The lysine-rich H1 histones from the slime moulds, Physarum polycephalum and Dictyostelium discoideum lack phosphorylation sites recognised by cyclic AMP-dependent protein kinase in vitro.

Calcium chloride-extracted histones were prepared from nuclei of the slime moulds, Physarum polycephalum and Dictyostelium discoideum, and phosphorylation by purified preparations of cyclic AMP-dependent protein kinase (cAMP-d PK) and growth-associated H1 histone kinase (HKG) examined and compared. Among the major histone fractions and other proteins in the two preparations, the H1 histones from both organisms were found to be effective and exclusive substrates for HKG. cAMP-d PK, which phosphorylates mammalian H1 histone and certain, in particular H2B, of the mammalian core histones, phosphorylated several of the core histones from both slime moulds but did not phosphorylate H1 histone from either. The slime mould H1s remained ineffective substrates for cAMP-d PK even after extensive alkaline phosphatase treatment of the histone preparations. Additional studies demonstrated that the lack of slime mould H1 phosphorylation by cAMP-d PK was not due to competition of the H1 molecules with the core histones for the kinase. Our studies suggest that H1 histones from these organisms, whilst clearly containing sites for phosphorylation by HKG, apparently lack phosphorylation sites recognised by cAMP-d PK. Thus, the mediation of specific nuclear functions by cAMP-dependent phosphorylation of H1 in higher organisms may not occur or be required in these lower eukaryotes.     

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).     

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.     

Identification of nuclear substrates of the cyclic AMP-dependent protein kinase in Dictyostelium discoideum

A search for nuclear substrates of the cAMP-dependent protein kinase (cAMP-d PK) of Dictyostelium discoideum led to the identification of several such proteins. Identification was based initially on increased phosphorylation of the proteins in nuclear extracts catalyzed by added cAMP-d PK. One protein of Mr 38,000 was phosphorylated also in intact nuclei and in vivo; the amount of phosphoprotein or the level of phosphorylation increased during development. Both the Mr 38,000 protein and another substrate of Mr 195,000 were found in the nuclei of prespore and prestalk cells. Phosphorylation of other potential substrates of the cAMP-d PK was either prespore or prestalk cell-specific.     

Mouse spleen cell nuclear protein kinases and the stimulating effect of dsDNA on NHP phosphorylation by cyclic AMP-independent protein kinase in vitro

cAMP-dependent (designated as enzyme I, about 68,000 daltons) and cAMP-independent protein kinase (designated as enzyme II, about 45,000 daltons) have been partially purified from the nuclei of mouse spleen cells. Both kinases phosphorylated calf thymus histones as well as non-histone proteins (NHP) and required Mg2+ (8 mM) or Mn2+ (2 mM) for maximal activity. NEM (0.5 mM), which is an inhibitor of SH-enzymes, inhibited the histone phosphorylating activity of enzyme II by more than 90%, whereas it inhibited the activity of enzyme I by less than 10%. Moreover, the activity of enzyme II was more sensitive to high temperature than that of enzyme I. Non-histone protein (CM-III protein) served as a more effective substrate for enzyme II than histones; the Km value for CM-III protein was 34.4 micrograms/ml whereas that for histone H2a (14,300 daltons) was 155 micrograms/ml (1.08 x 10(-5) M). CM-III protein phosphorylation by enzyme II in vitro was greatly stimulated by the addition of dsDNA, but not by single-stranded DNA or bacterial ribosomal RNA. However, the phosphorylation of CM-III protein by enzyme I was less than 50% of that of histones, and there was no stimulatory effect. SDS-gel electrophoresis showed that two distinct NHPs (about 13,000 and 19,000 daltons) prepared from calf thymus chromatin were preferentially phosphorylated by enzyme II in vitro in the presence of dsDNA. This finding suggests that these two NHPs may be specific phosphate acceptors of cAMP-independent protein kinase (enzyme II) in the nuclei of mouse spleen cells.     

DNA binding by cyclic adenosine 3',5'-monophosphate dependent protein kinase from calf thymus nuclei.

Cyclic adenosine 3',5'-monophosphate (cAMP) dependent protein kinase and proteins specifically binding cAMP have been extracted from calf thymus nuclei and analyzed for their abilities to bind to DNA. Approximately 70% of the cAMP-binding activity in the nucleus can be ascribed to a nuclear acidic protein with physical and biochemical characteristics of the regulatory (R) subunit of cAMP-dependent protein kinase. Several peaks of protein kinase activity and of cAMP-binding activity are resolved by affinity chromatography of nuclear acidic proteins on calf thymus DNA covalently linked to aminoethyl Sephrarose 4B. When an extensively purified protein kinase is subjected to chromatography on the DNA column in the presence of 10(-7) M cAMP, the R subunit of the kinase is eluted from the column at 0.05 M NaCl while the catalytic (C) subunit of the enzyme is eluted at 0.1-0.2 M NaCl. When chromatographed in the presence of histones, the R subunit is retained on the column and is eluted at 0.6-0.9 M NaCl. In the presence of cAMP, association of the C subunit with DNA is enhanced, as determined by sucrose density gradient centrifugation of DNA-protein kinase complexes. cAMP increases the capacity of the calf thymus cAMP-dependent protein kinase preparation to bind labeled calf thymus DNA, as determined by a technique employing filter retention of DNA-protein complexes. This protein kinase preparation binds calf thymus DNA in preference to salmon DNA, Escherichia coli DNA, or yeast RNA. Binding of protein kinases to DNA may be part of a mechanism for localizing cyclic nucleotide stimulated protein phosphorylation at specific sites in the chromatin.     

Spermine inhibits the phosphorylation of the 11,000- and 10,000-dalton nuclear proteins catalyzed by nuclear protein kinase NI in NB-15 mouse neuroblastoma cells

The nuclear protein kinase NI (NI kinase) was purified from NB-15 mouse neuroblastoma cells by phosphocellulose column and casein affinity column chromatography. The purified NI kinase exhibited (i) an apparent subunit molecular weight of about 37,000, (ii) autophosphorylation, and (iii) insensitivity to inhibition by heparin. When NI kinase was added to heat-treated neuroblastoma nuclei in the presence of [gamma-32P] ATP, two proteins with apparent subunit molecular weights of 11,000 and 10,000 were prominently phosphorylated. Other protein kinases tested including the nuclear protein kinase NII, Type I cAMP-dependent protein kinase, and protein kinase C did not catalyze the phosphorylation of these two proteins. The NI kinase-catalyzed phosphorylation of these two proteins was completely inhibited by 1 mM spermine. In contrast, 10 mM putrescine, 2 mM spermidine, 5 mM arginine, and 10 mM NH4Cl, had no inhibitory effect on this phosphorylation reaction. Our study also indicated that the phosphorylation of the 11,000- and 10,000-dalton proteins occurred in the nuclear matrix fraction but not in heterogeneous nuclear ribonucleoproteins, high mobility group proteins, or histone fractions. We have previously reported that spermine specifically inhibits the endogenous phosphorylation of an 11,000-dalton nuclear protein in various mammalian cell lines (Chen, K. Y., and Verma, R. (1984) Biochem. Biophys. Res. Commun. 118, 710-716). The present study suggests that the 11,000- and 10,000-dalton nuclear proteins may be native substrates of nuclear protein kinase NI and that their phosphorylation can be affected by physiological concentrations of spermine.     

The role of Ca2+ and cyclic AMP in the phosphorylation of rat-liver soluble proteins by endogenous protein kinases

Rat liver soluble proteins were phosphorylated by endogenous protein kinase with [gamma-32P]ATP. Proteins were separated in dodecyl sulphate slab gels and detected with the aid of autoradiography. The relative role of cAMP-dependent, cAMP-independent and Ca2+-activated protein kinases in the phosphorylation of soluble proteins was investigated. Heat-stable inhibitor of cAMP-dependent protein kinase inhibits nearly completed the phosphorylation of seven proteins, including L-type pyruvate kinase. The phosphorylation of eight proteins is not influenced by protein kinase inhibitor. The phosphorylation of six proteins, including phosphorylase, is partially inhibited by protein kinase inhibitor. These results indicate that phosphoproteins of rat liver can be subdivided into three groups: phosphoproteins that are phosphorylated by (a) cAMP-dependent protein kinase or (b) cAMP-independent protein kinase; (c) phosphoproteins in which both cAMP-dependent and cAMP-independent protein kinase play a role in the phosphorylation. The relative phosphorylation rate of substrates for cAMP-dependent protein kinase is about 15-fold the phosphorylation rate of substrates for cAMP-independent protein kinase. The Km for ATP of cAMP-dependent protein kinase and phosphorylase kinase is 8 microM and 38 microM, respectively. Ca2+ in the micromolare range stimulates the phosphorylation of (a) phosphorylase, (b) a protein with molecular weight of 130 000 and (c) a protein with molecular weight of 15 000. The phosphate incorporation into a protein with molecular weight of 115 000 is inhibited by Ca2+. Phosphorylation of phosphorylase and the 15 000-Mr protein in the presence of 100 microM Ca2+ could be completely inhibited by trifluoperazine. It can be concluded that calmodulin is involved in the phosphorylation of at least two soluble proteins. No evidence for Ca2+-stimulated phosphorylation of subunits of glycolytic or gluconeogenic enzymes, including pyruvate kinase, was found. This indicates that it is unlikely that direct phosphorylation by Ca2+-dependent protein kinases is involved in the stimulation of gluconeogenesis by hormones that act through a cAMP-independent, Ca2+-dependent mechanism.     

Phosphorylation of proteins in collecting tube cells under the effect of vasopressin]

Endogenous phosphorylation of proteins in cell suspensions of collecting tubes was studied. Using SDS disc electrophoresis in polyacrylamide gel with subsequent autoradiography, it was shown that vasopressin increases the 32P incorporation into two proteins with molecular masses of 15 kDa and 33 kDa, which serve as endogenous substrates for cAMP-dependent protein kinase. The hormone-dependent phosphorylation of these proteins was typical of the membrane fraction of collecting tube cells but was absent in the cytosolic fraction. The results obtained are suggestive of the direct involvement of vasopressin in the regulation of membrane protein phosphorylation by cAMP-dependent protein kinase which may increase the permeability of cells for H2O.     

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.     

Changes in the nuclear protein kinase activities in the regenerating liver of partially irradiated rat

X rays (4.8 Gy) inhibit both DNA synthesis and phosphorylation of histone H1 in the regenerating liver of the rat. To determine the cause of the inhibition of histone H1 phosphorylation, changes in the nuclear protein kinase activities during the prereplicative phase of regeneration were measured. The cAMP-dependent protein kinase activity was low during regeneration, and the changes in the activity were not statistically significant. The cAMP-independent protein kinase activity increased at 15 h, decreased at 18 h, and increased again at 24 h after partial hepatectomy. X irradiation prior to partial hepatectomy did not inhibit the increase at 15 h, but it did inhibit the increase at 24 h. The activity was not inhibited by isoquinolinesulfonamide inhibitors such as H-7, and it was activated by a commercial preparation of an inhibitor protein of the cAMP-dependent kinase. It was also inhibited by quercetin. The possibility that the radiation-sensitive nuclear protein kinase is a nuclear cAMP-independent protein kinase specific for histone H1 is considered.     

A DNA-activated protein kinase from HeLa cell nuclei.

A DNA-activated protein kinase (DNA-PK) was purified from nuclei of HeLa cells. Activity was associated with a single high-molecular-mass (approximately-300,000 Da) polypeptide when analyzed by gel filtration, denaturing polyacrylamide gel electrophoresis, and Western immunoblotting using a monoclonal antibody that also inhibits enzyme activity. Nuclear localization was indicated by subcellular fractionation and confirmed by immunofluorescence on whole cells. Double-stranded DNA stimulated phosphorylation of the 300-kDa polypeptide in purified preparations as well as phosphorylation of the exogenous substrates alpha-casein, simian virus 40 large T antigen, and the human heat shock protein hsp90. Autophosphorylation led to inactivation of the enzyme. The phosphorylation of casein was stimulated over 30-fold by DNA and was specific for serine and threonine residues. Bovine serum albumin and histone H1 were poor substrates for DNA-PK, and no phosphorylation of immunoglobulin G or histones other than H1 was observed. Supercoiled or heat-denatured DNA and synthetic double-stranded RNA or RNA-DNA copolymers did not stimulate casein phosphorylation by DNA-PK. Interaction of the enzyme with DNA in the absence of exogenous substrates was demonstrated by thermal inactivation and gel mobility shifts. These characteristics identify DNA-PK as distinct from other protein kinases described in the literature and suggest that activation by DNA is an important feature of the enzyme's in vivo function.     

In vivo phosphorylation of histones H1 and H5 in calf thymus and chicken erythrocyte as studied by 31P nuclear magnetic resonance spectroscopy

The 31P NMR method was first applied to characterize in vivo phosphorylation of H1 and H5 in calf thymus and chicken erythrocytes as well as in vitro phosphorylation of H1 and H5 by cAMP-dependent protein kinase. The amino acid residues phosphorylated in vivo in the histones were exclusively serine residues, and the mole fraction of phosphoserine was estimated to be 0.34 and 0.27 per molecule of calf thymus H1 and chicken erythrocyte H5, respectively. Interestingly, chicken erythrocyte H1 was not phosphorylated in vivo. Three H1 subtypes from calf thymus H1 varied in the 31P NMR spectra, and the bisected fragments of calf thymus H1 and chicken erythrocyte H5 exhibited characteristic spectral patterns, indicating that there are considerable diversities of the degree of phosphorylation and phosphorylation sites in very-lysine-rich histones. Furthermore, it was found that the microenvironment of phosphoserine residues phosphorylated in vivo in calf thymus H1 and chicken erythrocyte H5 is quite distinct from that of phosphoserine residues phosphorylated in vitro by bovine heart cAMP-dependent protein kinase.     

Cross-linking of proteins in nuclei and DNA-depleted nuclei from Friend erythroleukemia cells.

Reversible cross-linking of proteins in nuclei and DNA-depleted nuclei from Friend erythroleukemia cells was used as a probe to determine whether the protein structure was preserved following treatment with DNAase I. Interactions between histones were analyzed through cross-linking with 2-iminothiolane or dimethyl 3,3'-dithiobispropionimidate. No alterations in the interactions between intranucleosomal histone proteins resulted from digestion of the nuclear DNA. There was, however, a diminished extent of cross-linking of histone H1 to itself and to the intranucleosomal histones in DNA-depleted nuclei. The interactions of a group of nonhistone proteins with histone H3 could be monitored by cross-linking through the formation of disulfide bonds caused by oxidation of nuclei with H2O2. These interactions were not markedly affected by treatment of the nuclei with DNAase I. However, differences were observed in the extent of cross-linking of some of these proteins when cross-linking in nuclei from undifferentiated cells was compared to that in nuclei from cells which had been induced to differentiate with dimethylsulfoxide.     

Interaction of subunits of cAMP-dependent protein kinase with structural elements of the cell nucleus

Using the method of protein transfer from polyacrylamide gel to nitrocellulose filters with subsequent incubation of filter-adsorbed protein with [32P]DNA, it was found that the catalytic subunit of cAMP-dependent protein kinase from porcine brain is capable of interacting with DNA to form a stable complex. This complex is resistant even to 2 M NaCl. The ability of the catalytic subunit to interact with DNA depends on the degree of enzyme nativity. The regulatory subunit of cAMP-dependent protein kinase does not bind to DNA both in the presence and absence of cAMP. The 125I-labeled regulatory subunit can interact with some chromatin proteins, in particular, with histone H1 and core histones. An essential role in this binding belongs to electrostatic and hydrophobic interactions.     

Phosphorylation of histones by tissue transglutaminase

Tissue transglutaminase 2 (TG2) has recently been shown to have intrinsic serine/threonine kinase activity. Since histones are known to be cross-linked by TG2, we investigated whether histones are also substrates for TG2 kinase activity. TG2 was able to phosphorylate H1, H2A, H2B, H3, and H4 histones in vitro. Using peptide substrates and phosphospecific antibodies we demonstrated that TG2 phosphorylated Ser(10) in H3 and that this phosphorylation was reduced by acetylation, whereas phosphorylation of Ser(10) by TG2 enhanced acetylation. Furthermore we demonstrated that exogenous TG2 phosphorylated H1 and H3 in nucleosome preparations. We examined the abundance of TG2 in DNA-associated proteins from MCF-7 cells treated with phorbol ester (TPA) and 17beta-estradiol (E2). TG2 abundance was significantly reduced in E2-treated cells and enhanced in TPA-treated cells. In summary we have demonstrated that TG2 is able to phosphorylate purified histone proteins, and H3 and H1 in chromatin preparations, and it is associated with chromatin in breast cancer cells. These studies suggest a novel role for TG2 in the regulation of chromatin structure and function.     

Phosphohistidine is found in basic nuclear proteins of Physarum polycephalum

Nuclear extracts of the true slime mold, Physarum polycephalum, show protein histidine kinase activity towards exogenous histones [(1985) J. Biol. Chem. 260, 16106-16113]. Physarum microplasmodia were labeled with [32P]phosphate in vivo and two basic proteins containing alkali-stable phosphate were detected. The labeled proteins comigrated with Physarum histones H1 (approximately) and H2A and phosphoamino acid analysis showed that each protein contained [32P]-phosphohistidine. The H2A-like protein was also labeled in isolated nuclei incubated with [35S]thio-ATP. We conclude that some Physarum nuclear proteins contain phosphohistidine.     

Protein phosphorylation in normal and neoplastic hepatocytes]

Partially purified preparations of protein kinase were isolated from the cytoplasm and nuclei of rat liver and hepatoma 27 and characterized in terms of their substrate specificity. The protein kinases from normal liver and hepatoma revealed some differences in phosphorylating protein substrates. Hepatoma protein kinases were found to phosphorylate arginine-rich histones (H3, H4); differences in phosphorylation of histone H1 were revealed. Hepatoma protein kinases phosphorylated the C-terminal fragment of histone H1, whereas normal liver protein kinases produced no such effect. It was assumed that the phosphorylation of histone H1 in rapidly dividing and resting cells is operated through different channels.