated nuclear and cytoplasmic proteins were analysed by polyacrylamide gel electrophoresis and the stimulation index of each prominent peak measured. It was inferred that selective stimulation of the phosphorylation of individual proteins had occurred from: (1) the range of stimulation indices for different proteins, and (2) the appearance, after 8 h stimulation of an apparently newly phosphorylated non-histone chromatin protein of molecular weight 115 000. The pool size of ATP was monitored and showed only small changes during the first 24 h of exposure to lipopolysaccharide. Phosphatase activity was found to be associated with lymphocyte chromatin and nucleoplasm and may help to regulate the level of phosphorylation of non-histone chromatin proteins in vivo. To preserve phosphorylated proteins during their isolation phosphatase activity was inhibited by Na2MoO4. The selective changes in phosphorylation of nuclear proteins precede, and continue during, the stimulation of immunoglobulin and DNA synthesis. Our results are thus consistent with the hypothesis that phosphorylation of non-histone chromatin proteins plays a role in the regulation of gene expression in B lymphocytes.     

Perichromatin fibrils and chromatin ultrastructural pattern

The relationship between the synthesis of acidic nuclear proteins, phosphoproteins, RNA and chromatin ultrastructural pattern was studied in regenerating rat hepatocytes after partial hepatectomy. α-Amanitin induced, as early as 30 min after injection, a reduction of RNA synthesis to about 50% of the control level; the degree of inhibition had remained the same at 2 h after poisoning. No change was detected either in acidic nuclear protein synthesis or in phosphorylation for the whole time examined. The DNA-containing structures, demonstrated by the Gautier staining procedure, were in a dispersed pattern either in untreated regenerating hepatocytes or 30 min after α-amanitin administration to rats; but they did appear in a condensed form 1 h and more especially 2 h after toxin injection. In untreated regenerating hepatocytes, the regressive EDTA staining method for RNP revealed a large quantity of perichromatin fibrils which remained unchanged 30 min after α-amanitin treatment and were diminished at 1 h and strongly reduced 2 h thereafter.Cycloheximide treatment promptly reduced the synthesis of nuclear acidic proteins while leaving unchanged the synthesis of RNA; the quantity of perichromatin fibrils and the loosened appearance of DNA-containing structures were the same as in the control rat nuclei.Our results showed that the ultrastructural pattern of chromatin was not directly related either to the synthesis of RNA or to acidic nuclear proteins or to the phosphorylation of phosphoproteins; on the contrary, a strict relationship with the quantity of perichromatin fibrils was demonstrated. The possible interaction of perichromatin fibrils with other chromatin components was discussed as a possible regulatory mechanism of chromatin pattern.     

Sister subchromatid exchanged segments and chromosome structure

The relationship between the synthesis of acidic nuclear proteins, phosphoproteins, RNA and chromatin ultrastructural pattern was studied in regenerating rat hepatocytes after partial hepatectomy. α-Amanitin induced, as early as 30 min after injection, a reduction of RNA synthesis to about 50% of the control level; the degree of inhibition had remained the same at 2 h after poisoning. No change was detected either in acidic nuclear protein synthesis or in phosphorylation for the whole time examined. The DNA-containing structures, demonstrated by the Gautier staining procedure, were in a dispersed pattern either in untreated regenerating hepatocytes or 30 min after α-amanitin administration to rats; but they did appear in a condensed form 1 h and more especially 2 h after toxin injection. In untreated regenerating hepatocytes, the regressive EDTA staining method for RNP revealed a large quantity of perichromatin fibrils which remained unchanged 30 min after α-amanitin treatment and were diminished at 1 h and strongly reduced 2 h thereafter.Cycloheximide treatment promptly reduced the synthesis of nuclear acidic proteins while leaving unchanged the synthesis of RNA; the quantity of perichromatin fibrils and the loosened appearance of DNA-containing structures were the same as in the control rat nuclei.Our results showed that the ultrastructural pattern of chromatin was not directly related either to the synthesis of RNA or to acidic nuclear proteins or to the phosphorylation of phosphoproteins; on the contrary, a strict relationship with the quantity of perichromatin fibrils was demonstrated. The possible interaction of perichromatin fibrils with other chromatin components was discussed as a possible regulatory mechanism of chromatin pattern.     

Studies on histones and non-histone proteins from rats treated with dimethylnitrosamine.

A study has been made of the histone and non-histone chromosomal proteins of rat liver after treatment in vivo with dimethylnitrosamine (DMN) (2 mg/kg). DMN was found not to affect histone turnover, as measured by 3H-labelled amino-acids incorporation. A decrease was observed in specific activity of the histones with time after injection of [14C]DMN or [14C]-formate and this was attributable to demethylation of both abnormal and normal methylation sites in these proteins. In the case of the non-histone proteins, DMN was found to increase greatly the turnover of those non-histone proteins loosely associated with chromatin DNA and RNA; turnover of those non-histone proteins tightly bound to chromatin DNA and RNA was unaffected. Demethylation of both normal and abnormal methylation sites was found to take place from both non-histone protein fractions. In the case of the loosely bound non-histone proteins a lower rate of demethylation was observed after DMN treatment.     

Changes in chromatin of Daucus carota cells during embryogenesis

Cells of carrot (Daucus carota var. Rote Riesen) were cultured on media inductive and non-inductive for embryogenesis and analyzed for differences in their chromosomal proteins and chromatin template activity. Non-histone proteins were prepared from dehistonized chromatin and their properties were investigated. Non-histone proteins proved to be acidic and associated easily with calf thymus histone. Non-histone proteins were able to counteract the inhibitory effect of histone on DNA-directed RNA synthesis in vitro. Almost the same rate of restoration occurred regardless of the interaction between DNA and protein, when sufficient amounts of non-histone proteins were added. However, once the histone-DNA complex was established, the restoration by non-histone proteins at comparably lower concentration was poor. Another acidic protein, bovine serum albumin, had no effect on histone inhibited RNA synthesis. Also non-histone proteins enhanced the chromatin directed RNA synthesis more than 100%. The template activity of chromatin changed after the inductive treatment of embryo formation and induced cells showed higher template activity than non-indiiced controls after embryo cells were formed. Histone components were the same in inductive and non-inductive cells. On the other hand, there was a correlation between template activity and the stimulation by non-histone proteins of histone-inhibited RNA synthesis.     

Regulation of mouse preimplantation development: inhibitory effect of genistein, an inhibitor of tyrosine protein phosphorylation, on cleavage of one-cell embryos

We investigated the effects of genistein, an inhibitor of tyrosine protein phosphorylation, on mouse 1-cell embryos, since in response to mitogenic stimuli tyrosine protein phosphorylation in somatic cells is implicated in initiation of DNA synthesis. Genistein inhibits cleavage of 1-cell embryos in a concentration-dependent and reversible manner; biochanin A, which is a less potent inhibitor of tyrosine protein phosphorylation, is a less potent inhibitor of cell cleavage. Genistein does not inhibit [35S]methionine incorporation, but does inhibit [3H]thymidine incorporation. Consistent with genistein's ability to inhibit cleavage by inhibiting DNA synthesis is that the loss of genistein's ability to inhibit cleavage corresponds with exit of the 1-cell embryos from S phase. Genistein is likely to inhibit tyrosine protein phosphorylation in situ, since it reduces by 80% the relative amount of [32P]phosphotyrosine present in 1-cell embryos; genistein does not inhibit either [32P]orthophosphate uptake or incorporation. As anticipated, genistein has little effect on inhibiting changes in the pattern of phosphoprotein synthesis during the first cell cycle, since tyrosine protein phosphorylation constitutes a small percentage of total protein phosphorylation. Alkalai treatment of [32P]radiolabeled phosphoproteins transferred to Immobilon reveals a base-resistant set of phosphoproteins of Mr = 32,000 that displays cell-cycle changes in phosphorylation. Although these properties suggest that these phosphoproteins may be related to the p34cdc2 protein kinase, phosphoamino acid analysis of [32P]radiolabeled phosphoproteins reveals that they are not enriched for phosphotyrosine; the inactive for p34cdc2 protein kinase contains a high level of phosphotyrosine. Results of these experiments suggest that tyrosine protein phosphorylation in response to the fertilizing sperm may be involved in initiating DNA synthesis in the 1-cell embryo, as well as converting a meiotic cell cycle to a mitotic one.