Phosphorylation Of Nuclear Non-histone Proteins Research Articles

The effect of starvation on the diurnal variation of amylase secretion from rat parotid glands.

The purpose of this study was to investigate the effect of starvation on the cellular function of rat parotid glands in relation to the diurnal variation of amylase secretion from the tissue. Salivary amylase secreted from the glands of rats starved for 24 h showed diurnal variation, with two peaks at 13 and 21 h. The peak at 21 h was more notable than that in fed rats, because of the accumulation of a large amount of amylase in the tissue due to starvation. Maximal binding (Bmax) values of [3H]dihydroalprenolol in the parotid membrane of starved rats also showed diurnal variation, with marked peak at 13 and 21 h. However, phosphorylation of nuclear non-histone proteins in starved rat parotid glands showed diurnal variation, with peaks at 17 and 1 h. This phasing was delayed by 4 h in comparison to that of fed rats, suggesting that the delay of diurnal variation of amylase secretion commences at least a few days after the beginning of starvation. These results indicate that changes in salivary amylase secreted from parotid glands of rats starved for 24 h coupled with those in the number of beta-adrenoceptors in the tissue show the same diurnal variation as observed in fed rats. Starvation of rats for 24 h does not appear to influence the response of parotid gland cells to stimuli.

Age-dependent changes in the phosphorylation of nuclear proteins of submandibular glands in isoproterenol-treated rats

Age-dependent changes in the regulation of the phosphorylation of nuclear proteins were investigated in relation to DNA synthesis in rat submandibular glands after injection of isoproterenol (IPR). The level of phosphorylation of nuclear proteins in the tissue increased rapidly after birth, reaching a maximum at 4 weeks, and then decreased to the level of 52-week-old rats. The level of protein kinase activity in nuclei varied in parallel with that of phosphorylation of nuclear non-histone proteins after birth. The time after the injection of IPR required to initiate the phosphorylation of nuclear non-histone proteins of rat submandibular glands, which occurs prior to the onset of RNA synthesis that precedes the replication of DNA, increased with age. These results suggest that this delayed onset of phosphorylation plays a regulatory role in cell proliferation and cell function during aging.

Diurnal variation of amylase secretion is coupled with alterations of β-adrenoceptors in the rat parotid gland

Diurnal changes in the neurotransmitter receptors are important for studying the receptor function in neurophysiology. The purpose of this study is to gain an insight into the regulatory mechanisms of the diurnal variation of amylase secretion. Rat salivary amylase levels showed a diurnal variation with two peaks, a marked peak at 13 h and a lesser peak at 21 h. This increase in salivary amylase levels was completely inhibited by pretreatment of rats with the beta-adrenergic antagonist propranolol, but not by the alpha-adrenergic antagonist phentolamine. Amylase level in parotid tissue homogenate also showed a diurnal change, but there was only one peak, at 13 h. The number of maximal binding sites (Bmax) for [3H]dihydroalprenolol (DHA) in parotid membranes showed a diurnal variation with two marked peaks at 13 and 21 h, but the affinity of parotid beta-adrenoceptors for agonists or antagonists did not show any diurnal changes. Phosphorylation of nuclear non-histone proteins in the rat parotid gland showed diurnal variation with two marked peaks at 13 and 21 h. These results indicate that a diurnal variation in the number of rat parotid beta-adrenoceptors, which is presumably regulated by gene expression, is coupled with a change in salivary amylase secretion.

Rous sarcoma virus-induced changes in the pattern of phosphorylation of non-histone nuclear proteins

We here studied the protein kinase activity and in vitro phosphorylable sites of non-histone nuclear proteins, 0.4 M NaCl extracts (mostly chromosomal proteins) from chick embryo fibroblasts (CEF), infected or not with a Schmidt Ruppin strain subgroup A of Rous sarcoma virus (RSV). The infection and transformation of chick fibroblasts by RSV induced an increase in kinase activity and endogenous phosphorylation of non-histone chromosomal (NHC) proteins. The stimulation, by a change of medium, of the proliferation of dense cultures of normal chick fibroblasts also induced an increase in the kinase activity and endogenous phosphorylation of NHC proteins. However, two-dimensional gel electrophoresis of the 32P-phosphorylated proteins showed that stimulation due to a change of medium and that due to the expression of transformation were very different. The stimulation by a change of medium increased to a greater or lesser extent the phosphorylation of the different NHC proteins, with no fundamental variations in the pattern of protein phosphorylation. In contrast, RSV infection induced significant changes in the pattern of protein phosphorylation. One of the most striking feature was the large increase of amount and phosphorylation of high molecular weight (HMW) proteins in particular of phosphoproteins having an evaluated molecular weight (MW) of 78 K and 82 K and pI>8.2. The percent of phosphotyrosine residues in NHC proteins was clearly increased when the proteins were extracted from transformed cells instead of normal cells. But the alkaline treatment of two-dimensional gel electrophoresis indicated that the 80 K phosphoproteins did not contain phosphotyrosine residues, and thus cannot be considered as substrates for pp60 src kinase.

Effect of transformation of chicken cells by Rous sarcoma virus on in vitro phosphorylation of nuclear non-histone proteins

The use of virus temperature-sensitive (ts) mutants for transformation allows us to determine the effect of transformation on different alterations in cell morphology and cell functions. We studied the kinase activity of nuclear non-histone proteins (NHP) extracted from chicken embryo fibroblasts (CEF), both infected and non-infected with a ts mutant of Rous sarcoma virus (Schmidt Ruppin strain, subgroup A, T class) and cultivated at the restrictive (41°C) and permissive (37 °C) temperature for transformation.The endogenous in vitro phosphorylation of NHP extracted from stationary ts-infected cultures increased by 300% when the cells were cultivated at 37 instead of 41°C. This increase is probably a consequence of the expression of transformation, since it was not observed when NHP were extracted from non-infected CEF maintained either at 41 or 37 °C. The increase in endogenous phosphorylation of NHP does not seem to be entirely due to a change in the density-dependent inhibition of cell metabolism in transformed cells, as an increase in protein phosphorylation was observed when the ts cultures were in both exponential growth phase and plateau phase.The urea-polyacrylamide gel electrophoresis of 32P-phosphorylated proteins showed that the increase in phosphorylation observed when growing ts cells were maintained at 37 instead of 41 °C was mostly due to the stimulation of one fraction (fraction d), as if the increase in phosphorylation of this fraction was linked to the expression of transformation. When the ts cultures were in stationary phase, the enhancement of the phosphorylation of proteins observed when cells were maintained at 37 instead of 41 °C was due to stimulation of the phosphorylation of all protein fractions, and particularly of fractions d and b.When phosvitin was used as a substrate, the kinase activity of NHP extracted from ts-infected cells was increased 300% when the cells were cultivated at 37 instead of 41 °C. On the other hand, the phosvitin kinase activity of NHP extracted from non-infected cells decreased when the cells were maintained at 37 instead of 41 °C. Thus, the transformation of cells induced an increase in the phosvitin kinase activity of nuclear NHP.When ts cells were cultivated at 41 °C until saturation density and then transferred to 37 °C for 3 h before extraction of proteins, a 37% increase in phosvitin kinase activity of NHP was observed, as compared with the activity of proteins extracted from cells left at 41 °C. Thus, the change in kinase activity is an early event in the process of transformation.

Activation of cyclic AMP-dependent protein kinase activity during LPS stimulation of macrophage tumor cell line, J774.1

Stimulation of J774.1 macrophage cell line with lipopolysaccharide (LPS)- or 8Br-cyclic AMP induced phosphorylation of nonhistone nuclear proteins (NHP) particularly of size range 35,000 daltons. In vitro phosphorylation of nuclei isolated from LPS- or 8Br-cyclic AMP-stimulated cells also showed an increased incorporation of 32P into NHP indicating an activation of a NHP-specific protein kinase in nuclei. Incubation of normal nuclei with cytoplasmic extracts from LPS- or 8Br-cyclic AMP-stimulated cells increased NHP-specific phosphorylation in nuclei. Incubation of normal nuclei with normal cytoplasm in the presence of 1 × 10 −8 M cyclic AMP also induced an increased phosphorylation of NHP, suggesting that the LPS-induced increase of intracellular cyclic AMP activates a cyclic-AMP-dependent NHP-specific protein kinase in the cytoplasm, which moves into the nucleus to phosphorylate NHP. The increased phosphorylation of NHP proceeded in advance of the secretion of T-cell activating factor(s) and the optimum concentrations of LPS- or 8Br-cyclic AMP for the maximal induction of NHP-specific protein kinase activity were similar to those required for the induction of T-cell activating factor(s) in J774.1. These experiments suggest the intimate involvement of NHP phosphorylation in the induction processes of macrophage factor production.

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.

Adenosine 3′,5′-monophosphate-dependent protein kinases of myocardial non-histone nuclear proteins

Myocardial acidic non-histone nuclear proteins (NHPs) contain endogenous protein kinase activity. Phosphocellulose chromatography of purified NHPs identifies nine separate peaks of protein kinases which can phosphorylate both endogenous and exogenous substrates to a variable degree; endogenous NHPs are the best substrates. Cyclic AMP-stimulated protein kinase induced phosphorylation of endogenous and exogenous substrates; the extent of this stimulation varied according to the protein kinase fraction and substrate used. Cyclic AMP also enhanced NHP-indouced stimulation of RNA polymerase activity. This enhancement was dependent on protein kinase-induced phosphorylation of NHPs since it was prevented by alkaline phosphatase pretreatment. It is concluded that nuclear protein kinases regulate myocardial RNA synthesis by enhancing phosphorylation of NHPs and that this regulation is under cyclic AMP control.

Biphasic Effect of Cyclic AMP on IgG Production and on the Changes of Non-Histone Nuclear Proteins Induced with Anti-Immunoglobulin and Enhancing Soluble Factor

Abstract The relationship between the changes of non-histone nuclear proteins and the differentiation of B cells to IgG-producing cells was studied by analyzing the effect of exogenously administered dibutyryl cyclic AMP. Stimulation of B cells with anti-Ig or incubation of anti-Ig-stimulated cells with enhancing soluble factor (SF) induced an increase in the amounts and phosphorylation of non-histone nuclear proteins without any concomitant increase or decrease of histones. The addition of 5 × 10-4 M dibutyryl cyclic AMP or 5 × 10-5 M isoproterenol at 2 hr after anti-Ig stimulation showed the maximum augmentation of IgG production induced by the sequential stimulation of B cells with anti-Ig and SF. The addition of the same concentration of dibutyryl cyclic AMP within 24 hr after the initiation of the second stage of culture with SF inhibited the induction of IgG. The same biphasic effect of dibutyryl cyclic AMP was observed in the changes of non-histone nuclear proteins induced with anti-Ig or SF. 5 × 10-4 M dibutyryl cyclic-AMP augmented an increase of amounts or phosphorylation of non-histone nuclear proteins induced with anti-Ig, whereas an increase of amounts or phosphorylation of non-histone nuclear proteins induced with SF was suppressed by the addition of the same concentration of dibutyryl cyclic AMP. The same biphasic effect of dibutyryl cyclic AMP on IgG production and on the changes of non-histone nuclear proteins strongly suggests the intimate involvement of non-histone nuclear proteins in the differentiation process of B cells.

Nuclear non-histone proteins from rat ventral prostate cells undergoing hypertrophy or hyperplasia

The distribution of nuclear non-histone proteins and their labelling with [ 14C]amino acids or [ 32P]O 4 in rat ventral prostate cells undergoing hypertrophy (cell growth) or hyperplasia (cell division) were compared by SDS polyacrylamide gel electrophoresis. Marked quantitative changes in these proteins accompanied castration and replacement of hormone, but no absolute qualitative differences in their staining patterns were established. Preparations from cells undergoing hyperplasia were augmented in high molecular weight proteins and contained fewer proteins of <20000 D. The increased amounts of nuclear non-histone proteins from hormone-treated castrated rats were highly labelled by [ 14C]amino acids, but the molecular weights of radioactive proteins from cells undergoing hypertrophy were less diverse than those from replicating cells, Phosphorylation of nuclear non-histone proteins from short-term and long-term castrates, treated with testosterone propionate, was 170 and 60% greater than their controls. Proteins from 20–45 × 10 3 D were actively phosphorylated. Nuclear extracts from dividing cells contained additional radioactive high molecular weight proteins and fewer phosphorylated lower molecular weight components. The distribution of phosphorylated proteins and newly synthesized proteins was dissimilar. Quantitative and possible qualitative differences in staining of nuclear proteins isolated in N-ethylmalleamideurea-phosphate buffer from normal or hormone-treated castrated rats were accentuated when they were separated by charge at pH 2.8. In replicating cells, a more generalized synthesis of acidic nuclear proteins from all molecular weight classes occurred, which were not as highly phosphorylated as less heterogeneous nuclear proteins from cells undergoing hypertrophy. Examination of the immediate and the subsequent events following androgen-induced cellular hypertrophy or hyperplasia in the ventral prostate permits their comparison in the same tissue.

Differential Phosphorylation and Turnover of Nuclear Acidic Proteins during the Cell Cycle of Synchronized HeLa Cells

Abstract The phosphorylation of non-histone nuclear proteins has been studied in HeLa S-3 cells during synchronous growth. The uptake of [32P]orthophosphate into individual protein bands separated on sodium dodecyl sulfate polyacrylamide gels varies at different stages in the cell cycle while the banding patterns themselves are remarkably constant. Rates of phosphate uptake into most major phosphoprotein species are increased during the early G1 and early S phases and are minimal during the late G2 to M period. The turnover of previously incorporated phosphoryl groups during a cold chase following a 23-hour exposure to [32P]orthophosphate shows that some proteins lose their phosphoryl residues much more rapidly than do others. The halflives vary from 5 to 12 hours, with an over-all half-life of 6.7 hours. In contrast, the 14C specific activity of the proteins falls to 50% of the original activity in about 25 hours. The retention of [32P]phosphate is a more sensitive index of differential phosphoryl group turnover in different nuclear acidic proteins than is the uptake of [32P]phosphate in short term labeling experiments. If the nuclear phosphoproteins play a role in differential transcription during the cell cycle, changes in phosphorylation would appear to be more significant than changes in relative concentrations of individual protein species.

Modulation by Exogenous Histones of Phosphorylation of Non-Histone Nuclear Proteins in Isolated Rat Liver Nuclei

Abstract The phosphorylation of specific non-histone proteins in isolated rat liver nuclei is modified by the addition of exogenous histone fractions. The added histones are quantitatively adsorbed and their presence in the chromatin has been verified by electron microscope autoradiography. Histone fractions F1 and F2A1 stimulate phosphorylation of different nuclear phosphoproteins. F1 stimulates the phosphorylation of a protein fraction with a molecular weight of about 40,000, while F2A1 enhances phosphorylation of a protein fraction with a molecular weight of about 22,000. F1 and F2A1 each inhibit the phosphorylation of a specific low molecular weight nuclear protein fraction. Histones F2A2, F2B and F3 have little effect on the phosphorylation of non-histone proteins under these conditions. The results suggest that individual histones may be involved in the control of phosphorylation of specific non-histone nuclear proteins, particularly at times when the concentrations of nuclear proteins are changing relative to one another.