Characterization of maturation-activated histone H1 and ribosomal S6 kinases in sea star oocytes

DEAE-Sephacel chromatography of cytosolic extracts from sea star oocytes resolved at least two distinct peaks of maturation-activated protein kinase activity, each of which catalyzed the phosphorylation of histone H1, ribosomal protein S6, and Arg-Arg-Leu-Ser-Ser-Leu-Arg-Ala (RRLSSLRA), a synthetic peptide based on the sequence of a phosphorylation site in the latter protein. The first peak (elution conductivity approximately equal to 6 mmho) contained the major activated kinase with respect to the phosphorylation of histone H1, and the second peak (elution conductivity approximately equal to 10.5 mmho) contained the major activated kinase with respect to the phosphorylation of S6 and RRLSSLRA. These kinase activities were barely detectable in extracts from immature oocytes. The major stimulated histone H1 kinase exhibited an apparent Mr of approximately 90 000 on Sephacryl S-300 but eluted from TSK-400 with an apparent Mr of approximately 10 000. After DEAE-Sephacel fractionation, this kinase was shown to utilize both ATP (apparent Km approximately equal to 45 microM) and GTP (apparent Km approximately equal to 10 microM), although the Vmax was 8-fold higher with ATP than with GTP. The enzyme phosphorylated histone H1 with an apparent Km approximately equal to 50 micrograms/mL. Its properties resembled those of the growth-associated histone kinase. The major stimulated RRLSSLRA kinase had an apparent Mr of approximately 84 000 on Sephacryl S-300 and approximately 40 000 on TSK-400. After DEAE-Sephacel chromatography, this kinase selectively utilized ATP (apparent Km approximately equal to 25 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

In vivo phosphorylation and activation of ribosomal protein S6 kinases during Xenopus oocyte maturation

Previous studies have shown that increased ribosomal protein S6 kinase activity in unfertilized Xenopus eggs can be resolved by DEAE-Sephacel chromatography into two peaks, designated S6 kinase I and S6 kinase II. We show here that antibody against bacterially expressed S6 kinase II cross-reacts with S6 kinase I. Both S6 kinases undergo marked phosphorylation when they are activated during oocyte maturation, and both become deactivated and dephosphorylated upon activation of eggs. Immunoblotting of extracts of oocytes reveals that all S6 kinase molecules undergo a decrease and increase in electrophoretic mobility upon activation and deactivation, respectively. The increase in electrophoretic mobility can be produced in vitro by incubation of activated S6 kinase with purified phosphatases. Phosphoamino acid analysis of S6 kinase II labeled in vivo during maturation reveals both phosphoserine and phosphothreonine, and phosphopeptide maps suggest that several kinases may phosphorylate and activate S6 kinase II in vivo. These results demonstrate that, during oocyte maturation and early development, S6 kinase activation and deactivation are regulated by phosphorylation and dephosphorylation, suggesting a probable mechanism for S6 kinase regulation in other mitogenically stimulated cells.     

Activation of ribosomal S6 kinase (RSK) during porcine oocyte maturation

The normal kinetics of ribosomal S6 kinase (RSK) during the meiotic maturation of porcine oocytes were examined. The phosphorylation states of RSK and extracellular signal-regulated kinase (ERK), major mitogen-activated protein (MAP) kinases in maturating porcine oocytes, were detected by Western blotting analysis. The S6 protein kinase activity was assayed using a specific substrate peptide which contained the major phosphorylation sites of S6 kinase. Full phosphorylation of RSK was correlated with ERK phosphorylation and was observed before germinal vesicle breakdown. S6 kinase activity was low in both freshly isolated and 20 h cultured oocytes. S6 kinase activity was significantly elevated in matured oocytes to a level about 6 times higher than that in freshly isolated oocytes. Furthermore, full phosphorylation of RSK was inhibited when oocytes were treated with U0126, a specific MAP kinase kinase inhibitor, in dose-dependent manner, indicating that RSK is one of the substrates of MAP kinase. These results suggest that the activation of RSK is involved in the regulation of meiotic maturation of porcine oocytes.     

Distribution and expression of phosphorylated histone H3 during porcine oocyte maturation

Phosphorylation modification of core histones is correlated well with diverse chromatin-based cell activities. However, its distribution pattern and primary roles during mammalian oocyte meiosis are still in dispute. In this study, by performing immunofluorescence and Western blotting, spatial distribution and temporal expression of phosphorylated serine 10 or 28 on histone H3 during porcine oocyte meiotic maturation were examined and distinct subcellular distribution patterns between them were presented. Low expression of phosphorylated H3/ser10 was detected in germinal vesicle. Importantly, following gradual dephosphorylation from germinal vesicle (GV) to late germinal vesicle (L-GV) stage, a transient phosphorylation at the periphery of condensed chromatin was re-established at early germinal vesicle breakdown (E-GVBD) stage, and then the dramatically increased signals covered whole chromosomes from pre-metaphase I (Pre-MI) to metaphase II (MII). Similarly, hypophosphorylation of serine 28 on histone H3 was also monitored from GV to E-GVBD, indicating dephosphorylation of histone H3 maybe involved in the regulation of meiotic resumption. Moreover, the rim staining on the chromosomes and high levels of H3/ser28 phosphorylation were observed in Pre-MI, MI, and MII stage oocytes. Based on above results, such stage-dependent dynamics of phosphorylation of H3/ser 10 and 28 may play specific roles during mammalian oocyte maturation.     

Functions and regulation of the 70kDa ribosomal S6 kinases

The 70kDa ribosomal protein S6 kinases, S6K1 and S6K2 are two highly homologous serine/threonine kinases that are activated in response to growth factors, cytokines and nutrients. The S6 kinases have been linked to diverse cellular processes, including protein synthesis, mRNA processing, glucose homeostasis, cell growth and survival. Studies in model organisms have highlighted the roles that S6K activity plays in a number of pathologies, including obesity, diabetes, ageing and cancer. The importance of S6K function in human diseases has led to the development of S6K-specific inhibitors by a number of companies, offering the promise of improved tools with which to study these enzymes and potentially the effective targeting of deregulated S6K signalling in patients. Here we review the current literature on the role of S6Ks in the regulation of cell growth, survival and proliferation downstream of various signalling pathways and how their dysregulation contributes to the pathogenesis of human diseases.     

Tyrosyl phosphorylation and activation of the myelin basic protein kinase p44mpk during sea star oocyte maturation.

The most prominent tyrosyl-phosphorylated protein in maturing sea star oocytes was identified as the 44 kDa myelin basic protein (MBP) kinase p44mpk. Immunoblotting studies with anti-phosphotyrosine PY-20 antibody and phosphoamino acid analysis of in vivo [32P]phosphate-labelled p44mpk showed that the tyrosyl phosphorylation of the kinase correlated with a greater than 10-fold stimulation of its MBP phosphotransferase activity. The activation of p44mpk was reversed almost completely by purified preparations of the protein-tyrosyl phosphatases CD45 and 1B. Purified p44mpk has previously been shown to undergo autophosphorylation in vitro on seryl residues and this was associated with further enhancement of its MBP phosphorylating activity (Sanghera et al. (1991) J. Biol. Chem. 266, 6700-6707). p44mpk also underwent seryl phosphorylation during oocyte maturation, and the protein-seryl/threonyl phosphatase 2A reversed partially the maturation-associated stimulation of its MBP kinase activity. The properties of p44mpk resemble the murine 42 kDa mitogen-activated protein kinase (p42mapk). While p44mpk may feature the phosphorylatable tyrosyl residue that is critical for activation in p42mapk, it lacks the upstream threonyl phosphorylation site that is also required for p42mapk activity (Payne et al. (1991) EMBO J: 10, 885-892). These findings indicate partial differences in the regulatory mechanisms that govern the activities of these isozymes.     

Activation of myelin basic protein kinases during echinoderm oocyte maturation and egg fertilization

At least five activated protein kinases were detectable in soluble extracts from maturing as compared to immature sea star oocytes. These kinases could be distinguished on the basis of the time courses of their activation following exposure of the oocytes to 1-methyladenine, their substrate specificities, and their chromatographic properties on DEAE-Sephacel and Sephacryl S-200. A histone H1 kinase (HH1K) (Mr 110,000) underwent maximal activation near the time of 1-methyladenine-induced germinal vesicle breakdown (GVBD). When myelin basic protein (MBP) was used as a substrate, HH1K and two additional kinases (MBPK-I and MBPK-II) were detectable. MBPK-II (Mr 110,000) was fully activated at the time of GVBD, whereas peak activation of MBPK-I (Mr 45,000) occurred after this event. Two "ribosomal protein S6 kinases" (S6K-I and S6K-II) could be detected with a synthetic peptide (RRLSSLRA), which was patterned after a major phosphorylation site in S6. The two S6 kinases (Mr 110,000 for both) underwent activation post-GVBD. HH1K and S6K-I coeluted from DEAE-Sephacel at a conductivity of 5.5-6.0 mmho, whereas MBPK-I, MBPK-II, and S6K-II coeluted from this resin in a second peak at a conductivity = 10-11 mmho. The HH1K and MBPK-II activities both declined prior to the emission of the first polar body (i.e., meiotic cell division), but the MBPK-I, S6K-I, and S6K-II activities remained elevated during this time. The activities of these kinases were also examined during the early cell divisions in sea urchin embryos. Within 5 min after fertilization, the high level of MBPK-I activity in sea urchin eggs rapidly declined. However, along with the HH1K and MBPK-II activities, the MBPK-I activity was transiently increased prior to each cell division. No appreciable postfertilization changes in the S6K-I and S6K-II activities were apparent during the first three cycles of cell division.     

Vanadium salts stimulate mitogen-activated protein (MAP) kinases and ribosomal S6 kinases

Effect of several vanadium salts, sodium orthovanadate, vanadyl sulfate and sodium metavanadate on protein tyrosine phosphorylation and serine/threonine kinases in chinese hamster ovary (CHO) cells overexpressing a normal human insulin receptor was examined. All the compounds stimulated protein tyrosine phosphorylation of two major proteins with molecular masses of 42 kDa (p42) and 44 kDa (p44). The phosphorylation of p42 and p44 was associated with an activation of mitogen activated protein (MAP) kinase as well as increased protein tyrosine phosphorylation of p42^mapk and p44^mapk. Vanadinm salts also activated the 90 kDa ribosomal s6 kinase (p90^rsk) and 70 kDa ribosomal s6 kinase (p70^s6k). Among the three vanadium salts tested, vanadyl sulfate appeared to be slightly more potent than others in stimulating MAP kinases and p70^s6k activity. It is suggested that vanadium-induced activation of MAP kinases and ribosomal s6 kinases may be one of the mechanisms by which insulin like effects of this trace element are mediated.Abbreviations eIF-4 eukaryotic protein synthesis initiation factor-4 - GRB-2 growth factor receptor bound protein-2 - GSK-3 Glycogen Synthase Kinase-3 - IRS-1 insulin receptor substrate-1 - ISPK insulin stimulated protein kinase - MAPK mitogen activated protein kinase, also known as - ERK extracellular signal regulated kinase - MAPKK mitogen activated protein kinase kinase, also known as-MEK, MAPK or ERK kinase - PHAS-1 phosphorylated heat and acid stable protein regulated by insulin - PI3K phosphatidyl inositol 3-kinase - PP1-G protein phosphatase-glycogen bound form - PTK protein tyrosine kinase - PTPase protein tyrosine phosphatase - rsk ribosomal s6 kinases - shc src homology domain containing protein - SOS son of sevenless     

Changes in histone acetylation during oocyte meiotic maturation in the diabetic mouse

Although there is considerable evidence that diabetes can adversely affect meiosis in mammalian oocytes, acetylation status of oocytes in a diabetic environment remains unclear. The objective was to determine acetylation or deacetylation patterns (based on immunostaining) of H3K9, H3K14, H4K5, H4K8, H4K12, and H4K16 sites at various stages during meiosis in murine oocytes from control and diabetic mice. According to quantitative real time polymerase chain reaction (qPCR), mean ± SEM relative expression of Gcn5 (1.70 ± 0.14 at metaphase [M]I and 1.27 ± 0.01 at MII, respectively), Ep300 (1.74 ± 0.04 at MI and 1.80 ± 0.001 at MII), and Pcaf (2.01 ± 0.03 at MI and 1.41 ± 0.18 at MII) mRNA in oocytes from diabetic mice were higher than those from controls (P < 0.05), whereas there was no difference (P > 0.05) during the germinal vesicle (GV) stage between the two groups (1.23 ± 0.04 for Gcn5, 0.82 ± 0.06 for Ep300, and 0.80 ± 0.07 for Pcaf). Conversely, relative mRNA expression concentrations of Hdac1, Hdac2, Hdac3, Sirt1 and Sirt2 during the germinal vesicle stage were lower in oocytes of diabetic mice (0.24 ± 0.03 for Hdac1, 0.11 ± 0.001 for Hdac2, 0.31 ± 0.03 for Hdac3, 0.28 ± 0.02 for Sirt1, and 0.55 ± 0.02 for Sirt2; P < 0.05). Similarly, the expression concentrations of these genes at the MI stage were lower in oocytes from diabetic mice (0.79 ± 0.12 for Hdac1, 0.72 ± 0.001 for Hdac2, 0.02 ± 0.001 for Sirt1, and 0.84 ± 0.08 for Sirt2; P < 0.05). Their expression concentrations at the MII stage were also lower in oocytes from diabetic mice (0.46 ± 0.03 for Hdac1, 0.93 ± 0.01 for Hdac2, 0.56 ± 0.01 for Hdac3, 0.01 ± 0.002 for Sirt1, and 0.84 ± 0.04 for Sirt2; P < 0.05). At the MI stage, however, there was no difference in the expression of Hdac3 between the two groups of oocytes (0.96 ± 0.03; P > 0.05). Taken together, diabetes altered the intracellular histone modification system, which may have contributed to changes in histone acetylation, and may be involved in the compromised maturation rate of oocytes in diabetic humans.     

Chromatin condensation and histone H1 kinase activity during growth and maturation of rabbit oocytes

Fully grown rabbit oocytes, isolated from preovulatory follicles, exhibit highly condensed bivalents within an intact germinal vesicle while a very low level of histone H1 kinase activity could be detected in their extracts. Chromatin condensation started in growing oocytes isolated from antral follicles presenting a diameter of 0.5 mm. This event was accompanied by a transient rise in histone H1 kinase activity which culminated in large antral follicles measuring 0.75 to 1 mm in diameter. However, the extent of histone H1 kinase activity observed in these growing oocytes remained far less important than that recorded in extracts prepared from in vitro cultured metaphase I and metaphase II oocytes. Moreover, this activity was insufficient to induce germinal vesicle breakdown which will only occur with an increasing efficiency, following in vitro culture of medium, large, and fully grown antral follicles. © 1994 Wiley-Liss, Inc.     

At least two kinases phosphorylate the MPM-2 epitope during Xenopus oocyte maturation

MPM-2 antigens, a discrete set of phosphoproteins that contain similar phosphoepitopes (the MPM-2 epitope), are associated with various mitotically important structures. The central mitotic regulator cdc2 kinase has been proposed to induce M-phase by phosphorylating many proteins which might include the MPM-2 antigens. To clarify the relationship of cdc2 kinase and the MPM-2 antigens, we developed an in vitro assay that enabled us to specifically detect the kinases that phosphorylate the MPM-2 epitope (ME kinases) in crude cell extracts. Two different ME kinase activities were identified in unfertilized Xenopus eggs, neither of which was cdc2 kinase, but both appeared to be activated by the introduction of cdc2 kinase into oocytes or oocyte extract. The two ME kinases differed in molecular size, substrate specificity, peptide components, and MPM-2 reactivity. The larger one, ME kinase-H, phosphorylated several MPM-2 antigens, while the smaller one, ME kinase-L, phosphorylated mainly one. We purified ME kinase-L to near homogeneity by sequential chromatography and showed that it has the characteristics of the 42-kD microtubule-associated protein (MAP) kinase. Our results support the previous finding that MAP kinase is activated during Xenopus oocyte maturation and suggest that MAP kinase may contribute to oocyte maturation induction by phosphorylating one subtype of MPM-2 epitope.     

Cyclic activation of histone H1 kinase during sea urchin egg mitotic divisions

Fertilized sea urchin eggs undergo a series of rapid and synchronized mitotic divisions. Extracts were made at various times throughout the first three mitotic divisions and assayed for phosphorylating activity toward histone H1. Histone H1 kinase (HH1K) undergoes a transient activation (8- to 10-fold increase) 20 min before each cleavage. The amplitude of the HH1K peak strongly depends on the synchrony of the egg population. Concomitant cytological observations show that the time-course of HH1K correlates with the time-course of nuclear envelope breakdown and of metaphase. This correlation is observed at each cell division cycle. HH1K from each of the three first mitoses show identical time- and concentration-dependence curves as well as identical dose-inhibition curves with 6-dimethylaminopurine and quercetin, suggesting that the same (group of) kinase(s) is (are) activated before each cleavage. Ionophore A23187 does not trigger, but inhibits, HH1K activation; however, partial activation of the eggs with ammonia at pH 9.0 (but not at pH 8.0) triggers the transient HH1K activation. Appearance of the HH1K cycle requires protein synthesis since it is completely abolished in emetine-treated eggs. Although cytochalasin B blocks egg cleavage, it does not inhibit HH1K activation nor nuclear divisions. A prolonged HH1K activation cycle is observed in eggs arrested in metaphase with colchicine or nocodazole. Despite the existence of a cycle in cAMP concentration during mitosis, forskolin, an activator of adenylate cyclase, does not modify the time-course of HH1K activation and of cell division. The cycling HH1K is independent of calcium-calmodulin, calcium-phospholipids, or cyclic AMP. It clearly resembles the mammalian "growth-associated histone kinase." The relationship between the transient activation of HH1K and the intracellular mitotic factors driving the cell cycle is discussed.     

Characterization of fertilization-modulated myelin basic protein kinases from sea star: regulation of Mapk.

The myelin basic protein (MBP)-phosphorylating enzymes present during maturation and early embryogenesis of the sea star (Pisaster ochraceus) were investigated. The major maturation-activated MBP kinase (p45 Mapk) was molecularly cloned based on tryptic sequence information obtained with the purified enzyme and shown to be highly related to human Erk1 with 76% amino acid identity. Kinase assays and immunoblotting studies revealed that Mapk remained highly active until 12 h post-fertilization (PF), after which it declined. By 4 days PF, Mapk protein was no longer detectable. At 3 h PF, about half of the detectable MBP phosphotransferase activity could be attributed to a 75 kDa protein kinase that was distinct from Mapk. Like Mapk, this protein phosphorylated MBP mostly on threonine residues, but it failed to phosphorylate a peptide (APRTPGGRR) based upon the Thr-97 MAP kinase phosphorylation site in MBP. Rather, it phosphorylated a peptide (AAQKRPSQRTKYLA) patterned after the N-terminus of MBP. Our studies also showed a dramatic increase in MBP phosphotransferase activity occurred by 4 days PF that arose from a third kinase that phosphorylated MBP solely on serine residues. This kinase exhibited the following substrate substrate preference: AAQKRPSQRTKYLA, peptide substrate for S6 kinases (AKRRRLSSLRASTSKSESSQK) > MBP > histone H1 > prota-mine > casein > APRTPGGRR. This kinase was not appreciably affected by addition of phosphatidylserine/diacylglycerol, or the staurosporine analogue Roche Compound 3, but it was partly inhibited by a protein kinase C pseudosubstrate peptide. Gel filtration analysis revealed an apparent molecular mass of 41 kDa for the enzyme. Therefore, at least two novel MBP-phosphorylating enzymes distinct from Mapk are preferentially activated following fertilization and early embryogenesis of the sea star.     

Histone modifications during mammalian oocyte maturation: Dynamics,regulation and functions

Histone modifications are associated with many fundamental biological processes in cells. An emerging notion from recent studies is that meiosis stage-dependent histone modifications are crucial for the oocyte development in mammals. In this paper, we review the changes and regulation as well as functions of histone modifications during meiotic maturation of mammalian oocyte, with particular emphasis on histone acetylation, phosphorylation and methylation. In general, dynamic and differential modification patterns have been revealed during oocyte maturation, indicative of functional requirement. Disruption of histone modifications leads to defective chromosome condensation and segregation, delayed maturation progression and even oocyte aging. Although several histone-modifying enzymes have been identified in mammalian oocytes, more works are necessary to determine how they direct histone modifications globally and individually in oocytes. Studies on chromatin modification during oocyte development will have implications for our understanding of the mechanisms controlling nuclear architecture and genomic stability in female germ line.