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1.
Sea urchin protease specific to the SPKK motif in histone 总被引:1,自引:0,他引:1
2.
M Suzuki 《The EMBO journal》1989,8(3):797-804
A new DNA-binding unit of a protein different from the alpha-helix, the beta-sheet and the Zn-finger is proposed based on the analysis of the structure of the N-terminus of sea urchin spermatogenous histone H1. DNA-binding arms of the sea urchin spermatogenous histones, H1 and H2B, are composed of repeats of Ser-Pro-Lys(Arg)-Lys(Arg) (SPKK) residues. A six-times repeat of SPKK (S6 peptide) was isolated from H1 and the competition of S6 for DNA binding with a DNA-binding dye, Hoechst 33258, was analysed. The S6 peptide is shown to be a competitive inhibitor of Hoechst 33258, and it is concluded that the SPKK repeat binds to DNA in its minor groove with a binding constant, KS6 = 1.67 X 10(10) M-1. The circular dichroism (CD) spectrum of a synthetic peptide, SPRKSPRK (S2 peptide), is quite different from those of both the alpha-helix and the beta-sheet and resembles that of a random coil. From statistical consideration of protein structures it is proposed that SPKK forms a compact beta-turn stabilized by an additional hydrogen bond. Since a repeated chain of such turn of SPKK offers a repeat of amides of Ser residues at a distance similar to that of DNA-binding amides of the drugs, Hoechst 33258 and netropsin, and since the amides of these drugs bind to DNA replacing the spine of hydration in a minor groove, it is proposed that a repeat of SPKK binds to DNA in the minor groove using similar hydrogen bonds. 相似文献
3.
Protein synthesis in sea urchin eggs is stimulated dramatically upon fertilization. We previously demonstrated that this stimulation is primarily due to an increase in the rate of polypeptide chain initiation which in turn may be regulated at the level of recycling of eukaryotic initiation factor 2 (eIF-2) (Colin, A. M., Brown, B. D., Dholakia, J. N., Woodley, C. L., Wahba, A. J., and Hille, M. B. (1987) Dev. Biol. 123, 354-363). We have now purified eIF-2 from sea urchin Strongylocentrotus purpuratus blastulae to apparent homogeneity by chromatography on DEAE-cellulose, phosphocellulose, Mono Q, Mono P, and Mono S columns. The factor, which differs from mammalian eIF-2, is composed of three non-identical subunits with apparent molecular weights of 40,000-alpha; 47,000-beta, and 58,000-gamma as estimated by sodium dodecyl-polyacrylamide gel electrophoresis. Antibodies raised against rabbit reticulocyte eIF-2 do not cross-react with sea urchin eIF-2. The binding of Met-tRNA(f) to sea urchin eIF-2 is totally dependent on GTP. A 4-fold stimulation in the rate of protein synthesis in unfertilized sea urchin egg extracts is observed by the addition of 1 micrograms of purified eIF-2. The factor also binds GDP to form a binary (eIF-2.GDP) complex which is stable in the presence of Mg2+. GDP binding to sea urchin eIF-2 inhibits ternary (eIF-2-GTP.[35S]Met-tRNA(f) complex formation. The rabbit reticulocyte guanine nucleotide exchange factor (GEF) catalyzes the exchange of GDP bound to sea urchin eIF-2 for GTP and stimulates ternary complex formation. The requirement of GEF for the recycling of eIF-2 suggests that protein synthesis in sea urchins is similar to that in mammalian systems and may also be regulated at the level of GEF activity. The reticulocyte heme-controlled repressor phosphorylates the alpha-subunit of eIF-2 from both sea urchins and rabbit reticulocytes. However, casein kinase II which phosphorylates the beta-subunit of the reticulocyte factor specifically phosphorylates the alpha-subunit of sea urchin eIF-2. In this respect, the sea urchin factor is similar to eIF-2 isolated from other nonmammalian sources. Since both heme controlled repressor and casein kinase II phosphorylate the alpha-subunit of sea urchin eIF-2 caution should be exercised when interpreting the significance of eIF-2(alpha) phosphorylation in sea urchins. 相似文献
4.
Stephens S Beyer B Balthazar-Stablein U Duncan R Kostacos M Lukoma M Green GR Poccia D 《Molecular reproduction and development》2002,62(4):496-503
Decondensation of compact and inactive sperm chromatin by egg cytoplasm at fertilization is necessary to convert the male germ cell chromatin to an active somatic form. We studied decondensation of sea urchin sperm nuclei in a cell-free extract of sea urchin eggs to define conditions promoting decondensation. We find that egg cytosol specifically phosphorylates two sperm-specific (Sp) histones in vitro in the same regions as in vivo. This activity is blocked by olomoucine, an inhibitor of cdc2-like kinases, but not by chelerythrine, an inhibitor of protein kinase C (PKC). PKC phosphorylates and solubilizes the sperm nuclear lamina, one requirement for decondensation. Olomoucine, which does not inhibit lamina removal, blocks sperm nuclear decondensation in the same concentration range over which it is effective in blocking Sp histone phosphorylation. In a system free of other soluble proteins, neither PKC nor cdc2 alone elicit sperm chromatin decondensation, but the two act synergistically to decondense sperm nuclei. We conclude that two kinases activities are sufficient for sea urchin male pronuclear decondensation in vitro, a lamin kinase (PKC) and a cdc2-like Sp histone kinase. 相似文献
5.
Two species of histones in sea urchin sperm (Sp H1 and Sp H2B) are chimeric molecules whose highly basic amino-terminal domains are dephosphorylated at the last stage of sperm cell differentiation, and rephosphorylated immediately following fertilization. The phosphorylated regions consist largely of repeating tetrapeptides with two basic residues flanking Ser-Pro residues ('SPKK' motifs) and are predicted to have beta-turn secondary structures. Alteration of the charge and structure of the SPKK sites may play a role in the unusually dense DNA packaging of the mature sperm chromatin. The motif resembles the target site of cell-cycle-associated cdc2 kinases and is found in several other proteins whose nucleic acid affinities may be altered during the cell cycle. 相似文献
6.
Progression through mitosis requires the inactivation of the protein kinase activity of the p34cdc2-cyclin complex by a mechanism involving the degradation of cyclin. We have examined the stability in Xenopus egg extracts of radiolabeled Xenopus or sea urchin B-type cyclins synthesized in reticulocyte lysates. Xenopus cyclin B2 and sea urchin cyclin B were stable in metaphase extracts from unfertilized eggs but were specifically degraded following addition of Ca2+ to the extracts. The degradation of either cyclin was inhibited by the addition of an excess of unlabeled Xenopus cyclin B2 but not by the addition of a number of control proteins. A truncated protein containing only the amino terminus of Xenopus cyclin B2, including sequences known to be essential for cyclin degradation in other species, also inhibited cyclin degradation, even though the truncated protein was stable in extracts following Ca2+ addition. The addition of the truncated protein did not stimulate histone H1 kinase activity in extracts but prevented the loss of H1 kinase activity that normally follows Ca2+ addition to metaphase extracts. When the amino-terminal fragment was added to extracts capable of several cell cycles in vitro, progression through the first mitosis was inhibited and elevated histone H1 kinase activity was maintained. These results indicate that although the amino terminus of cyclin does not contain all of the information necessary for cyclin destruction, it is capable of interacting with components of the cyclin destruction pathway and thereby preventing the degradation of full-length cyclins. 相似文献
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8.
High-mobility-group proteins P1, I and Y as substrates of the M-phase-specific p34cdc2/cyclincdc13 kinase 总被引:4,自引:0,他引:4
L Meijer A C Ostvold S I Walass T Lund S G Laland 《European journal of biochemistry》1991,196(3):557-567
All dividing cells entering the M phase of the cell cycle undergo the transient activation of an M-phase-specific histone H1 kinase which was recently shown to be constituted of at least two subunits, p34cdc2 and cyclincdc13. The DNA-binding high-mobility-group (HMG) proteins 1, 2, 14, 17, I, Y and an HMG-like protein, P1, were investigated as potential substrates of H1 kinase. Among these HMG proteins, P1 and HMG I and Y are excellent substrates of the M-phase-specific kinase obtained from both meiotic starfish oocytes and mitotic sea urchin eggs. Anticyclin immunoprecipitates, extracts purified on specific p34cdc2-binding p13suc1-Sepharose and affinity-purified H1 kinase display strong HMG I, Y and P1 phosphorylating activities, demonstrating that the p34cdc2/cyclincdc13 complex is the active kinase phosphorylating these HMG proteins. HMG I and P1 phosphorylation is competitively inhibited by a peptide mimicking the consensus phosphorylation sequence of H1 kinase. HMG I, Y and P1 all possess the consensus sequence for phosphorylation by the p34cdc2/cyclincdc13 kinase (Ser/Thr-Pro-Xaa-Lys/Arg). HMG I is phosphorylated in vivo at M phase on the same sites phosphorylated in vitro by H1 kinase. P1 is phosphorylated by H1 kinase on sites different from the sites of phosphorylation by casein kinase II. The three thermolytic phosphopeptides of P1 phosphorylated in vitro by purified H1 kinase are all present in thermolytic peptide maps of P1 phosphorylated in vivo in proliferating HeLa cells. These phosphopeptides are absent in nonproliferating cells. These results demonstrate that the DNA-binding proteins HMG I, Y and P1 are natural substrates for the M-phase-specific protein kinase. The phosphorylation of these proteins by p34cdc2/cyclincdc13 may represent a crucial event in the intense chromatin condensation occurring as cells transit from the G2 to the M phase of the cell cycle. 相似文献
9.
CAMP-dependent protein kinase of sea urchin sperm phosphorylates sperm histone H1 on a single site 总被引:1,自引:0,他引:1
The phosphorylation of sperm specific histone H1 in the sea urchin Strongylocentrotus purpuratus occurs both in vivo and in vitro on a single serine site in the sequence Arg-Lys-Gly-Ser(P)-Ser-Asn-Ala-Arg. This is a preferred sequence for cAMP-dependent protein kinase. The in vitro phosphorylation is completely dependent on cAMP and is inhibited by the peptide protein kinase inhibitor. The protein kinase inhibitor H-8 blocks the in vivo phosphorylation of H1 without damaging motility, the acrosome reaction or the ability of sperm to fuse with and activate eggs. 相似文献
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11.
Tumor promoters and diacylglycerol activate the Na+/H+ antiporter of sea urchin eggs 总被引:4,自引:0,他引:4
Various tumor promoters (TPA, lyngbyatoxin and aplysiatoxin) and diacylglycerol induced cytoplasmic alkalinization of sea urchin eggs independently of intracellular Ca2+ release. This response stimulated protein synthesis and was blocked by amiloride or a lack of extracellular Na+, procedures which inhibit the Na+/H+ antiporter. These results suggest that the antiporter which is responsible for cytoplasmic alkalinization in sea urchin eggs is activated directly or indirectly by protein kinase C in a Ca2+-independent manner. 相似文献
12.
In a previous study, we demonstrated that caulerpenyne (Cyn), a natural sesquiterpene having an antiproliferative potency, blocked the mitotic cycle of sea urchin embryos at metaphase and inhibited the phosphorylation of several proteins, but did not affect histone H1 kinase activation (Pesando et al, 1998, Eur. J. Cell Biol. 77, 19-26). Here, we show that concentrations of Cyn that blocked the first division of the sea urchin Paracentrotus lividus embryos in a metaphase-like stage (45 microM) also inhibited the stimulation of mitogen-activated protein kinase (MAPK) activity in vivo as measured in treated egg extracts using myelin basic protein (MBP) as a substrate (MBPK). However, Cyn had no effect on MBP phosphorylation when added in vitro to an untreated egg extract taken at the time of metaphase, suggesting that Cyn acts on an upstream activation process. PD 98059 (40 microM), a previously characterized specific synthetic inhibitor of MAPK/extracellular signal-regulated kinase-1 (MEK1), also blocked sea urchin eggs at metaphase in a way very similar to Cyn. Both molecules induced similar inhibitory effects on MBP kinase activation in vivo, but had no direct effect on MBP kinase activity in vitro, whereas they did not affect H1 kinase activation neither in vivo nor in vitro. As a comparison, butyrolactone 1 (100 microM), a known inhibitor of H1 kinase activity, did inhibit H1 kinase of sea urchin eggs in vivo and in vitro, and blocked the sea urchin embryo mitotic cycle much before metaphase. Immunoblots of mitotic extracts, treated with anti-active MAP-kinase antibody, showed that both Cyn and PD 98059 reduced the phosphorylation of p42 MAP kinase (Erk2) in vivo. Our overall results suggest that Cyn blocks the sea urchin embryo mitotic cycle at metaphase by inhibiting an upstream phosphorylation event in the MBPK activation pathway. They also show that H1 kinase and MBPK activation can be dissociated from each other in this model system. 相似文献
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14.
Nucleosomal structure of sea urchin and starfish sperm chromatin. Histone H2B is possibly involved in determining the length of linker DNA. 总被引:6,自引:3,他引:3
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Comparison has been made between sea urchin and starfish sperm chromatin. The only protein by which chromatins from these sources differ significantly is histone H2B. Sea urchin sperm H2B is known to contain an elongated N-terminal region enriched in Arg. Analysis of the micrococcal nuclease digests of sea urchin and starfish nuclei in one- and two-dimensional electrophoresis has shown that sperm chromatin of both animals consists of repeated units similar in general features to those of rat thymus or liver. However, DNA repeat length in chromatin of sea urchin sperm (237 bp) is higher than that of starfish sperm (224 bp), while the core DNA length does not differ and is the same as in the chromatin of rat liver or thymus. A suggestion has been made that the N-terminal region of histone H2B is associated with the linker DNA and is responsible for the increased length of sea urchin linker DNA. 相似文献
15.
Microinjection of a bacterially expressed stable delta 90 sea urchin cyclin B into Xenopus prophase oocytes, in absence or presence of cycloheximide, provokes the activation of histone H1 kinase and the tyrosine dephosphorylation of p34cdc2. Unexpectedly, when prophase oocytes are submitted to a treatment known to elevate the intracellular cAMP level (3-isobutyl-1-methylxanthine and cholera toxin), delta 90 cyclin has no effect and the oocytes remain blocked in prophase. This inhibition is reverted by the microinjection of the inhibitor of cAMP-dependent protein kinase. When delta 90 cyclin is microinjected into oocytes depleted of endogenous cyclins (cycloheximide-treated metaphase I) and in the presence of a high intracellular concentration of cAMP, p34cdc2 kinase is tyrosine rephosphorylated. Altogether, our results indicate that in Xenopus oocyte, cAMP-dependent protein kinase (A-kinase) controls the formation of the cyclin B/p34cdc2 complex which remains inactive and tyrosine phosphorylated. 相似文献
16.
1,2-Diacylglycerols mimic phorbol 12-myristate 13-acetate activation of the sea urchin egg 总被引:7,自引:0,他引:7
Phorbol diesters have been reported to stimulate the Na+/H+ antiport of a variety of cells including sea urchin eggs. Since stimulation of the Na+/H+ antiport is necessary for metabolic derepression during fertilization and protein kinase C is a target of phorbol diesters, enhanced Na+/H+ exchange during fertilization may be a result of protein kinase C activity. Protein kinase C is probably physiologically activated by diacylglycerols, which are derived from hydrolysis of phosphatidylinositol. Treatment of sea urchin eggs with 1,2-diacylglycerols was found to stimulate the Na+/H+ antiport. The 1,3-isomers were without effect. Further, the effects of 1,2-diacylglycerol and phorbol diester are not additive with respect to Na+/H+ exchange. While a direct participation of protein kinase C activity during fertilization remains to be demonstrated, these data support the hypothesis that protein kinase C activity plays a role in fertilization. However, the cytotoxic effect of protein kinase C activators suggests effects associated with their pleiotropic nature. 相似文献
17.
Multifunctional Ca2+/calmodulin-dependent protein kinase is necessary for nuclear envelope breakdown 总被引:14,自引:7,他引:7
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《The Journal of cell biology》1990,111(5):1763-1773
The role of multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase) in nuclear envelope breakdown (NEB) was investigated in sea urchin eggs. The eggs contain a 56-kD polypeptide which appears to be a homologue of neuronal CaM kinase. For example, it undergoes Ca2+/calmodulin-dependent autophosphorylation that converts it to a Ca2(+)-independent species, a hallmark of multifunctional CaM kinase. It is homologous to the alpha subunit of rat brain CaM kinase. Autophosphorylation and substrate phosphorylation by the sea urchin egg kinase are inhibited in vitro by CaMK(273-302), a synthetic peptide corresponding to the autoinhibitory domain of the neuronal CaM kinase. This peptide inhibited NEB when microinjected into sea urchin eggs. Only one mAb to the neuronal enzyme immunoprecipitated the 56-kD polypeptide. Only this antibody blocked or significantly delayed NEB when microinjected into sea urchin eggs. These results suggest that sea urchin eggs contain multifunctional CaM kinase, and that this enzyme is involved in the control of NEB during mitotic division. 相似文献
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19.
Tosuji H Fusetani N Seki Y 《Comparative biochemistry and physiology. Toxicology & pharmacology : CBP》2003,135(4):415-424
Calyculin A is known to inhibit the type-1 and type-2A phosphatases. We previously reported that calyculin A induces contractile ring formation in unfertilized sea urchin eggs, an increase in histone H(1) kinase activity, and chromosome condensation in the calyculin A-treated unfertilized eggs, and the changes induced by calyculin A are not affected by emetine, an inhibitor of protein synthesis. These observations suggest that the mechanism by which histone H(1) kinases are activated by calyculin A is different from that of maturation-promoting factor (MPF), which is activated by a molecular modification of existed cdc2 and newly synthesized cyclin B. We report here that no cyclin B was detected by immunoblotting of unfertilized calyculin A-treated eggs. In addition, no DNA synthesis was induced by calyculin A. As well, butyrolactone I (an inhibitor of cdc2 and cdk2 kinase) had no effect on the increase in histone H(1) kinase activity nor the chromosome condensation, both of which were induced by calyculin A. Thus, we conclude that calyculin A induces histone H(1) phosphorylation in an MPF-independent manner through inhibition of type-1 phosphatase, and that the chromosome condenses as a result of histone H(1) phosphorylation. 相似文献
20.
Addition of [gamma -32P]ATP to a 2% Brij-78 40,000g supernatant of sea urchin sperm results in the cAMP-dependent phosphorylation of eight to ten proteins. One phosphoprotein of Mr 190 kD is sperm adenylate cyclase (AC). An antiserum to the AC immunoprecipitates the Mr 190 kD protein. Peptide maps of immunoprecipitates show that the AC is the only phosphoprotein present in the Mr 200 kD range. With respect to the in vitro phosphorylation of AC, the endogenous kinase has a Km for ATP of 5.2 microM and is maximally stimulated by 4-8 microM cAMP. The protein kinase inhibitors H8 (9 microM) and PKI (30 U/ml) inhibit the phosphorylation of the AC. The catalytic subunit of bovine cAMP-dependent protein kinase phosphorylates the AC on the same peptides as the endogenous protein kinase. Cyanogen bromide generated peptide maps of the phosphorylated AC show a minimum of five sites of phosphorylation. No change in the Km or Vmax of the sperm AC resulted from the additional phosphorylation by bovine kinase. Calcium ions at submicromolar concentrations completely block the in vitro phosphorylation of the AC, suggesting the presence in the preparation of a Ca2(+) -activated protein phosphatase. To our knowledge, this is the first report of the phosphorylation of an AC by cAMP-dependent protein kinase. 相似文献