Sea urchin embryo as a model for analysis of the signaling pathways linking DNA damage checkpoint, DNA repair and apoptosis.

DNA integrity checkpoint control was studied in the sea urchin early embryo. Treatment of the embryos with genotoxic agents such as methyl methanesulfonate (MMS) or bleomycin induced the activation of a cell cycle checkpoint as evidenced by the occurrence of a delay or an arrest in the division of the embryos and an inhibition of CDK1/cyclin B activating dephosphorylation. The genotoxic treatment was shown to induce DNA damage that depended on the genotoxic concentration and was correlated with the observed cell cycle delay. At low genotoxic concentrations, embryos were able to repair the DNA damage and recover from checkpoint arrest, whereas at high doses they underwent morphological and biochemical changes characteristic of apoptosis. Finally, extracts prepared from embryos were found to be capable of supporting DNA repair in vitro upon incubation with oligonucleotides mimicking damage. Taken together, our results demonstrate that sea urchin early embryos contain fully functional and activatable DNA damage checkpoints. Sea urchin embryos are discussed as a promising model to study the signaling pathways of cell cycle checkpoint, DNA repair and apoptosis, which upon deregulation play a significant role in the origin of cancer.

Sea urchin elongation factor 1delta (EF1delta) and evidence for cell cycle-directed localization changes of a sub-fraction of the protein at M phase.

Eukaryotic elongation factor 1 (eEF1) is a translational multimolecular complex reported in higher eukaryotes to be a target of CDK1/cyclin B, the universal regulator of M phase, but whose role in the cell cycle remains to be determined. A specific polyclonal antibody was produced and used to characterize the delta subunit of sea urchin elongation factor 1 (SgEF1delta) in early embryos, a powerful model for investigating cell cycle regulation. The SgEF1delta protein was present in unfertilized eggs as two isoforms of 35 and 37 kDa, issued from two different mRNAs. The two canonical eEF1delta partners, eEF1gamma and eEF1beta, were shown to co-immunoprecipitate with the SgEF1delta isoforms. Both isoforms were associated in a macromolecular complex, which resolved upon gel filtration chromatography at a molecular weight > 400 kDa, suggesting association with other yet unidentified partners. After fertilization, the amount as well as the ratio of both SgEF1delta isoforms remained constant during the first cell division as judged by Western blotting. Immunofluorescence analysis showed that a pool of the protein concentrated as a ring at the embryo nuclear location around the period of nuclear envelope breakdown and was visualized later as two large spheres around the mitotic spindle poles. Thus, the eEF1delta protein shows cell cycle-specific localization changes in sea urchin embryos.

Protein translation during early cell divisions of sea urchin embryos regulated at the level of polypeptide chain elongation and highly sensitive to natural polyamines.

Protein synthesis was analysed following fertilisation in sea urchin. Fluctuations in the accumulation of neo-synthesised proteins were observed during the first cell cycles. Accurate translation analyses were performed from lysates prepared from early embryos. The lysates readily translated endogenous pre-initiated mRNAs allowing the determination of elongation rates in the absence of re-initiation in vitro. The translation capacity of embryo lysates increased 18-fold from 0 to 90 min after fertilisation, reflecting the increase in the amount of pre-initiated mRNAs during early development. Kinetics analysis at a short time interval during the course of early development (240 min) showed an overall increase in the elongation rate (> 10-fold) which is regulated by pauses in synchrony with the cell divisions. Elongation activity in the lysates was highly sensitive to the natural polyamines, spermine (ID50 = 0.2 mM) and spermidine (ID50 = 1.8 mM), indicating high potential regulation by the intracellular level of polyamines in embryos. The regulation in the elongation changes associated with the early embryo cell divisions is discussed in the light of the physiological fluctuations in polyamine concentrations.

eIF4E association with 4E-BP decreases rapidly following fertilization in sea urchin.

The eukaryotic translation initiation factor (eIF) 4F facilitates the recruitment of ribosomes to the mRNA 5' end. The 4E-BPs are small proteins with hypophosphorylated forms that interact with the cap binding protein eIF4E, preventing its interaction with eIF4G, thereby preventing ribosome interaction with mRNA. In sea urchin, fertilization triggers a rapid rise in protein synthesis. Here, we demonstrate that a 4E-BP homologue exists and is associated with eIF4E in unfertilized eggs. We also show that 4E-BP/eIF4E association diminishes a few minutes following fertilization. This decrease is correlated with a decrease in the total amount of 4E-BP in combination with an increase in the phosphorylation of the protein. We propose that 4E-BP acts as a repressor of protein synthesis in unfertilized sea urchin eggs and that 4E-BP/eIF4E dissociation plays an important role in the rise in protein synthesis that occurs shortly following fertilization.

Evidence for regulation of protein synthesis at the elongation step by CDK1/cyclin B phosphorylation.

Eukaryotic elongation factor 1 (eEF-1) contains the guanine nucleotide exchange factor eEF-1B that loads the G protein eEF-1A with GTP after each cycle of elongation during protein synthesis. Two features of eEF-1B have not yet been elucidated: (i) the presence of the unique valyl-tRNA synthetase; (ii) the significance of target sites for the cell cycle protein kinase CDK1/cyclin B. The roles of these two features were addressed by elongation measurements in vitro using cell-free extracts. A poly(GUA) template RNA was generated to support both poly(valine) and poly(serine) synthesis and poly(phenylalanine) synthesis was driven by a poly(uridylic acid) template. Elongation rates were in the order phenylalanine > valine > serine. Addition of CDK1/cyclin B decreased the elongation rate for valine whereas the rate for serine and phenylalanine elongation was increased. This effect was correlated with phosphorylation of the eEF-1delta and eEF-1gamma subunits of eEF-1B. Our results demonstrate specific regulation of elongation by CDK1/cyclin B phosphorylation.

Transient increase of a protein kinase activity identified to CK2 during sea urchin development.

Using GST-EF-1 delta as an exogenous substrate, and EF-1 delta kinase activity was shown to increase transiently during early development of sea urchin embryos. The basal activity of EF-1 delta kinase in unfertilized eggs was 150 fmoles/min/mg protein. The activity began to increase 10 h after fertilization and reached its maximum level (8.4 x basal) at 24 h. The activity then declined to twice the basal value at 72 h post-fertilization. The EF-1 delta kinase activity was identified to a CK2-type enzyme on the basis of its substrate specificity for EF-1 delta, crude casein and beta casein, its inhibition by heparin, DRB, 2,3-bisphosphoglycerate, and its stimulation by spermine, spermidine, and polylysin. Furthermore, the activity was inhibited by the synthetic peptide RRREEETEEE specific for CK2. DRB (200 microM) and 2,3-bisphosphoglycerate (2.5 mM) blocked or delayed the transition from blastula to gastrula of the embryos, suggesting a role for the kinase in early development.

Multiple phosphorylation sites and quaternary organization of guanine-nucleotide exchange complex of elongation factor-1 (EF-1betagammadelta/ValRS) control the various functions of EF-1alpha.

The eukaryotic guanine-nucleotide exchange factor commonly called elongation factor-1 betagammadelta (EF-1betagammadelta), comprises four different subunits including valyl-tRNA synthetase (EF-1betagammadelta/ValRS). The factor is multiply-phosphorylated by three different protein kinases, protein kinase C, casein kinase II and cyclin dependent kinase 1 (CDKI). EF-1betagammadelta/ValRS is organized as a macromolecular complex for which we propose a new structural model. Evidence that EF-1betagammadelta/ValRS is a sophisticated supramolecular complex containing many phosphorylation sites, makes it a potential regulator of any of the functions of its partner EF-1alpha, not only involved in protein synthesis elongation, but also in many other cellular functions.

Developmental regulation of elongation factor-1 delta in sea urchin suggests appearance of a mechanism for alternative poly(A) site selection in gastrulae.

Elongation factor-1 delta gene expression was analyzed during sea urchin development. EF-1 delta mRNA is present as a single 2.7-kb transcript in unfertilized eggs and in rapidly dividing cleavage stage embryos. It decreases rapidly 6 h after fertilization and then reappears at the gastrula stage as two transcripts of 2.7 and 2.0 kb. cDNA clones encoding the 2.7- and 2.0-kb transcripts were isolated from a sea urchin embryos library. The two cDNAs originate from alternative poly(A) site selection from a unique precursor. Both cDNAs are terminated by a poly(A) tail and were shown to encode for the same protein identified as EF-1 delta. Thus, EF-1 delta gene expression undergoes developmental regulation in early embryos leading to the presence of two poly(A) forms of the transcript. Since the 2.0-kb polyadenylated form of the EF-1 delta transcript appears at gastrula stage, our results suggest that a mechanism for alternative poly(A) site selection of the EF-1 delta transcript appears during embryonic development.

mRNP3 and mRNP4 are phosphorylatable by casein kinase II in Xenopus oocytes, but phosphorylation does not modify RNA-binding affinity.

mRNP3 and mRNP4 (also called FRGY2) are two mRNA-binding proteins which are major constituents of the maternal RNA storage particles of Xenopus laevis oocytes. The phosphorylation of mRNP3-4 has been implicated in the regulation of mRNA masking. In this study, we have investigated their phosphorylation by casein kinase II and its consequence on their affinity for RNA. Comparison of the phosphopeptide map of mRNP3-4 phosphorylated in vivo with that obtained after phosphorylation in vitro by purified Xenopus laevis casein kinase II strongly suggests that casein kinase II is responsible for the in vivo phosphorylation of mRNP3-4 in oocytes. The phosphorylation occurs on a serine residue in a central domain of the proteins. The affinity of mRNP3-4 for RNA substrates remained unchanged after the treatment with casein kinase II or calf intestine phosphatase in vitro. This suggests that phosphorylation of these proteins does not regulate their interaction with RNA but rather controls their interactions with other proteins.

Major intracellular localization of elongation factor-1.

Polyclonal antibodies directed against the two components of EF-1, the G-protein EF-1 alpha and the guanine-nucleotide exchange complex EF-1 beta gamma delta, were used for the analysis of EF-1. We show that Xenopus oocytes as well as Xenopus A6 cultured cells contain comparable ratios around 2:1 of EF-1 alpha versus EF-1 beta gamma delta. Immunolocalization of EF-1 was analyzed in A6 cultured cells. Both components appeared to be mainly localized in the cytoplasmic compartment, as a granulous diffuse network forming a gradient from the nucleus to the periphery of the cells. The major fraction of EF-1 was correlated to endoplasmic reticulum localization and not to the microtubule network. Co-localization of EF-1 with the endoplasmic reticulum is consistent with the function of EF-1 in peptide chain elongation.

The guanine-nucleotide-exchange complex (EF-1 beta gamma delta) of elongation factor-1 contains two similar leucine-zipper proteins EF-1 delta, p34 encoded by EF-1 delta 1 and p36 encoded by EF-1 delta 2.

We have cloned and sequenced a Xenopus cDNA referred to as EF-1 delta 2. The cDNA is homologous to EF-1 delta 1 encoding for EF-1 delta a protein of the guanine-nucleotide exchange complex of elongation factor-1 (EF-1). The protein sequence deduced from the cDNA, contains the two characteristic features of EF-1 delta protein, the leucine-zipper domain and the guanine-nucleotide exchange domain. In vitro and in vivo translation leads to the production of a 36-kDa protein from EF-1 delta and a 34-kDa protein from EF-1 delta 1. The clone EF-1 delta 2 therefore encodes for authentic p36 protein of EF-1 beta gamma delta complex, while EF-1 delta 1 encodes for a newly characterised p34 protein of the leucine zipper family. Both EF-1 delta proteins are simultaneously present in oocytes extracts, at a molecular ratio around 1:10 for p34 versus p36 proteins. Both are associated in a macromolecular structure that is greater than 750 kDa upon gel filtration. The two proteins are targets for Cdc2 kinase in meiotic maturation.

Brefeldin A provokes indirect activation of cdc2 kinase (MPF) in Xenopus oocytes, resulting in meiotic cell division.

Brefeldin A, a fungal metabolite which disrupts protein traffic, provokes indirect activation of cdc2 protein kinase in Xenopus oocytes. Cdc2 protein kinase activation was judged by MPF (M-phase factor) transfer activity, histone H1 kinase activity, and phosphorylation in vivo of the guanine-nucleotide exchange complex EF-1 beta gamma delta. Oocytes resumed complete meiosis upon brefeldin A treatment. Cdc2 protein kinase, MAP kinase, cyclin B, MPF, and protein synthesis changes were all comparable in brefeldin A-treated oocytes and in progesterone-induced oocytes. ED50 for brefeldin A was 0.6 microM. Brefeldin A activation of cdc2 protein kinase occurs with a long time course. Simultaneous treatment of the oocytes at a subthreshold concentration of 1 nM progesterone and 30 microM brefeldin A considerably shortened the kinetics of maturation. Brefeldin A induction of maturation was sensitive to drugs that act on cAMP metabolism. ID50 for IBMX was 0.1 mM, compared to 1 mM for progesterone-treated oocytes. Brefeldin A inhibited protein traffic in oocytes as determined from protein export experiments. ID50 was between 0.1 and 1 microM. Our results give new insights into the possible mechanism of induction of meiotic maturation and further demonstrate that brefeldin A acts on cell cycle regulatory elements.

Phosphorylation of elongation factor-1 (EF-1) by cdc2 kinase.

Elongation factor-1 (EF-1) is a major substrate for cdc2 kinase in Xenopus oocytes. The guanine-nucleotide exchange factor EF-1 beta gamma delta, appears to have a highly complex macromolecular structure containing several GTP/GDP exchange proteins, valyl-tRNA synthetase, and a putative anchoring protein EF-1 gamma. During meiotic cell division, the factor becomes phosphorylated by cdc2 kinase, not only on EF-1 gamma, but also on two different phospho-acceptors on EF-1 delta. Phosphorylation is concomitant with changes in protein synthesis in vivo. Xenopus oocytes, and potentially all cells, contain a multitude of heteromeric forms of the complex which postulates that EF-1 beta gamma delta is not a "house keeping" factor but a sophisticated regulatory element.

Elongation factor EF-1 delta, a new target for maturation-promoting factor in Xenopus oocytes.

A new physiological target for Cdc2 protein kinase has been identified. It corresponds to a protein EF-1 delta, a constituent of the nucleotide exchange factor EF-1 beta gamma delta, involved in the elongation step of protein synthesis. EF-1 delta is phosphorylated by Cdc2 kinase on threonine and serine residues. Threonine has been identified as Thr122 in the sequence VQVTPAAK. During oocyte maturation, Thr122 is phosphorylated at metaphase, when p34cdc2 is active. Phosphorylation studies revealed the presence of two post-translational regulated forms of EF-1 delta protein. Identification of two isoforms of the delta protein, together with the presence of two guanine-nucleotide exchange proteins (beta and delta) and physiologically regulated phosphorylation sites by Cdc2 kinase on gamma and delta proteins, implicate that EF-1 beta gamma delta exists in the cell under a multitude of macromolecular forms which suggests that EF-1 beta gamma delta is a sophisticated regulatory factor rather than a "housekeeping" element of the cell.

cdc2 kinase sets a memory phosphorylation signal on elongation factor EF-1 delta during meiotic cell division, which perdures in early development.

EF-1 delta is a physiological substrate for cdc2 protein kinase in Xenopus oocytes. The protein is part of the nucleotide exchange factor EF-1 beta gamma delta, involved in the elongation step of protein synthesis. We show that EF-1 delta exists under four isoforms in the prophase oocyte, all phosphorylable by casein kinase II. Each of the prophase isoforms was further separated into a 36 and a 38 kDa form upon phosphorylation by cdc2 kinase which therefore reveals the existence of eight different isoforms. Phosphorylation by cdc2 kinase can be monitored as the electrophoretic mobility dedoublement 36/38 kDa. Developmental regulation of EF-1 delta was analyzed. The cdc2 kinase-induced change occures at meiotic division, after complete oogenesis and perdures during early development. It is therefore a phosphorylation memory signal for early development.

Expression of elongation factor 1 alpha (EF-1 alpha) and 1 beta gamma (EF-1 beta gamma) are uncoupled in early Xenopus embryos.

In the amphibian Xenopus laevis, the elongation factor 1 alpha proteins (EF-1 alpha) synthesised in oocytes and somatic cells correspond to distinct gene products. Furthermore, the somatic EF-1 alpha gene (EF-1 alpha S) produces one of the most highly expressed early zygotic transcripts in the embryo. The functional recycling of EF-1 alpha (conversion of EF-1 alpha-GDP to EF-1 alpha-GTP) is assured by the EF-1 beta gamma complex. We show here that in Xenopus laevis embryos, contrary to the situation for EF-1 alpha, EF-1 beta, and EF-1 gamma mRNAs are transcribed from the same genes in oocytes and somatic cells. In addition, the onset of transcription of the EF-1 beta and EF-1 gamma genes from the zygotic genome occurs several hours after that of the somatic EF-1 alpha S gene. Therefore, during early Xenopus development the expression of these three elongation factors is not co-ordinated at the transcriptional level. The consequences of this uncoupling on the efficiency of translational elongation in the early Xenopus embryo are discussed.

Phosphorylation of Xenopus elongation factor-1 gamma by cdc2 protein kinase: identification of the phosphorylation site.

The cdc2 protein kinase phosphorylates elongation factor-1 gamma (EF-1 gamma) during meiotic maturation of Xenopus oocytes. A synthetic peptide P2: PKKETPKKEKPA matching the cDNA-deduced sequence of EF-1 gamma was an in vitro substrate for cdc2 protein kinase and inhibited phosphorylation of EF-1 gamma. Tryptic hydrolysis of EF-1 gamma and the P2 peptide, both phosphorylated by cdc2 protein kinase, resulted in multiple partial digestion products generated by the presence of barely hydrolysable bonds. The two peptides obtained from the hydrolysis of EF-1 gamma comigrated exactly in two-dimensional separation with two of the P2 peptide hydrolysates. EF-1 gamma therefore contains one unique phosphoacceptor for cdc2 protein kinase, identified as threonine-230.