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 Ca^2–/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.Abbreviations NHP Non-Histone Protein - PMSF Phenylmethylsulfonyl Fluoride - DTT Dithiothreitol - SDS Sodium Dodecyl Sulfate     

The protein composition of Friend cell nuclear matrix stabilized by various treatments. Different recovery of nucleolar proteins B23 and C23 and nuclear lamins

Summary— Using two-dimensional polyacrylamide gels stained with Coomassie blue we have studied the protein composition of the nuclear matrix obtained from mouse erythroleukemic nuclei kept at O°C throughout the isolation procedure to prepare the high ionic strength resistant fraction (control matrix) or stabilized in vitro or in vivo by different procedures prior to subfractionation (ie 37°C incubation of isolated nuclei; sodium tetrathionate exposure of purified nuclei; heat shock of intact cells). When the matrix obtained from 37°C incubated nuclei was compared with the control matrix, striking differences in the polypeptide pattern were seen if the protein was obtained in both cases from an equivalent number of nuclei. On the other hand, if the same amount of protein for both the samples was applied to the gels the differences were less evident. Sodium tetrathionate stabilization of isolated nuclei and heat shock of intact cells produced a matrix protein pattern that was very similar and differed from that of the in vitro heat-exposed matrix. Using specific polyclonal antisera, we demonstrate that nucleolar proteins B23/numatrin and C23/nucleolin were very abundant in the matrix obtained from chemically-treated nuclei or in vivo heat-stabilized nuclei but were recovered in very small amounts (B23) or completely absent (C23) in the matrix prepared from nuclei heated to 37°C in vitro. Differences were seen also in the recovery of nuclear lamins, and especially lamin B, that was poorly represented in the sodium tetrathionate-stabilized matrix. The results demonstrate that in mouse erythroleukemia cells the increased recovery of nuclear matrix protein that is seen after in vitro heating of isolated nuclei is predominantly due to an additional recovery of the same types of polypeptides that are detected also in the absence of such a treatment. The data also indicate that in vivo heat shock of intact cells produces a nuclear matrix protein pattern that is more similar to the pattern seen after stabilization of purified nuclei with sodium tetrathionate and differs significantly from that obtained by exposing nuclei to 37°C in vitro, unlike to that what previous reports have indicated.     

Cell cycle-related transfer of proteins between the nucleus and cytoplasm of Physarum polycephalum

The proteins of wild-type and polyploid plasmodia of P. polycephalum were prelabelled with [³H]leucine and [¹⁴C]leucine. The two types of plasmodia were then fused for 2 h. Following fusion the nuclei were isolated and the smaller wild-type cell nuclei separated from the larger polyploid cell nuclei. The proteins were isolated from the recipient cell nuclei and the recipient nuclear proteins extracted. Ratios of ³H/¹⁴C in the various nuclear protein fractions show that during fusion differential transfer of labelled preformed proteins from the donor cell into the recipient cell nucleus occurs. The quantity of proteins transferred varies among the different fractions and with the phase of the cell cycle. Isotopic dilution experiments indicate that these differences in protein transfer are, in part, due to a high rate of synthesis and turnover of the nuclear proteins.     

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.     

Protein Phosphorylation in Polysomes of Pumpkin Cotyledons after Coumarin Treatment

Abstract: The natural compound, coumarin, caused a change in protein pattern and influenced the phosphorylation status of some ribosome-associated proteins of pumpkin seedlings in vivo and in vitro. Low concentrations of coumarin stimulated ribosome-associated protein phosphorylation only in cotyledons but not in roots and stems. Two phosphoproteins whose phosphorylation state was influenced upon coumarin treatment could be isolated and characterized by their relative molecular weight of about 58 and 65 kDa and pl-values at 5.2 and 5.7, respectively. These phosphoproteins are not major constituents of small or large subunits of ribosomes. We did not find any influence of coumarin on phosphorylation of ribosomal proteins S6, LAO and LAI–3.     

Protein phosphorylation in human peripheral nerve: Altered phosphorylation of a 25-kDa glycoprotein in leprosy

Protein phosphorylation in a low speed supernatant of human peripheral nerve (tibial and sural) homogenate was investigated. The major phosphorylated proteins had molecular mass in the range of 70, 55, 45, and 25 kDa. Mg²⁺ or Mn²⁺ was essential for maximum phosphorylation although Zn²⁺, Co²⁺, and Ca²⁺, could partially support phosphorylation. External protein substrates casein and histone were also phosphorylated. The protein phosphatase inhibitor orthovanadate enhanced the phosphorylation of the 45 and 25 kDa proteins significantly. Concanavalin A-Sepharose chromatography of the phosphorylated peripheral nerve proteins showed that the 25 kDa protein was a glycoprotein. Protein phosphorylation of peripheral nerves from leprosy affected individuals was compared with normals. The phosphorylation of 25 kDa protein was decreased in most of the patients with leprosy.     

2,4-Dichlorophenoxyacetic Acid-enhanced Phosphorylation of Soybean Nuclear Proteins

In vitro nuclear protein phosphorylation is enhanced in nuclei isolated from 2,4-dichlorophenoxyacetic acid (2,4-d)-treated mature soybean (Glycine max) hypocotyl relative to nuclei from untreated tissue. Increased nuclear protein phosphorylation correlates with increased levels of nuclear protein kinase activity. These changes generally parallel previously reported 2,4-d-enhanced RNA polymerase activity of these nuclei and the in vivo levels of RNA synthesis. Phosphate incorporation represents bona fide protein phosphorylation, with 87% of the label being identified as phosphoserine and 7% as phosphothreonine. Label from [γ-³²P]adenosine 5′-triphosphate is incorporated primarily into various nonhistone fractions with the greatest accumulation in loosely associated fractions (either released during incubation with ATP or removed by 0.15 m Nacl). Although electrophoretic analysis on sodium dodecyl sulfate gels shows no differences in the protein profiles of the loosely associated or sodium dodecyl sulfate-soluble nonhistone proteins, there are changes in the pattern of phosphorylation of other proteins, after 2,4-d treatment. Acid-soluble basic nuclear proteins are phosphorylated to a much lower extent than are the other nuclear protein fractions. While histone F₁ is subject to slight phosphorylation when nuclei are labeled in vitro, phosphorylation of the other histones is undetectable. One acid-soluble protein shows a substantial increase in quantity and in phosphorylation after 2,4-d treatment. This protein is similar in electrophoretic mobility to pea histone F₁ but its identity is unknown. Urea-acetic acid gels of the acid-soluble nuclear proteins show that auxin treatment results in increased quantities and in increased phosphorylation of various low mobility nonhistone basic nuclear proteins.     

Light-Induced Changes in Phosphorylation Status of Low Molecular Weight Wheat Nuclear Proteins

A large number of polypeptides were phosphorylated when in vitro protein phosphorylation was carried out in nuclei isolated from dark-grown seedlings. For studying the effect of light, dark-grown seedlings were exposed to light and the isolated nuclear proteins phosphorylated in vitro. Although 4 h of white light was sufficient to alter the phosphorylation status of at least two polypeptides of 19 and 17 kD but the effect of light was more pronounced after irradiation for 8 h or more, leading to virtual disappearance of a 19 kD and emergence of a 17 kD phosphopolypeptide. Studies using norflurazon, a bleaching herbicide, indicate that, in addition to 19 and 17 kD phosphopolypeptides, another 21 kD phosphopolypeptide may be involved in the de-etiolation process. However, the nature of the photoreceptor involved in these light-induced changes in nuclear protein phosphorylation remains to be established.     

Ca(2+)-dependent in vivo protein phosphorylation and encystment induction in the ciliated protozoan Colpoda cucullus

Encystment induction of Colpoda cucullus is promoted by an increase in external Ca²⁺ and overpopulation of Colpoda vegetative cells. Using phos-tag detection assays, the present study revealed that the in vivo phosphorylation level in several proteins [33 kDa, 37 kDa, 37.5 kDa, 43 kDa, 47 kDa, 49 kDa, etc.] was raised when the vegetative cells were stimulated by overpopulation to encyst in a medium containing 0.1 mM Ca²⁺ or without the addition of Ca²⁺. Both overpopulation-mediated encystment induction and protein phosphorylation were suppressed by the addition of EGTA. Ca²⁺/overpopulation-stimulated encystment induction and protein phosphorylation were also suppressed by the addition of BAPTA-AM. These results suggest that the Ca²⁺ inflow promoted by cell-to-cell stimulation due to overpopulation may activate signaling pathways involving protein phosphorylation and encystment induction. In the presence of cAMP-AM, the phosphorylation levels of 33 kDa, 37 kDa, 37.5 kDa, 43 kDa, 47 kDa and 49 kDa proteins were enhanced, and encystment induction was promoted. Enzyme immunoassays (EIAs) showed that intracellular cAMP concentration was raised prior to encystment when the cells were stimulated by overpopulation. These results suggest that cAMP/PKA-dependent protein phosphorylation, which is an event on Ca²⁺-triggered signaling pathways, may be involved in encystment induction.     

Phosphorylation of nuclear matrix proteins in isolated regenerating rat liver nuclei

The phosphorylation of nuclear matrix proteins from normal and regenerating rat liver nuclei was examined using an $\text{in vitro}$ system of isolated nuclei and γ-³²P-ATP. Phosphorylation of the nuclear matrix proteins was 2–3 fold higher than that of the total nuclear proteins in normal nuclei. The level of phosphorylation of the matrix proteins was enhanced an additional three fold at a period in liver regeneration (12 hours) just preceding the onset of DNA synthesis.     

Identification of New Amine Acceptor Protein Substrate Candidates of Transglutaminase in Rat Liver Extract: Use of 5-(Biotinamido) Pentylamine as a Probe

Transglutaminases (TGs) are a family of enzymes that catalyze Ca²⁺-dependent post-translational modification of proteins by introducing protein-protein crosslinks (between specific glutamine and lysine residues), amine incorporation, and site-specific deamidation. In this study, new amine acceptor protein substrates of TG were isolated from rat liver extract and identified using 5-(biotinamido) pentylamine, a biotinylated primary amine substrate, as a probe. TG protein substrate candidates labeled with biotin by endogenous TG activity were isolated and recovered by avidin column chromatography. Proteins with molecular masses of 40, 42, and 45 kDa were the main components of the labeled proteins. Determination of their partial amino acid sequences and immunoblotting analyses were done to identify them. The 45-kDa protein was identical with betaine-homocysteine S-methyltransferase (EC 2.2.2.5), which was identified in our previous study. The 40- and 42-kDa proteins were identified as arginase-I (EC 3.5.3.1) and fructose-1,6-bisphosphatase (EC 3.1.3.11) respectively. TG catalyzed incorporation of 5-(biotinamido) pentylamine into both arginase-I and fructose-1,6-bisphosphatase purified from rat liver was confirmed in vitro. These results suggest that these two enzymes are the new protein substrate candidates of TG and that they can be modified post-translationally by cellular TG.     

The CD95 (Fas/APO-1) receptor is phosphorylatedin vitro andin vivo and constitutively associates with several cellular proteins

Different CD95 (Fas/APO-1) isoforms and phosphory lated CD95 species were identified in human T and B cell lines. We had shown previously that the CD95 intracellular domain (IC), expressed as a glutathione S-transferase (GST) fusion protein in murine L929 fibroblasts, was phosphorylatedin vivo. GST-CD95IC was phosphorylatedin vitro by a kinase present in extracts from the human lymphocytic cell lines Jurkat and MP-1 and from murine L929 cells. Phosphoamino acid analysis indicated that phosphorylation occurred at multiple threonine residues and also at tyrosine (Tyr232 and Tyr291) and serine. Amino acids 191 to 275 of CD95 were sufficient for phosphorylation at threonine, tyrosine and serine and also mediated interaction with a 35 kDa cellular protein. Immuno-precipitation of CD95 and chemical cross-linking revealed CD95-associated proteins of approximately 35, 45 and 75 kDa. GST-CD95IC affinity chromatography detected binding of the 35 and 75 kDa protein species. The 75 kDa species may correspond to the CD95-associated proteins RIP or FAF1 and the 35 kDa protein may represent a TRADD analogue. These data indicate that several cellular proteins interact with CD95, possibly in a multi-protein complex, and that a kinase activity is associated with CD95 not onlyin vitro but alsoin vivo. Therefore, receptor phosphorylation may play a role in CD95 signal transduction. This work was in part supported by a grant from the Health Research Council of New Zealand (to JW).     

Protein phosphorylation in isolated nuclei from etiolated Avena seedlings. Effects of red/far-red light and cholera toxin

We have studied the phosphorylation/dephosphorylation of several nuclear proteins in isolated nuclei from etiolated Avena seedlings as a function of red/far-red light. The effect of stimulatory (ADP-ribosylation by cholera toxin) or inhibitory (GDP beta S) conditions for GTP-binding proteins was also studied. Red or far-red light enhanced the phosphorylation level of 2 nuclear proteins with molecular masses of 75 and 60 kDa. The phosphorylation pattern was affected by the addition of cholera toxin or GDP beta S to the isolated nuclei. At least 2 proteins with molecular masses of 24 and 75 kDa cross-reacted by Western blot with GTP-binding protein antibodies.     

Cellular Immune Responses to Cell Wall Peptidoglycan Associated Protein Antigens in Tuberculosis Patients and Healthy Subjects

We have isolated cell wall peptidoglycan associated proteins (CW-Pr) of Mycobacterium tuberculosis H₃₇Ra by chemical treatment with trifluoromethanesulfonic acid:anisole (2:1), which further resolved into 71, 60 and 45 kDa proteins on SDS-PAGE. A study was carried out to investigate the immunoreactivity of these proteins with blood samples from 4 categories, including 15 tuberculous patients (TB), 5 tuberculous patients on ATT (TBT), 10 PPD non-reactive healthy controls (HPPD^?) and 11 PPD reactive healthy controls (HPPD⁺). Comparing the proliferative responses to cell wall protein antigens, it was observed that the 71 kDa protein gave maximum stimulation with PBMCs from the TB and HPPD⁺ groups. The adherent PBMCs from the TB group also demonstrated enhanced phagocytosis, particularly in the presence of 71 and 45 kDa proteins, and the phagocytic index was significantly higher (P < 0.05) than the TBT group. However, PBMCs from of the groups recognized the 60 kDa cell wall antigen. Our results suggest that the 71 kDa protein from the cell wall of M. tuberculosis is highly immunogenic.     

Thiophosphorylation and phosphorylation of saponin-permeabilized cultured chromaffin cells

There is increasing evidence that phosphorylation of cellular proteins plays a role in the control of events surrounding secretion in neurons and chromaffin cells. In previous studies, we have used thiophosphorylation of cell proteins as a means of fixing cellular phosphorylation reactions in the phosphorylated state. Thiophosphorylation of permeabilized chromaffin cells with adenosine-5′-O-(3-thiotriphosphate) results in irreversible inhibition of secretion. Thiophosphate is incorporated primarily by two cellular proteins of 58 and 47 kDa. Calcium enhanced thiophosphorylation of the 47 kDa protein but not the 54 kDa protein. This pattern of thiophosphorylation differed markedly from that for phosphorylation under similar treatment conditions. The phosphoprotein composition of the cells depended upon the medium calcium and ATP concentration. In the absence of exogenous ATP, fewer phosphoproteins were seen in calcium stimulated cells than in unstimulated cells. Proteins labelled with ³²P or ³⁵S migrated to the same position on polyacrylamide gels containing sodium dodecyl sulfate. In the presence of exogenous ATP, ³²P incorporation was similar for both control and calcium-stimulated cells and was found primarily in a 64 kDa protein. Incorporation of [³²P]phosphate by calcium-stimulated cells was reduced to the same extent by pretreatment of the cells with either adenosine-5′-O-(3-thiotriphosphate) or ATP.The different electrophoretic banding patterns for thiophosphorylation and phosphorylation are likely due to the irreversibility of the thiophosphorylation reaction and reversibility of the phosphorylation reaction. The inability to turn over thiophosphate groups, in association with changes in secretion, may permit identification of those phosphoproteins that are putatively involved in secretion.     

Elevated levels of glycoprotein gp200 in progeria fibroblasts

The glycosylation of proteins in fibroblasts from people with the premature ageing disease Hutchinson-Gilford Progeria Syndrome (progeria) was investigated.Protein was prepared from fibroblast cell lines established from skin biopsy taken from progeria patients and control donors. Glycoproteins were labelled by the covalent attachment of the steroid hapten digoxygenin to the sugar group. After separation of total protein by SDS-PAGE and electroblotting onto Immobilon-P, glycoproteins were detected by enzyme immunoassay.We have observed a glycoprotein of M_r 200 kDa which is consistently present in protein preparations from progeria fibroblasts and which is absent, or markedly reduced, in preparations from control fibroblasts. This suggests that it may be useful as a marker for progeria. Similar analysis of progeria lymphoblast and control lymphoblast cultures did not show this altered pattern of glycosylated proteins, indicating that it may be cell-type specific.Glycoproteins were also detected by labelling fibroblastsin vitro with D-[6-³H] glucosamine hydrochloride followed by SDS-PAGE of isolated protein and subsequent fluorography. Profiles of glycoproteins from progeria and control fibroblasts were consistent with those obtained from labelling of carbohydrate groups with digoxygenin. Protease digestion of cell protein verified that the band at M_r 200 kDa contains a protein core.Characteristic features of progeria primarily involve the connective tissue and include wrinkled and loose skin, loss of soft tissue, thin limbs and stiff joints. Death of progeria patients is usually a result of cardiovascular abnormalities. The most consistent manifestations thus involve the connective tissue.The glycoprotein of M_r 200 kDa which we have observed in progeria fibroblastsin vitro could reflect a perturbation in glycosylation which may underly the connective tissue defects seen in progeria.Abbreviations EMEM Eagle's Minimum Essential Medium with Earle's Salts - FCS Fetal Calf Serum - PBS Ca²⁺- and Mg²⁺-free Phosphate-Buffered Saline - PDL Population Doubling Level - SDS Sodium Dodecyl Sulphate - SDS-PAGE Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis     

Phosphorylation mediates the nuclear targeting of the maize Rab17 protein

The maize abscisic acid-responsive Rab17 protein localizes to the nucleus and cytoplasm in maize cells. In-frame fusion of Rab17 to the reporter protein β-glucuronidase (GUS) directed GUS to the nucleus and cytoplasm in transgenic Arabidopsis thaliana and in transiently transformed onion cells. Analysis of chimeric constructs identified one region between amino acid positions 66–96, which was necessary for targeting GUS to the nucleus. This region contains a serine cluster followed by a putative consensus site for protein kinase CK2 phosphorylation, and a stretch of basic amino acids resembling the simian virus 40 large T antigen-type nuclear localization signal (NLS). Mutation of two basic amino acids in the putative NLS had a weak effect on nuclear targeting in the onion cell system and did not modify the percentage of nuclear fusion protein in the Arabidopsis cells. The mutation of three amino acids in the consensus site for CK2 recognition resulted in the absence of in vitro phosphorylated forms of Rab17 and in a strong decrease of GUS enzymatic activity in isolated nuclei of transgenic Arabidopsis. These results suggest that phosphorylation of Rab17 by protein kinase CK2 is the relevant step for its nuclear location, either by facilitating binding to specific proteins or as a direct part of the nuclear targeting apparatus.     

Purification of N-terminally truncated histone H2A-monoubiquitin conjugates from leukemic cell nuclei: probable proteolytic products of ubiquitinated H2A

To gain insight into the significance of nuclear ubiquitinated proteins, two serial extracts prepared from various leukemic cells were analysed by western blotting with anti-ubiquitin antibody. Two previously unidentified ubiquitinated proteins with molecular masses of 10 and 17 kDa were found in 8 M urea-soluble extracts, obtained from Tris-buffer-insoluble materials, of acute myeloid leukemia OCI/AML 1a cells and the cells from the leukemia patients. Both proteins were successfully purified from the OCI/AML 1a cells and identified as monoubiquitin-truncated H2A conjugates, the 10 kDa ubiquitinated H2A(115-129) and the 17 kDa ubiquitinated H2A(54-129), suggesting that both proteins were produced by limited proteolysis of an intact form (23 kDa) of ubiquitinated H2A(1-129). The 17 kDa protein as well as the 23 kDa ubiquitinated histone H2A were localised in chromatin fractions of the OCI/AML cells and released by high concentrations of salt in a micrococcal nuclease-sensitive manner, suggesting their association with chromatin. In contrast, the 10 kDa protein remained insoluble even when the nuclei were treated with nuclease under high salt concentrations, presumably due to binding to the nuclear matrix. An antibody recognising H2A(70-81) also detected the 17 kDa protein in anti-ubiquitin immunoprecipitates obtained from the OCI/AML cell nuclei. In addition, the 17 kDa protein levels in THP-1 cells were transiently increased, concomitant with a decrease in the 23 kDa ubiquitinated H2A, by treatment with phorbol 12-myristate 13-acetate or all-trans-retinoic acid, both of which induce differentiation. This is the first report of probable proteolytic products of ubiquitinated H2A, which might have a role in nuclear functions.     

Influence of chromatin structure, antibiotics, and endogenous histone methylation on phosphorylation of histones H1 and H3 in the presence of protein kinase A in rat liver nuclei in vitro

In vitro phosphorylation of histones H1 and H3 by cAMP-dependent protein kinase A and endogenous phosphokinases in the presence of [γ-³²P]ATP was studied in isolated rat liver nuclei with different variants of chromatin structural organization: condensed (diameter of fibrils 100–200 nm; N-1) and partly decondensed (diameter of fibrils ～30 nm; N-2). In the N-1 state histone, H1 is phosphorylated approximately twice as much than histone H3. Upon the decondensation of the chromatin in the N-2 state, 1.5-fold decrease of total phosphorylation of H1 is observed, while that of H3 does not change, although the endogenous phosphorylation of both histones is reduced by half. Changes in histone phosphorylation in the presence of low or high concentrations of distamycin and chromomycin differ for H1 and H3 in N-1 and N-2. It was found that distamycin (DM) stimulates the phosphorylation of tightly bound H1 fraction, which is not extractable by polyglutamic acid (PG), especially in N-1. Chromomycin (CM) increases the phosphorylation of both histones in PG extracts and in the nuclear pellets, particularly in N-2. At the same time, in N-1 one can detect phosphorylation of a tightly bound fraction of histones H1 whose N-termini are located on AT-rich sites that become inaccessible for protein kinase in the process of chromatin decondensation in N-2. At the same time, in N-2 the accessibility for protein kinase A of tightly bound H1 fractions, whose N-termini are located on GC-rich sites, increases dramatically. High concentrations of both CM and DM in N-1 and N-2 stimulated phosphorylation of the non-extractable by PG fraction of H1 whose N-termini are located on sites where AT ≈ GC. CM at high concentration stimulated 4–7 times the phosphorylation of a small fraction of H3, which is extracted by PG from both types of nuclei. We detected an effect of endogenous methylation of histones H1 and H3 in the nuclei on their subsequent phosphorylation depending on the chromatin structure, histone-chromatin binding strength, and concentration of DM.