Increased Ser-10 phosphorylation of histone H3 in mitogen-stimulated and oncogene-transformed mouse fibroblasts.

When the Ras mitogen-activated protein kinase (MAPK) signaling pathway of quiescent cells is stimulated with growth factors or phorbol esters, the early response genes c-fos and c-myc are rapidly induced, and concurrently there is a rapid phosphorylation of histone H3. Using an antibody specific for phosphorylated Ser-10 of H3, we show that Ser-10 of H3 is phosphorylated, and we provide direct evidence that phosphorylated H3 is associated with c-fos and c-myc genes in stimulated cells. H3 phosphorylation may contribute to proto-oncogene induction by modulating chromatin structure and releasing blocks in elongation. Previously we reported that persistent stimulation of the Ras-MAPK signaling pathway in oncogene-transformed cells resulted in increased amounts of phosphorylated histone H1. Here we show that phosphorylated H3 is elevated in the oncogene-transformed mouse fibroblasts. Further we show that induction of ras expression results in a rapid increase in H3 phosphorylation. H3 phosphatase, identified as PP1, activities in ras-transformed and parental fibroblast cells were similar, suggesting that elevated H3 kinase activity was responsible for the increased level of phosphorylated H3 in the oncogene-transformed cells. Elevated levels of phosphorylated H1 and H3 may be responsible for the less condensed chromatin structure and aberrant gene expression observed in the oncogene-transformed cells.     

Involvement of histone phosphorylation in thymocyte apoptosis by protein phosphatase inhibitors

Incubation of rat thymocytes with the inhibitors of protein phosphatase such as calyculin A and okadaic acid resulted in an increase in DNA fragmentation. These effects were dependent on the concentration of the inhibitors and the incubation time. Analyses of the fragmented DNA revealed the production of approximately 50 kbp of DNA and a 180 bp DNA ladder. In addition, a laser scanning-microscopic analysis showed that these compounds caused nuclear condensation. Thus, these results demonstrated that protein phosphatase inhibitors induced thymocyte apoptosis. The inhibitors of protein phosphatase increased the phosphorylation of proteins of approximately 15 kDa. The phosphorylation of proteins preceded the DNA fragmentation induced by these inhibitors. Judging from acetic acid-urea-Triton X-100 gel electrophoresis, the phosphorylated proteins were histone H1 and H2A/H3. Therefore, these results suggest that phosphorylation of histones triggers the DNA fragmentation of thymocytes undergoing apoptosis.     

Hyperphosphorylation of histone H2A.X and dephosphorylation of histone H1 subtypes in the course of apoptosis.

Chromatin condensation paralleled by DNA fragmentation is one of the most important nuclear events occurring during apoptosis. Histone modifications, and in particular phosphorylation, have been suggested to affect chromatin function and structure during both cell cycle and cell death. We report here that phosphate incorporation into all H1 subtypes decreased rapidly after induction of apoptosis, evidently causing a strong reduction in phosphorylated forms of main H1 histone subtypes. H1 dephosphorylation is accompanied by chromatin condensation preceding the onset of typical chromatin oligonucleosomal fragmentation, whereas H2A.X hyperphosphorylation is strongly correlated to apoptotic chromatin fragmentation. Using various kinase inhibitors we were able to exclude some of the possible kinases which can be involved directly or indirectly in phosphorylation of histone H2A.X. Neither DNA-dependent protein kinase, protein kinase A, protein kinase G, nor the kinases driven by the mitogen-activated protein kinase (MAP) pathway appear to be responsible for H2A.X phosphorylation. The protein kinase C activator phorbol 12-myristate 13-acetate (PMA), however, markedly reduced the induction of apoptosis in TNFalpha-treated cells with a simultaneous change in the phosphorylation pattern of histone H2A.X. Hyperphosphorylation of H2A.X in apoptotic cells depends indirectly on activation of caspases and nuclear scaffold proteases as shown in zVAD-(OMe)-fmk- or zAPF-cmk-treated cells, whereas the dephosphorylation of H1 subtypes seems to be influenced solely by caspase inhibitors. Together, these results illustrate that H1 dephosphorylation and H2A.X hyperphosphorylation are necessary steps on the apoptotic pathway.     

Chromosome condensation induced by fostriecin does not require p34cdc2 kinase activity and histone H1 hyperphosphorylation, but is associated with enhanced histone H2A and H3 phosphorylation.

Chromosome condensation at mitosis correlates with the activation of p34cdc2 kinase, the hyperphosphorylation of histone H1 and the phosphorylation of histone H3. Chromosome condensation can also be induced by treating interphase cells with the protein phosphatase 1 and 2A inhibitors okadaic acid and fostriecin. Mouse mammary tumour FT210 cells grow normally at 32 degrees C, but at 39 degrees C they lose p34cdc2 kinase activity and arrest in G2 because of a temperature-sensitive lesion in the cdc2 gene. The treatment of these G2-arrested FT210 cells with fostriecin or okadaic acid resulted in full chromosome condensation in the absence of p34cdc2 kinase activity or histone H1 hyperphosphorylation. However, phosphorylation of histones H2A and H3 was strongly stimulated, partly through inhibition of histone H2A and H3 phosphatases, and cyclins A and B were degraded. The cells were unable to complete mitosis and divide. In the presence of the protein kinase inhibitor starosporine, the addition of fostriecin did not induce histone phosphorylation and chromosome condensation. The results show that chromosome condensation can take place without either the histone H1 hyperphosphorylation or the p34cdc2 kinase activity normally associated with mitosis, although it requires a staurosporine-sensitive protein kinase activity. The results further suggest that protein phosphatases 1 and 2A may be important in regulating chromosome condensation by restricting the level of histone phosphorylation during interphase, thereby preventing premature chromosome condensation.     

Increases in intracellular calcium dephosphorylate histone H3 at serine 10 in human hepatoma cells: potential role of protein phosphatase 2A-protein kinase CbetaII complex

We present evidence that increases in intracellular calcium, induced by treatment with calcium ionophore A23187 or the endoplasmic reticulum calcium-ATPase inhibitor thapsigargin, dephosphorylated histone H3 at serine10 (histone H3-Ser10) in a dose-dependent manner in human hepatoma HepG2 cells. Inhibition of p42/44MAPK, pp90RSK, or p38MAPK did not affect the ability of A23187 to dephosphorylate histone H3-Ser10. This response is significantly blocked by okadaic acid, indicating a requirement for protein phosphatase 2A (PP2A). A23187 increased the activity of PP2A towards phosphorylated histone H3-Ser10. Furthermore, pretreatment with calphostin C, a selective protein kinase C (PKC) inhibitor, blocked A23187-dependent dephosphorylation of histone H3-Ser10, and coimmunoprecipitation analysis showed PP2A association with the PKCbetaII isoform. Unlike untreated cells, coimmunoprecipitated complex from A23187-treated cells showed greater dephosphorylation of histone H3-Ser10 in a PP2A-dependent manner. Inhibition of PP2A increased phosphorylation at Ser660 that determines calcium sensitivity and activity of PKCbetaII isoform, thus supporting a role for intracomplex regulation. Finally, chromatin immunoprecipitation assays following exposure to A23187 and okadaic acid revealed regulatory role of histone H3-Ser10 phosphorylation in selective gene induction. Altogether, our findings suggest a novel role for calcium in modulating histone H3-Ser10 phosphorylation level and led us to propose a model emphasizing PP2A activation, occurring downstream following perturbations in calcium homeostasis, as key event in dephosphorylating histone H3-Ser10 in mammalian cells.     

Sphingosine reverses growth inhibition caused by activation of protein kinase C in vascular smooth muscle cells.

In certain cell systems, including neonatal vascular smooth muscle (VSM) cells, phorbol esters are growth inhibitory. Here we show that 1,2-dioctanoyl-sn-glycerol (DiC8), when added 2 h after alpha-thrombin, reverses by greater than 95% the induction of DNA synthesis in VSM cells by alpha-thrombin. Sphingosine, a naturally occurring lysosphingolipid inhibitor of protein kinase C, and its synthetic analogues N-acetylsphingosine and C11-sphingosine were used to investigate this phenomenon further. Neither phorbol 12-myristate 13-acetate (PMA;200 ng/ml) nor sphingosine (up to 10 microM) alone had any effect upon basal DNA synthesis in VSM cells. Like DiC8, PMA totally blocked the induction of DNA synthesis by alpha-thrombin. This inhibitory effect of PMA was reversed by sphingosine in a dose-dependent manner with complete reversal at 10 microM. Neither N-acetylsphingosine nor C11-sphingosine exhibited any effect on DNA synthesis in VSM cells. The effect of sphingosine and its analogues on the activity of protein kinase C extracted from VSM cells was measured by histone III-S phosphorylation. Protein kinase C activity was inhibited 50% by 300 microM sphingosine, but less than 15% by similar concentrations of N-acetylsphingosine and C11-sphingosine. To assess the effects of sphingosine and analogues on protein kinase C in intact cells, we examined the effect of the lipids on [3H]phorbol dibutyrate binding. Sphingosine (at greater than 5 microM), but not N-acetylsphingosine or C11-sphingosine, blocked [3H]phorbol dibutyrate binding in a dose- and time-dependent fashion. Thus the mechanism of growth inhibition by DiC8 and PMA in neonatal VSM cells appears to be through activation of protein kinase C by these compounds. Sphingosine reverses this growth inhibition through interference with the binding to protein kinase C of phorbol esters or other activators of this enzyme.     

Alpha-thrombin, epidermal growth factor, and okadaic acid activate the Na+/H+ exchanger, NHE-1, by phosphorylating a set of common sites.

The ubiquitous and amiloride-sensitive Na+/H+ exchanger (NHE-1), a plasma membrane phosphoglycoprotein that regulates intracellular pH, is rapidly activated by growth factors. We showed previously that epidermal growth factor (EGF), alpha-thrombin, or serum stimulates Na+/H+ exchange activity in growth-arrested Chinese hamster lung fibroblasts (ER22 cells) in a time-dependent manner which correlates with increased phosphorylation of NHE-1 at serine residues (Sardet, C., Counillon, L., Franchi, A., and Pouysségur, J. (1990) Science 247, 723-726). Here we show that the tumor promoter, okadaic acid, a potent in vivo inhibitor of serine/threonine protein phosphatases 1 (PP1) and 2A (PP2A), stimulates Na+/H+ exchange in G0-arrested ER22 cells and in exchanger-deficient fibroblasts transfected with the human NHE-1 cDNA. Okadaic acid effects are maximal at 1 microM (EC50 = 500 nM), detected in 2 min, complete within 15-20 min, and are additives when combined with EGF or alpha-thrombin. Parallel to the pHi-induced rise, okadaic acid alone or together with growth factors stimulated the phosphorylation of NHE-1. More importantly tryptic phosphopeptide maps of NHE-1, immunoprecipitated from cells treated with EGF, alpha-thrombin, or okadaic acid, show a common pattern of phosphorylation. This pattern consists of five major 32P-labeled peptides (P1-P5) present in lower amounts in resting cells. One of them, P5, barely detectable in resting cells is increased up to 15-fold in mitogen-stimulated cells. Taken together these results reinforce the notion that phosphorylation of NHE-1 controls the set point value of the exchanger and suggest that: (i) the proximate step in Na+/H+ exchange activation is mediated by as yet unidentified growth factor-activatable serine "NHE-1 kinase(s)" and (ii) this NHE-1 kinase(s), partly active in resting cells, integrate signals from receptor tyrosine kinases and G protein-coupled receptors.     

Studies of the differentiation of promyelocytic cells by phorbol ester. II. A methylation inhibitor, 3-deazaadenosine, inhibits the induction of specific differentiation proteins. Lack of effect on early and late phosphorylation events

Cells of the promyelocytic leukemic line, HL-60, differentiate into macrophage-like cells in response to phorbol-12-myristate-13-acetate (phorbol ester). We have shown that this process is associated with expression of motility functions and induction of specific changes in the synthesis of discrete proteins. In the present study we show that a methylation inhibitor, 3-deazaadenosine, inhibits the phorbol ester induction of the differentiation functions, adherence and motility. These effects were associated with interference in the expression of membrane proteins indicative of the acquisition of macrophage-like characteristics by HL-60 cells. Of particular importance was the observation that the methylation inhibitor preferentially arrested the induction of one differentiation, protein m10 (pI approximately equal to 7, 28 kDa). The selective effect of 3-deazaadenosine is further supported by observations in this study which show that the methylation inhibitor did not affect other cellular and biochemical effects of phorbol ester in these cells. Most significantly, 3-deazaadenosine did not inhibit the induction of phosphorylation in pp17 and pp27, which was previously shown to be a rapid event associated with initiation of differentiation in HL-60 cells by phorbol ester (Feuerstein, N. and Cooper, H. (1983) J. Biol. Chem. 258, 10786-10793). Furthermore, later elevated phosphorylation of other proteins as well as the arrest of cell growth induced by phorbol ester were also unimpeded by the methylation inhibitor. These results indicate that the methylation inhibitor did not interfere with the transduction of the phorbol ester signal at an early step so as to block all the subsequent events in this sequence, but rather its effect was selectively associated with interference in later events in the sequence of differentiation. It is proposed that transmethylation events might be specifically involved in the induction and expression of discrete proteins during the differentiation of these leukemic cells but not in the initial propagation of the signal produced by phorbol ester binding.     

Phosphorylation of nonhistone proteins during the HeLa cell cycle. Relationship to DNA synthesis and mitotic chromosome condensation

Cell cycle variations in the phosphorylation of chromatin-associated nonhistones were determined. Cells were radiolabeled with [32P]orthophosphate and chromatin was obtained by mild digestion of nuclei with micrococcal nuclease. The experiments were performed in the presence of a substrate inhibitor of alkaline phosphatase, beta-glycerophosphate. The results show that, while similar molecular weight species of phosphorylated nonhistones are associated with interphase chromatin through the HeLa cell cycle, the incorporation (32P cpm/micrograms of protein) profiles of selected major phosphononhistones show substantial changes. The most prominent peaks of specific radioactivity occur in the DNA synthesis phase (S phase). The phosphorylation states of the proteins of isolated metaphase chromosomes were also determined. Nonhistone proteins of isolated metaphase chromosomes are strikingly dephosphorylated, especially in comparison to histone H1. The phosphorylation of the major phosphononhistone of chromatin, which has a molecular weight of 55,000, was further characterized by techniques that included one-dimensional peptide mapping in sodium dodecyl sulfate-polyacrylamide gels and nonequilibrium pH gradient slab gel electrophoresis. Phosphoproteins are also components of the nuclear scaffold, and cell cycle variations in these proteins were investigated. The primary phosphorylated species has a molecular weight of 119,000. As with chromatin-associated nonhistones, this nuclear scaffold protein shows substantial incorporation of 32P in S phase, and a high level of incorporation also occurs close to mitosis.     

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.     

The protein phosphatase inhibitor, okadaic acid, potentiates the stimulatory effect of phorbol ester on phosphatidylcholine synthesis, but not on phospholipid hydrolysis, in fibroblasts.

The potent protein phosphatase inhibitor, okadaic acid, was used to determine the possible role of protein phosphorylation reaction(s) in phorbol ester-induced synthesis and hydrolysis of phosphatidylcholine (PtdCho) in NIH 3T3 fibroblasts. Okadaic acid (2 microM) was found to enhance the stimulatory effects of lower concentrations (2.5-25 nM) of phorbol 12-myristate 13-acetate (PMA) on PtdCho synthesis, but not on PtdCho hydrolysis, after treatments for 30-60 min. These data support a view that in fibroblasts PMA stimulates only PtdCho synthesis, and not PtdCho hydrolysis, by a protein phosphorylation-dependent mechanism.     

Stimulatory effect of okadaic acid, an inhibitor of protein phosphatases, on nuclear envelope breakdown and protein phosphorylation in mouse oocytes and one-cell embryos.

Treatment of one-cell mouse embryos with okadaic acid (OA), which is an inhibitor of protein phosphatases 1 and 2A, induces a concentration-dependent precocious nuclear envelope breakdown (NEBD) of the pronuclei; at 10 microM okadaic acid, NEBD starts to occur after 1 hr and the embryos become committed to NEBD after about 45 min. Correlated with NEBD is the conversion of a protein of Mr 32,000 (p32) to more highly phosphorylated forms. One-cell embryos cultured continuously in OA-containing medium do not cleave, whereas one-cell embryos incubated for 15-60 min prior to transfer to OA-free medium reveal a time-dependent inhibition in their ability to cleave. OA treatment of oocytes that are arrested from resuming spontaneous maturation by either a phosphodiesterase inhibitor or biologically active phorbol diester results in germinal vesicle breakdown and the maturation-associated changes in the pattern of protein phosphorylation, which include the apparent phosphorylation of p32. Results of these experiments implicate protein phosphatases in the G2 to M transition of the cell cycle in both meiotic and mitotic cells.