Nonhistone chromatin proteins of B-lymphocytes stimulated by lipopolysaccharide Two-dimensional gel electrophoresis analysis of proteins synthesized and phoshorylated during the initiation of lymphocyte differentiation

Synthesis and phosphorylation of nonhistone chromatin and nucleoplasmic proteins during the first 24 h of activation of mouse B-lymphocytes by the B-cell mitogen lipopolysaccharide have been studied by two-dimensional gel electrophoretic analysis. Although little change occurs in the nucleoplasmic proteins, it has been shown that the incorporation of [³⁵S]methionine into nonhistone chromatin proteins is selectively stimulated. The degree of stimulation and the kinetics of synthesis are characteristic for each individual protein; some proteins exhibit increased incorporation only 4 h after addition of mitogen, while others are synthesized de novo between 8 and 24 h. After 72 h stimulation, the majority of nonhistone chromatin protein synthesis occurs in the highly differentiated lymphoblasts and plasma cells actively secreting IgM, very little synthesis taking place in the small lymphocytes. Analysis of nuclear proteins from lymphocytes stimulated for 2 h showed no selective stimulation of phosphorylation. These observations suggest that nonhistone chromatin proteins play an important role in the regulation of gene expression in B-lymphocytes.     

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

The synthesis of non-histone chromatin proteins and nucleoplasmic proteins has been followed during lipopolysaccharide-induced division and differentiation of murine B lymphocytes. Synthesis was measured by pulse labelling with [3H]leucine, extraction of proteins was under conditions designed to prevent proteolysis and analysis of labelled proteins was by polyacrylamide gel electrophoresis. The average specific activity of non-histone chromatin proteins increased 3-fold, to a maximum, after culture for 24 h with lipopolysaccharide. Comparison of the relative synthesis of individual proteins (stimulation index) reveals three distinct responses: (1) those in the largest group show low stimulation indices, generally less than two; (2) a group of four proteins have indices between 4 and 5; (3) two proteins (molecular weights 21 000 and 22 000) both show an index of 5 at 24 h rising to between 7 and 8 by 48 h when the average specific activity is falling, coinciding with the period of rapid differentiation to high rate IgM secretion. Additionaly at this time, a newly labelled protein (Mr = 36 500) appears in the nucleoplasm followed by a second protein (Mr = 63 000) appearing between 48 and 72 h. The patterns of change are consistent with an overall increase in non-histone chromatin proteins synthesis, necessary for cell division, with superimposed specific changes in synthesis of non-histone chromatin proteins which could be related to regulation of cell differentiation.     

Phosphorylation of nuclear proteins

Many nuclear proteins are phosphorylated: they range from enzymes to several structural proteins such as histones, non-histone chromosomal proteins and the nuclear lamins. The pattern of phosphorylation varies through the cell cycle. Although histone H1 is phosphorylated during interphase its phosphorylation increases sharply during mitosis. Histone H3, chromosomal protein HMG 14 and lamins A, B and C all show reversible phosphorylation during mitosis. Several nuclear kinases have been characterized, including one that increases during mitosis and phosphorylates H1 in vitro. Factors have been demonstrated in maturing amphibian oocytes and mitotic mammalian cells that induce chromosome condensation and breakdown of the nuclear membrane. The possibility that they are autocatalytic protein kinases is considered. The location of histone phosphorylation sites within the nucleosome is consistent with a role for phosphorylation in modulating chromatin folding.     

Changes in the phosphorylation of non-histone chromatin proteins during the cell cycle of HeLa S 3 cells

The phosphorylation of non-histone chromatin proteins in synchronized HeLa S₃ cells was studied in 5 phases of the cell cycle: mitosis, G₁, early and late S, and G₂. The rate of non-histone chromatin protein phosphorylation was found to be maximal during G₁ and G₂, somewhat decreased during S phase, and almost 90% depressed during mitosis. Analysis of the phosphorylated non-histone chromatin proteins by SDS-acrylamide gel electrophoresis showed a heterogeneous pattern of phosphorylation as measured by labeling with ³²P. Significant variations in the labeling pattern were seen during different stages of the cell cycle, and particular unique species appeared to be phosphorylated selectively during certain stages of the cycle.     

Stimulatory effect of Bestatin,a new specific inhibitor of aminopeptidases,on the blastogenesis of guinea pig lymphocytes

A potent protease-inhibitor of Actinomycetes origin, Bestatin. which is of dipeptide nature and inhibits aminopeptidase B and leucine-aminopeptidase competitively, strongly stimulates blastogenesis of small lymphocytes triggered with polyclonal mitogen. such as phytohemagglutinin (PHA), concanavalin A (Con A), pokeweed mitogen (PWM) and lipopolysaccharide of Escherichiae coli (LPS), whereas it inhibits DNA synthesis of normal resting lymphocytes. The stimulatory effect is non-selective with respect to the category of small lymphocytes, i.e. T- and B-lymphocytes, but strikingly selective with respect to the stage of blastogenesis: the stimulation is greatest at a relatively early stage, diminishes as mitogen-activation proceeds, and is not appreciable at a later stage of lymphocyte blastogenesis.The pattern of Bestatin stimulation on lymphocyte blastogenesis is specific for the mitogen used: in T-lymphocyte activation with PHA or Con A, the stimulation first increases and then decreases with increase in mitogen concentrations, whereas in B-lymphocyte activation with LPS, with increasing concentrations of the mitogen, the stimulation increases to a plateau at approximately 100 μg/ml of mitogen. The optimum concentration of Bestatin was found to be approximately 50 μg/ml (0.16 mM) for either PHA or Con A activation, and 50 to 75 μg/ml for B-cell activation with LPS. Bestatin must remain in cultures of T- and B-lymphocytes with polyclonal mitogens for at least about 24 and 16 hr, respectively, to exert its stimulatory effect on blastogenesis.Biochemical results, together with those from autoradiographic analyses, indicate that Bestatin increases the number of blastoid-transformed lymphocytes with polyclonal stimulants. It is suggested that aminopeptidases, possibly located at the cell surface, may play a role in the control of lymphocyte activation during immune responses.     

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.     

Phosphorylation of rat thymus histones, its control and the effects thereon of gamma-irradiation.

The phosphate content of rat thymus histones was determined. As expected for a replicating tissue, histones 1 and 2B were more phosphorylated and had higher 32P uptakes than did histones from resting liver nuclei; the other histones all showed 32P uptake, but the phosphate content and uptake of histone 2A was about half that for liver histone 2A. When thymus nuclei were incubated in a slightly hypo-osmotic medium, non-histone proteins and phosphorylated histones were released into solution; this was enhanced if ATP was present in the medium. [gamma-32P]ATP was incorporated into non-histone proteins, including protein P1, and into the ADP-ribosylated form of histone 1; negligible 32P was incprporated into the other, bound, histones. Histones 1 and 2B added to the incubation medium were extensively, and histones 2A and 4 slightly, phosphorylated. Histones released by increasing the ionic strength of the medium were phosphorylated. Added lysozyme and cytochrome c were neither bound nor phosphorylated, but added non-histone protein P1 was phosphorylated, causing other histones to be released from the nuclei, especially histones 2A and 3. The released histones were phosphorylated. gamma-Irradiation decreased 32P uptake into the non-ADP-ribosylated histones 1 and 4; phosphorylation of histone 1 in vitro was unaffected. The importance of non-histone proteins, ATP availability and nuclear protein kinases to the control of histone phosphorylation in vivo is discussed.     

Increases in microtubule assembly and in tubulin content in mitogenically stimulated mouse splenic T lymphocytes

We have examined the changes in the microtubule and tubulin contents in populations of mouse splenic T lymphocytes stimulated by the mitogen concanavalin A. Indirect immunofluorescence staining with antiserum to tubulin indicated that a more extensive microtubule network was assembled from the centrosome in those cells which had increased in size in response to the mitogen. Direct counts of microtubules from electron micrographs of the centrosome regions of cells showed approximately a 2-fold increase in microtubule number in 48 h stimulated populations and up to a 5-fold increase in the large, fully stimulated, blast cells. Determinations of tubulin and actin contents were made by the measurement of peptides specific to those proteins. As a percentage of total cell protein both of these cytoskeletal proteins increased during the first 24 h of stimulation. Tubulin increased 50% by 24 h and remained high in populations stimulated for 48 h. The tubulin content per cell increased 2.5-fold, from 0.20 to 0.51 μg/10⁶ cells, in the 48 h stimulated population. An increase in tubulin content was also seen following the stimulation of nude mouse B lymphocyte populations and of total splenic lymphocyte populations. Our results show that during lymphocyte stimulation there is a large increase in the numbers of microtubules assembled which is correlated with, and appears dependent on, a similar large increase in the cellular tubulin content.     

Early Events in Lymphocyte Transformation by Phytohaemagglutinin

Synthesis and phosphorylation of three main nuclear protein fractions were studied in human lymphocytes stimulated by phytohaemagglutinin (PHA). The first fraction to be synthesized and phosphorylated after induction was that of the acidic proteins, followed by that containing the soluble proteins. Synthesis of histories commenced 24 h after exposure to PHA. Distinctive patterns of both synthesis and phosphorylation of the acidic proteins were recorded at different times in the cell cycle, which may reflect activation or suppression of specific cellular functions. Phosphorylation of the histones also occurred, as an early event during lymphocyte transformation and also much later, at the time of DNA synthesis.     

Nuclear protein kinase activities during the cell cycle of HeLa S3 cells

To ascertain the activity and substrate specificity of nuclear protein kinases during various stages of the cell cycle of HeLa S3 cells, a nuclear phospho-protein-enriched sample was extracted from synchronised cells and assayed in vitro in the presence of homologous substrates. The nuclear protein kinases increased in activity during S and G2 phase to a level that was twice that of kinases from early S phase cells. The activity was reduced during mitosis but increased again in G1 phase. When the phosphoproteins were separated into five fractions by cellulose-phosphate chromatography each fraction, though not homogenous, exhibited differences in activity. Variations in the activity of the protein kinase fractions were observed during the cell cycle, similar to those observed for the unfractionated kinases. Sodium dodecyl sulfate polyacrylamide gel electrophoretic analysis of the proteins phosphorylated by each of the five kinase fractions demonstrated a substrate specificity. The fractions also exhibited some cell cycle stage-specific preference for substrates; kinases from G1 cells phosphorylated mainly high molecular weight polypeptides, whereas lower molecular weight species were phosphorylated by kinases from the S, G2 and mitotic stages of the cell cycle. Inhibition of DNA and histone synthesis by cytosine arabinoside had no effect on the activity or substrate specificity of S phase kinases. Some kinase fractions phosphorylated histones as well as non-histone chromosomal proteins and this phosphorylation was also cell cycle stage dependent. The presence of histones in the in vitro assay influenced the ability of some fractions to phosphorylate particular non-histone polypeptides; non-histone proteins also appeared to affect the in vitro phosphorylation of histones.     

Differential kinase systems are involved in the rapidly turning over phosphorylation of prominent nuclear proteins

The activity of endogenous nuclear protein kinases has been probed in an vitro assay system of isolated nuclei from Chironomus salivary gland cells. The phosphorylation of a set of seven prominent rapidly phosphorylated non-histone proteins and of histones H3, H2A and H4 was analyzed using ATP or GTP as phosphoryl donor and heparin as protein kinase effector. The core histones H2A and H3 both incorporate 32P from [gamma-32P]ATP as well as from [gamma-32P]GTP but their phosphorylation is differentially affected by heparin. The phosphorylation of H2A is blocked by heparin while that of H3 is even stimulated in the presence of heparin when ATP is used as phosphate donor. H4 is unable to incorporate phosphate groups from GTP but its ATP-based phosphorylation is heparin sensitive. Of the non-histone protein kinase substrates, we could only detect two, the 44-kDa and 115-kDa proteins, which are heparin sensitive with either ATP or GTP and, thus, strictly meet the criteria for casein kinase type II-specific phosphorylation. The investigated histones and non-histone proteins can be grouped into three broad categories on the basis of their phosphorylation properties. (A) Proteins very likely affected by casein kinase NII. (B) Proteins phosphorylated by strictly ATP-specific protein kinases. (C) Proteins phosphorylated by ATP as well as GTP utilizing protein kinase(s) other than casein NII. Category B proteins can be subdivided into proteins phosphorylated in a heparin-resistant (B1) and heparin-sensitive (B2) manner. The phosphorylation of category C proteins may be heparin sensitive with ATP only (C1), heparin sensitive with GTP only (C2), heparin insensitive with both ATP and GTP (C3) or stimulated by heparin (C4).     

Serine and tyrosine phosphorylation of 28- and 35-kDa proteins of human T lymphocytes stimulated by streptococcal M protein

Purified polypeptide fragments of certain surface M proteins of group A streptococci stimulate blastogenesis and the differentiation of cytotoxic T lymphocytes of normal human lymphocytes. The biochemical basis of lymphocyte stimulation by a type M5 protein polypeptide fragment (pep M5) was investigated. Optimal blastogenic doses of pep M5 or phytohemagglutinin stimulated the phosphorylation of several cellular proteins. However, pep M5 but not phytohemagglutinin induced the phosphorylation of 28- and 35-kDa proteins. The 28-kDa protein was shown to be phosphorylated only at serine residues, whereas the 35-kDa protein was phosphorylated only at tyrosine residues. Stimulation of peripheral blood lymphocytes with pep M5 caused a two-fold increase in the CD8+ and CD4+ 4B4+ subpopulations of T lymphocytes. The phosphorylation of the 28-kDa protein appeared to be confined to the CD4+ T cell subpopulation.     

Influence of thyroid hormone on ADP-ribosylation of nuclear proteins in cultured GH1 cells

We present evidence that T3 can alter the ADP-ribosylation of chromatin associated proteins. Nuclei from GH1 cells were incubated with [adenylate-32P]NAD and the radioactivity incorporated into histone and non-histone proteins was quantitated and analyzed by gel electrophoresis and autoradiography. Incubation of GH1 cells for 24 h with T3 lowered by 40-70% the [32P]ADP-ribose incorporated into nuclear proteins. However, incubation for 3 h with T3 resulted in a stimulation instead of a decrease of in vitro [32P]ADP-ribose incorporation. The major ADP-ribosylated component electrophoresed as a 120,000 molecular mass non-histone protein, and radiolabeled histones were also observed. The same protein species were observed for all the experimental groups and T3 affected the extent of ADP-ribosylation but did not alter the sedimentation of the [32P]ADP-ribosylated components excised from chromatin after micrococcal nuclease digestion.     

The binding of [3H]chlorambucil to nuclear proteins of the Yoshida ascites sarcoma.

The binding of [3H]chlorambucil to nuclear proteins, extracted from Yoshida ascites sarcoma cells at 6 h and 24 h after administration of 3H-labelled drug to tumour-bearing animals, has been examined. Both covalent and non-covalent binding was detected. Considerably more drug was found associated with the proteins isolated from the tumour sensitive to the effects of the drug compared with similar proteins isolated from the tumour with an acquired resistance to the effects of alkylating agents. The two-fold difference in binding to total cell protein is attributed to a higher intranuclear protein binding in sensitive cells. In particular the soluble nuclear sap fraction from sensitive cells bound at least five times as much drug as the corresponding fraction from resistant cells. Low levels of binding to histones were demonstrated compared with that to the non-histone chromatin proteins. Binding to the nuclear sap and non-histone chromatin proteins was principally to high molecular weight protein species; these did not appear to represent aggregation products as scans of stained polyacrylamide gels of the extracted protein fractions were unaltered by the treatment of tumour-bearing animals with chlorambucil. Binding to the nuclear proteins from sensitive cells tended to persist over a 24-h period, whereas it was considerably reduced in resistant cells.     

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.     

Importance of substrate conformation in the phosphorylation of chromatin-associated proteins by exogenous protein kinase C.

Protein kinase C (PKC)-mediated phosphorylation of chromatin-associated proteins was studied in vitro. HL-60 and HeLa nuclear proteins were notably unresponsive to exogenously added brain PKC. In contrast, 3T3 fibroblasts and lymphocytes from primary cultures exhibited PKC-dependent phosphorylation of chromatin-associated proteins when chromatin was induced to expand. Unexpanded nuclei in all cell lines were unresponsive. Responsiveness was particularly obvious in the decondensed chromatin of primary lymphocytes, where a large number of proteins were phosphorylated in response to exogenous PKC. DNAase-I and micrococcal nuclease strongly modulated these phosphorylation patterns indicating that the substrates were DNA-associated. It was concluded that although substrate conformation, i.e. condensation state, was the primary determining factor in control of PKC-dependent nuclear protein phosphorylation, different cell lines greatly differ in their overall responsiveness to exogenous PKC.     

Differential effects of cyclic adenosine-3',5'-monophosphate on phosphorylation of rat liver nuclear acidic proteins

The effects of cyclic AMP on the phosphorylation of different acidic proteins of rat liver nuclei were examined in vivo and in vitro. N⁶,O²′-dibutyryl cyclic AMP selectively stimulated in vivo phosphorylation of specific nuclear proteins more than twofold within 15 min after injection. Cyclic AMP caused only a small stimulation of phosphorylation of acidic proteins in isolated nuclei but the stimulation was selective for specific proteins. When isolated nuclear acidic proteins were incubated with a soluble cyclic AMP-dependent protein kinase, the cyclic nucleotide stimulated total phosphorylation about 1.7-fold. These results support the view that the regulatory effects of cyclic AMP may involve phosphorylation of acidic proteins associated with DNA in the chromatin.     

The phosphorylation of nuclear proteins in the regenerating and premalignant rat liver and its significance for cell proliferation.

In liver regeneration or neoplastic transformation, phosphorylation of nuclear proteins is stimulated. In the regenerating liver all main histone fractions are involved in this process. The type of histone phosphorylated seems to be dependent on the position of the partially synchronized cells within the generation cycle. At a time when most cells are exhibiting maximum HnRNA-synthesis, histone F2a2 belongs to those fractions with highly stimulated phosphate incorporation. Phosphorylation of this fraction alone is stimulated by cyclic AMP in parallel to a stimulation of HnRNA-synthesis. The preneoplastic liver is characterized by oscillating phosphorylation and dephosphorylation reactions of nearly all histone fractions during the first days of N-nitroso-diethylamine administration. After 2 months of carcinogen feeding a 50-150% stimulation of the phosphorylation of Fl subfractions is observed. The phosphate content of the other histones, however, has returned to the original level. A series of further proteins, isolated together with the histones, show very similar phosphorylation characteristics. These proteins are mostly of non-histone origin. It is suggested that some of them are responsible for the transport of RNA with messenger properties within the cell.     

Biogenesis of chromatin in animal cells. Stimulation of nuclear protein and DNA synthesis in hepatocytes after brief inhibition of translation by cycloheximide]

A drastic and brief inhibition of protein synthesis (up to 95% for 3--6 hrs) by cycloheximide in the liver of rats starved for 24 hrs results in a recovery and subsequent marked stimulation of non-histone proteins, histone chromosomal proteins and DNA. The stimulation of non-histone protein synthesis was observed after 1 hr (inhibition) 12--24 hrs (recovery and stimulation of protein synthesis) and 48--60 hrs (stimulation of DNA synthesis) following the administration of cycloheximide. Two periods of histone biosynthesis were observed. The first one (24--36 hrs) was not coupled and the second one (48--60 hrs) was coupled with DNA replication. During the recovery and stimulation of protein synthesis acetylation of the histone and non-histone proteins proceeds at an increased rate. Possible applicability of the model in question for investigations of chromatin biogenesis is discussed.