Possible role of H1 histone in the differentiation of mouse erythroleukemia cells

Serum-starved mouse erythroleukemia cells, stationary phase cells or cells cultured in dibutyryl cAMP (1 mM) can be induced to differentiate by addition of 20% fetal calf serum plus cycloheximide. Culturing unstarved log phase cells in 20% fetal calf serum plus low levels of cycloheximide and histone H1 also causes a significant level of differentiation. These same concentrations of cycloheximide and H1 histone employed separately with 20% fetal calf serum do not induce differentiation. The role these procedures may have in causing an accumulation of histone H1 and cell differentiation is discussed.     

A cyclic AMP-dependent phosphorylation of spectrin dimer

In contrast to the properties of spectrin obtained from [³²P]phosphate-labeled red cells, purified spectrin dimer could be phosphorylated by a cAMP-dependent protein kinase from bovine heart. Both spectrin bands were phosphorylated. Spectrin band 2 contained in addition to autophosphorylated peptides several phosphopeptides that were distinct from autophosphorylated ones. The cAMP-dependent phosphorylation of spectrin band 1 was modulated by reducing agent and the concentration of spectrin. At high concentrations spectrin band 2 was predominantly labeled. The cAMP-dependent phosphoform of spectrin band 2 had a pI slightly higher than that of autophosphorylated spectrin band 2, but lower than that of ankyrin.     

Properties of the cyclic AMP-dependent protein kinases in mouse mastocytoma cells

The properties of type I and type II protein kinases in PY815 mouse mastocytoma cells were shown to change following growth inhibition by prostaglandin E1 and 3-isobutyl-1-methylxanthine. These changes included a large reduction in type I protein kinase consistent with a role for this isoenzyme as a positive effector of growth, a decrease in free cyclic AMP binding protein and an increase in type II protein kinase. Some properties of the fully activated isoenzymes are presented that may be important in determining their activity in vivo.     

Differential phosphorylation of histone H1 subfractions in vivo.

A study was made of the phosphorylation of chromatographically purified histone H1 subfractions from the liver of premetamorphic tadpoles ($\text{Rana}\text{catesbeiana}$). Two H1 subfractions were obtained which differed in terms of net incorporation of [³²P]phosphate $\text{in}\text{vivo}$. Analysis of N-bromosuccinimide cleavage products further revealed that the two subfractions also differed in the relative distribution of [³²P]phosphate in N- and C-terminal regions of the molecule. Incorporation of [³²P]phosphate into both regions of the molecule occurred virtually exclusively in serine residues.     

Mitotic histone H3 phosphorylation by vaccinia-related kinase 1 in mammalian cells

Mitotic chromatin condensation is essential for cell division in eukaryotes. Posttranslational modification of the N-terminal tail of histone proteins, particularly by phosphorylation by mitotic histone kinases, may facilitate this process. In mammals, aurora B is believed to be the mitotic histone H3 Ser10 kinase; however, it is not sufficient to phosphorylate H3 Ser10 with aurora B alone. We show that histone H3 is phosphorylated by vaccinia-related kinase 1 (VRK1). Direct phosphorylation of Thr3 and Ser10 in H3 by VRK1 both in vitro and in vivo was observed. Loss of VRK1 activity was associated with a marked decrease in H3 phosphorylation during mitosis. Phosphorylation of Ser10 by VRK1 is similar to that by aurora B. Moreover, expression and chromatin localization of VRK1 depended on the cell cycle phase. Overexpression of VRK1 resulted in a dramatic condensation of nuclei. Our findings collectively support a role of VRK1 as a novel mitotic histone H3 kinase in mammals.     

Regulation of maxi-K+ channels on pancreatic duct cells by cyclic AMP-dependent phosphorylation

Summary Using the patch-clamp technique we have identified a Ca²⁺-sensitive, voltage-dependent, maxi-K⁺ channel on the basolateral surface of rat pancreatic duct cells. The channel had a conductance of 200 pS in excised patches bathed in symmetrical 150mm K⁺, and was blocked by 1mm Ba²⁺. Channel openstate probability (P _o ) on unstimulated cells was very low, but was markedly increased by exposing the cells to secretin, dibutyryl cyclic AMP, forskolin or isobutylmethylxanthine. Stimulation also shifted theP _o /voltage relationship towards hyperpolarizing potentials, but channel conductance was unchanged. If patches were excised from stimulated cells into the inside-out configuration,P _o remained high, and was not markedly reduced by lowering bath (cytoplasmic) Ca²⁺ concentration from 2mm to 0.1 m. However, activated channels were still blocked by 1mm Ba²⁺. ChannelP _o was also increased by exposing the cytoplasmic face of excised patches to the purified catalytic subunit of cyclic AMP-dependent protein kinase., We conclude that cyclic AMP-dependent phosphorylation can activate maxi-K⁺ channels on pancreatic duct cells via a stable modification of the channel protein itself, or a closely associated regulatory subunit, and that phosphorylation alters the responsiveness of the channels to Ca²⁺. Physiologically, these K⁺ channels may contribute to the basolateral K⁺ conductance of the duct cell and, by providing a pathway for current flow across the basolateral membrane, play an important role in pancreatic bicarbonate secretion.     

Activation and phosphorylation of the 'dense-vesicle' high-affinity cyclic AMP phosphodiesterase by cyclic AMP-dependent protein kinase.

Incubation of a hepatocyte particulate fraction with ATP and the isolated catalytic unit of cyclic AMP-dependent protein kinase (A-kinase) selectively activated the high-affinity 'dense-vesicle' cycle AMP phosphodiesterase. Such activation only occurred if the membranes had been pre-treated with Mg2+. Mg2+ pre-treatment appeared to function by stimulating endogenous phosphatases and did not affect phosphodiesterase activity. Using the antiserum DV4, which specifically immunoprecipitated the 51 and 57 kDa components of the 'dense-vesicle' phosphodiesterase from a detergent-solubilized membrane extract, we isolated a 32P-labelled phosphoprotein from 32P-labelled hepatocytes. MgCl2 treatment of such labelled membranes removed 32P from the immunoprecipitated protein. Incubation of the Mg2+-pre-treated membranes with [32P]ATP and A-kinase led to the time-dependent incorporation of label into the 'dense-vesicle' phosphodiesterase, as detected by specific immunoprecipitation with the antiserum DV4. The time-dependences of phosphodiesterase activation and incorporation of label were similar. It is suggested (i) that phosphorylation of the 'dense-vesicle' phosphodiesterase by A-kinase leads to its activation, and that such a process accounts for the ability of glucagon and other hormones, which increase intracellular cyclic AMP concentrations, to activate this enzyme, and (ii) that an as yet unidentified kinase can phosphorylate this enzyme without causing any significant change in enzyme activity but which prevents activation and phosphorylation of the phosphodiesterase by A-kinase.     

Ultraviolet B-induced phosphorylation of histone H3 at serine 28 is mediated by MSK1.

N-terminal tail phosphorylation of histone H3 plays an important role in gene expression, chromatin remodeling, and chromosome condensation. Phosphorylation of histone H3 at serine 10 was shown to be mediated by RSK2, mitogen- and stress-activated protein kinase-1 (MSK1), and mitogen-activated protein kinases depending on the specific stimulation or stress. Our previous study showed that mitogen-activated protein kinases MAP kinases are involved in ultraviolet B-induced phosphorylation of histone H3 at serine 28 (Zhong, S., Zhong, Z., Jansen, J., Goto, H., Inagaki, M., and Dong, Z., J. Biol. Chem. 276, 12932-12937). However, downstream effectors of MAP kinases remain to be identified. Here, we report that H89, a selective inhibitor of the nucleosomal response, totally inhibits ultraviolet B-induced phosphorylation of histone H3 at serine 28. H89 blocks MSK1 activity but does not inhibit ultraviolet B-induced activation of MAP kinases p70/85(S6K), p90(RSK), Akt, and protein kinase A. Furthermore, MSK1 markedly phosphorylated serine 28 of histone H3 and chromatin in vitro. Transfection experiments showed that an N-terminal mutant MSK1 or a C-terminal mutant MSK1 markedly blocked MSK1 activity. Compared with wild-type MSK1, cells transfected with N-terminal or C-terminal mutant MSK1 strongly blocked ultraviolet B-induced phosphorylation of histone H3 at serine 28 in vivo. These data illustrate that MSK1 mediates ultraviolet B-induced phosphorylation of histone H3 at serine 28.     

Morphine inhibits cyclic AMP-dependent protein kinase and ornithine decarboxylase activities in neuroblastoma X glioma hybrid cells.

The effect of morphine on neuroblastoma x glioma hybrid cells is not limited to the inhibition of adenylate cyclase activity. It is demonstrated that in morphine-treated cells there is a marked reduction in the activity of both cyclic AMP-dependent protein kinase and ornithine decarboxylase (ODC) in response to stimulation by PGE₁. The effect of morphine is to block a cascade of events which may be crucial for the normal biochemical processes in these cells.     

NADP increases the level of histone H2AX phosphorylation in mouse heart cells after ionizing radiation

Phosphorylation of replaceable histone H2AX occurs in megabase chromatin domains around DNA double-strand breaks (DSBs), and this modification called gamma-H2AX can be used as an effective marker for DSBs repair and DNA damage response. Using Western blotting and immunohistochemistry techniques we have studied here the influence of exogenous nicotinamide adenine dinucleotide phosphate (NADP) which could potentially increase the intracellular level of NAD+ and on the level of gamma-H2AX formation in mouse heart cells after ionizing radiation (IR). We have found that injection of NAD+ in different doses immediately after IR causes an increased level of gamma-H2AX in mouse heart cells 20 min after IR at the dose of 3 Gy compared to control mice after IR exposure. It indicates that it could be a relationship between intracellular NAD+ content and DNA damage response in vivo.     

Plasma membrane cyclic AMP-dependent protein phosphorylation system in L6 myoblasts.

Plasma membranes can be isolated without disruption of cells by the plasma membrane vesiculation technique (Scott, R.E. (1976) Science 194, 743-745). A major advantage of this technique is that it avoids contamination of plasma membranes with intracellular membrane components. Using this method, we prepared plasma membranes from L6 myoblasts grown in tissue culture and studied the characteristics of the protein phosphorylation system. We found that these plasma membrane preparations contain protein kinase which is tightly bound to the membrane and cannot be removed by washing in EDTA or in high ionic strength salt solutions. This protein kinase activity can catalyze the phosphorylation of several exogenous substrates with decreasing efficiency as acceptors of phosphate: calf thymus histones f2b, protamine and caseine. Cyclic AMP causes a dose-dependent stimulation of protein kinase activity; the highest stimulation (4-fold) is achieved at concentration 10(-5) M cyclic AMP. Cyclic AMP-dependent stimulation can be completely inhibited by heat-stable protein kinase inhibitor isolated from rabbit skeletal muscle. On the other hand, cyclic GMP does not affect the activity of protein kinase. Plasma membrane-bound protein kinase also catalyzes the phosphorylation of endogenous membrane protein substrates and this is also stimulated by addition of cyclic AMP. Analysis of plasma membrane proteins by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis showed that specific polypeptides are phosphorylated by cyclic AMP-independent and by cyclic AMP-dependent protein kinase systems. The results of these studies demonstrate the presence of endogenous cyclic AMP-dependent and -independent protein phosphorylating systems (enzyme activity and substrates) in purified plasma membrane preparations. These data provide a basis for further investigations on the role of plasma membrane phosphorylation as a regulator of membrane functions including those that may control cellular differentiation.     

Chromosomal protein HMGN1 modulates histone H3 phosphorylation

Here we demonstrate that HMGN1, a nuclear protein that binds to nucleosomes and reduces the compaction of the chromatin fiber, modulates histone posttranslational modifications. In Hmgn1-/- cells, loss of HMGN1 elevates the steady-state levels of phospho-S10-H3 and enhances the rate of stress-induced phosphorylation of S10-H3. In vitro, HMGN1 reduces the rate of phospho-S10-H3 by hindering the ability of kinases to modify nucleosomal, but not free, H3. During anisomycin treatment, the phosphorylation of HMGN1 precedes that of H3 and leads to a transient weakening of the binding of HMGN1 to chromatin. We propose that the reduced binding of HMGN1 to nucleosomes, or the absence of the protein, improves access of anisomysin-induced kinases to H3. Thus, the levels of posttranslational modifications in chromatin are modulated by nucleosome binding proteins that alter the ability of enzymatic complexes to access and modify their nucleosomal targets.