Mass fingerprinting and MS/MS analysis demonstrated that Xyk, a 57-kDa Src family tyrosine kinase that is activated within minutes of Xenopus egg fertilization, comprises a mixture of two Src proteins, Src1 and Src2. However, the Xenopus Src protein, denoted as xSrc, is hardly detectable with mAb327, a universal Src-specific antibody, whose target sequence has not yet been determined. We show that a point amino acid substitution in the Src homology 3 domain of xSrc is critical for improvement of the low efficiency of its recognition by mAb327. Namely, a point-mutated xSrc, in which Arg-121 was replaced by His that is conserved among mAb327-reactive Src in mammals and chicken, showed increased recognition by mAb327. On the other hand, a mutant chicken Src, in which the His-122 residue is replaced by Arg, showed decreased recognition by mAb327. Genomic sequencing analysis also demonstrated that reptile Src proteins are of either the R-type (snake) or H-type (caiman, turtle, and tortoise). These studies revealed, for the first time, a critical amino acid in the Src SH3 domain for mAb327 recognition, and suggest a novel scheme for the molecular evolution of Src, in which the H-type Src(s) are monophyletic and derived from the R-type Src.

We demonstrate here that the intracellular environment of Xenopus oocytes is quite compatible with the requirements of T7 RNA polymerase (T7 RNAP)-mediated transcription. This reaction runs robustly in the oocyte cytoplasm for many hours. The coinjection of a T7 promoter-driven luciferase-encoding plasmid DNA and purified T7 RNAP into oocytes results in the prolonged production of luciferase protein. Thus, the efficient coupling of T7 RNAP-mediated transcription with the intrinsic oocyte translation machinery occurs in the oocyte cytoplasm. The coupled protein synthesis generates high expression yield, displays little variation in the expression level between individual oocytes, requires very limited amounts of DNA template and T7 RNAP, and does not affect the oocyte viability and functional status. Our detailed, quantitative comparison of the existing expression methods in Xenopus oocytes highlights the advantages of the technique based on the cytoplasmic coinjection of T7 RNAP and T7 promoter-driven plasmid DNA and demonstrates that it is greatly superior to the alternative methods of heterologous gene expression.

Pyruvate dehydrogenase kinase (PDK) inactivates the multienzyme mitochondrial pyruvate dehydrogenase complex by the phosphorylation of three seryl residues in the pyruvate dehydrogenase moiety, and thus plays an important role in the control of glucose homeostasis. Genetically and biochemically distinct PDK family isozymes have been identified in mammalian species. In the present study, we demonstrate that the complete family of expressed PDK family genes in the tissues of the African clawed frog, Xenopus laevis, consists of four members, which are divided into two evolutionary groups. Xenopus PDKs (xPDKs) share an overall homology of about 70% to the human isoforms of PDK. The abundance of mRNAs for the four xPDK isoforms was analyzed by the real-time reverse transcriptase PCR technique in the various tissues of Xenopus laevis, including heart, lung, spleen, liver, kidney, skin, testis, oocytes, and eggs. Our data suggest that one of the xPDK isozymes can be referred to as an oocyte-specific xPDK. Functional differences between the xPDK isoforms are discussed, based on their different tissue-specific distributions and phylogenetic similarities to human PDKs.

We reported previously that egg membrane rafts serve as a subcellular microdomain for sperm-dependent tyrosine kinase signaling in Xenopus fertilization. Moreover, we demonstrated that raft-associated Src tyrosine kinase was activated by sperm in vitro. Here we show that egg rafts incubated with sperm or hydrogen peroxide (H2O2) can promote Src-dependent phosphorylation of phospholipase Cgamma (PLCgamma) and transient calcium release in the extracts of unfertilized Xenopus eggs. In vivo egg activation by sperm or H2O2 also promotes tyrosine-phosphorylation and raft-translocalization of PLCgamma. Immunodepletion of PLCgamma from the egg extracts inhibits the raft-dependent calcium release. Rafts prepared from H2O2-activated eggs also promote Src-dependent dephosphorylation of p42 mitogen-activated protein kinase and cell cycle transition from metaphase II to interphase in egg extracts. PLCgamma phosphorylation and calcium release in egg extracts can be promoted by rafts prepared from COS-7 cells expressing the Xenopus Src gene. These results demonstrate that the signaling events elicited by fertilization in Xenopus eggs can be reconstituted in vitro. The development of such experimental platforms will allow us to dissect the molecular mechanism of sperm-dependent activation of raft-associated Src and subsequent up-regulation of PLCgamma and egg activation machinery in Xenopus eggs.

Protein-tyrosine phosphorylation plays an important role in egg activation signaling at fertilization. We show that in Xenopus, fertilization stimulates a rapid and transient tyrosine phosphorylation of egg proteins, as revealed by immunoblotting with anti-phosphotyrosine antibody. Immunofluorescent microscopic analysis demonstrated that the phosphorylation occurs in cortical area of the egg animal hemisphere. To further characterize subcellular compartment for fertilization-dependent tyrosine kinase signaling, we isolated low density detergent-insoluble membrane (LD-DIM) fraction from Xenopus eggs. The egg LD-DIM was enriched in cholesterol and GM1 ganglioside. It also contained signaling molecules such as Xyk (Xenopus egg Src), Gq alpha, Ras, integrin beta 1 and CD9. Fertilization stimulated tyrosine phosphorylation of Xyk and some other LD-DIM proteins. Remarkably, sperm stimulated tyrosine phosphorylation of the LD-DIM proteins in vitro. The sperm-dependent phosphorylation was sensitive to the tyrosine kinase inhibitors PP2 and genistein. We found that pretreatment of eggs with methyl-beta-cyclodextrin, a cholesterol-binding substance, led to a decrease in cholesterol, Xyk and sperm-induced tyrosine phosphorylation in LD-DIM. In methyl-beta-cyclodextrin-treated eggs, sperm-induced Ca(2+) transient and first cell division were also inhibited. These findings suggest that the egg LD-DIM might serve as subcellular microdomain for tyrosine kinase signaling in Xenopus egg fertilization.

The activity of c-Src protein-tyrosine kinase is up-regulated under a number of receptor signaling pathways. However, the activation mechanism of c-Src under physiological conditions has remained unclear. We show here that the Shc adaptor protein is a novel direct activator of c-Src in epidermal growth factor receptor signaling in A431 human epidermoid carcinoma cells. Among the three Shc isoforms, P66 and P52, but not P46, were found to interact with and activate c-Src in vitro and in vivo. Activation of c-Src accompanied autophosphorylation of c-Src in the activation segment, but the carboxyl-terminal dephosphorylation was not observed. We have identified the interaction sites between Shc and c-Src and constructed a point mutant of Shc that abolishes the c-Src activation. Using this mutant, we have confirmed that the Shc-mediated c-Src activation triggers Stat-p21/WAF1/Cip1 pathway that has been implicated in the cell cycle arrest and apoptosis of epidermal growth factor-stimulated A431 cells.

Mobilization of intracellular calcium is an indispensable step of fertilization-induced egg activation. Recently, this process has been shown to require the sequential activation of Src family tyrosine kinases, phospholipase Cgamma (PLCgamma), and inositol-1,4,5-trisphosphate (IP3)-dependent receptor of endoplasmic reticulum. In the present study, we made an attempt to recapitulate the early events of egg activation by stimulating Src kinase activity in the cell-free extracts of Xenopus eggs. We found that enhanced Src kinase activity can initiate calcium response of low magnitude in cytostatic factor (CSF)-arrested mitotic extracts without releasing them into interphase. The addition of catalytically active recombinant Src kinase, as well as the activation of endogenous Xenopus Src family kinase by hydrogen peroxide (H2O2), increased total tyrosine phosphorylation, tyrosine phosphorylation of PLCgamma, and IP3 production in the extracts. The treatment with the Src family kinase-specific inhibitor, PP1, or PLC inhibitor, U73122, or IP3 receptor antagonist, heparin, prevented calcium release in the extracts. We conclude, therefore, that possible mechanism of Src/H2O2 action in the extracts might involve tyrosine phosphorylation and activation of PLCgamma, accompanied by the increase in IP3 content and subsequent calcium release from IP3-regulated calcium stores. These results also suggest that monitoring calcium signals induced in the Xenopus egg extracts by various components of signaling pathways may provide a particularly useful approach to investigating their role in the signal transduction.

Cell cycle in various types of cells and in early embryos is often accompanied by transient changes in the concentration of free cytosolic calcium. In the present study, using fluorescent indicator fura-2, we demonstrate that Ca(2+) oscillates cyclically with an amplitude of about 100 nM and a period of mitotic cycle in cell-free Xenopus egg cycling extracts. It peaks in early metaphase just preceding mitotic reactivation of Cdc2 kinase and MAPK and reaches a minimum in interphase. The source of Ca(2+) in the extracts is a particulate fraction containing egg intracellular Ca(2+) stores, since the addition of a calcium-mobilizing second messenger, inositol 1,4,5-trisphosphate (IP3), induced a transient increase in Ca(2+). The inclusion of heparin, an IP3 receptor antagonist, or ultrafiltration of the extracts prevented Ca(2+)-releasing activity of IP3. The depletion of Ca(2+) in the extracts by the calcium chelator BAPTA resulted in the blockade of cell cycle at different stages, depending on the time of drug administration. The addition of BAPTA late in interphase blocked cell cycle at mitotic entry in prophase, whereas its application in anaphase or telophase blocked the extracts in early interphase. BAPTA administration in metaphase before transition to anaphase brought about a metaphase-like arrest in the cycling extracts. Inhibition of IP3-induced calcium release by heparin also arrested cell cycle progression in the cycling extracts.

Conference Abstract| October 01 2000 Protein-tyrosine phosphorylation in Xenopus egg cycling extracts A. A. Tokmakov; A. A. Tokmakov 1Laboratory of Molecular Biology, Biosignal Research Center, Kobe University, Kobe 657–8501 Search for other works by this author on: This Site PubMed Google Scholar K.-I. Sato; K.-I. Sato 1Laboratory of Molecular Biology, Biosignal Research Center, Kobe University, Kobe 657–8501 Search for other works by this author on: This Site PubMed Google Scholar T. Iwasaki; T. Iwasaki 1Laboratory of Molecular Biology, Biosignal Research Center, Kobe University, Kobe 657–8501 Search for other works by this author on: This Site PubMed Google Scholar K. Owada; K. Owada 2Institute of Molecular and Cellular Biology for Pharmaceutical Sciences, Kyoto Pharmaceutical University, Kyoto 607–8412 Search for other works by this author on: This Site PubMed Google Scholar Y. Fukami Y. Fukami 1Laboratory of Molecular Biology, Biosignal Research Center, Kobe University, Kobe 657–8501 Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (2000) 28 (5): A434. https://doi.org/10.1042/bst028a434 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Twitter LinkedIn Cite Icon Cite Get Permissions Citation A. A. Tokmakov, K.-I. Sato, T. Iwasaki, K. Owada, Y. Fukami; Protein-tyrosine phosphorylation in Xenopus egg cycling extracts. Biochem Soc Trans 1 October 2000; 28 (5): A434. doi: https://doi.org/10.1042/bst028a434 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search nav search search input Search input auto suggest search filter All ContentAll JournalsBiochemical Society Transactions Search Advanced Search This content is only available as a PDF. © 2000 Biochemical Society2000 Article PDF first page preview Close Modal You do not currently have access to this content.

Conference Abstract| October 01 2000 EGF-induced translocation of the adaptor protein She is involved in up-regulation of the tyrosine kinase c-Src in A431 cells K.-I. Sato; K.-I. Sato 1Lab. of Mol. Biol., Kobe Univ., Kobe 657–8501, Japan Search for other works by this author on: This Site PubMed Google Scholar M. Kimoto; M. Kimoto 1Lab. of Mol. Biol., Kobe Univ., Kobe 657–8501, Japan Search for other works by this author on: This Site PubMed Google Scholar M. Kakumoto; M. Kakumoto 1Lab. of Mol. Biol., Kobe Univ., Kobe 657–8501, Japan Search for other works by this author on: This Site PubMed Google Scholar D. Horiuchi; D. Horiuchi 1Lab. of Mol. Biol., Kobe Univ., Kobe 657–8501, Japan Search for other works by this author on: This Site PubMed Google Scholar T. Iwasaki; T. Iwasaki 1Lab. of Mol. Biol., Kobe Univ., Kobe 657–8501, Japan Search for other works by this author on: This Site PubMed Google Scholar A. A. Tokmakov; A. A. Tokmakov 1Lab. of Mol. Biol., Kobe Univ., Kobe 657–8501, Japan Search for other works by this author on: This Site PubMed Google Scholar Y. Fukami Y. Fukami 1Lab. of Mol. Biol., Kobe Univ., Kobe 657–8501, Japan Search for other works by this author on: This Site PubMed Google Scholar Author and article information Publisher: Portland Press Ltd Online ISSN: 1470-8752 Print ISSN: 0300-5127 © 2000 Biochemical Society2000 Biochem Soc Trans (2000) 28 (5): A432. https://doi.org/10.1042/bst028a432c Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn Email Cite Icon Cite Get Permissions Citation K.-I. Sato, M. Kimoto, M. Kakumoto, D. Horiuchi, T. Iwasaki, A. A. Tokmakov, Y. Fukami; EGF-induced translocation of the adaptor protein She is involved in up-regulation of the tyrosine kinase c-Src in A431 cells. Biochem Soc Trans 1 October 2000; 28 (5): A432. doi: https://doi.org/10.1042/bst028a432c Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search This content is only available as a PDF. © 2000 Biochemical Society2000 Article PDF first page preview Close Modal You do not currently have access to this content.

Autophosphorylation of recombinant mitogen-activated protein kinase (MAPK) on Tyr was found to be several-fold stimulated at weakly acidic pH (5.5–6.0), whereas the phosphorylation of a protein substrate, myelin basic protein, was greatly inhibited at pH below 6.0. In contrast to phosphorylation at pH 8.0, both MAPK autophosphorylation and MAPK phosphorylation with upstream MAPK kinase at low pH failed to stimulate essentially its kinase activity towards the exogenous protein substrate. Immunoprecipitation and ELISA with an activation segment-specific antibody, kinetic analysis, and reversible phosphorylation assay revealed a difference in the folding of MAPK activation segment at pH 5.5 and 8.0. The data suggest that a rearrangement of the activation segment at low pH promotes a stable low-activity conformation of the enzyme which is favorable for intramolecular autophosphorylation. In this conformation, the phosphorylation of the exogenous protein substrate is inhibited due to persistent blocking of the enzyme catalytic center by the activation segment.

A 58-kDa protein was detected inXenopusegg lysate by SDS-PAGE and immunoblotting with an antibody raised against adaptor protein Shc, a well known tyrosine kinase substrate in numerous biological events. Tyrosine phosphorylation of theXenopusShc protein (p58 xShc) was found to increase 2.3 ± 0.4-fold (n= 3) upon fertilization. Pretreatment of eggs with the tyrosine kinase inhibitor genistein effectively blocked the fertilization-dependent phosphorylation. Tyrosine phosphorylation of p58 xShc was also observed when eggs were activated parthenogenetically by an integrin-interacting RGDS-peptide which is known to cause egg activation accompanied by intracellular calcium release. On the other hand, other egg-activating treatments such as electrical shock and calcium ionophore, which directly induce the elevation of intracellular calcium, did not show such an effect. It is also suggested that the phosphorylated p58 xShc may play a role unique to the egg activation process because we found that there was no increase of Shc-Grb2 complex after fertilization. These results demonstrate that p58 xShc is a substrate of egg tyrosine kinases which may be activated by sperm-egg interaction and suggest that the phosphorylated p58 xShc may act upstream of the calcium-dependent pathway of egg activation.

The signal transduction pathway mediated by mitogen-activated protein kinases is an attractive target for the design of pharmacologically effective inhibitors. Two specific cell-permeant small molecule inhibitors of this pathway have been reported. However, under certain circumstances, nonpermeable inhibitors, such as neutralizing antibodies and peptide inhibitors, are also useful. We present here a novel approach for such peptide inhibitor design. The procedure is based on the synthesis of a structure-mimetic peptide corresponding to a short peptide segment in the target molecule. The results obtained so far show that a peptide designed in such a way is an effective inhibitor of the pathway. The possible application of such peptides and antipeptide antibodies as probes for protein kinase regulation mechanisms is also evaluated.

Recently, we have purified a Src-related tyrosine kinase, namedXenopustyrosine kinase (Xyk), from oocytes ofXenopus laevisand found that the enzyme is activated within 1 min following fertilization [Satoet al.(1996)J. Biol. Chem.271, 13250–13257]. A concomitant translocation of a part of the activated enzyme from the membrane fraction to the cytosolic fraction was also observed. In the present study, we show that parthenogenetic egg activation by a synthetic RGDS peptide [Y. Iwao and T. Fujimura, T. (1996)Dev. Biol.177, 558–567], an integrin-interacting peptide, but not by electrical shock or the calcium ionophore A23187 causes the kinase activation, tyrosine phosphorylation, and translocation of Xyk. A synthetic tyrosine kinase-specific inhibitor peptide was employed to analyze the importance of the Xyk activity in egg activation. We found that the peptide inhibits the kinase activity of purified Xyk at IC50of 8 μM. Further, egg activation induced by sperm or RGDS peptide but not by A23187 was inhibited by microinjection of the peptide. In the peptide-microinjected eggs, penetration of the sperm nucleus into the egg cytoplasm and meiotic resumption in the egg were blocked. Indirect immunofluorescence study demonstrates that Xyk is exclusively localized to the cortex ofXenopuseggs, indicating that Xyk can function in close proximity to the sperm–egg or RGDS peptide–egg interaction site. Taken together, these data suggest that the tyrosine kinase Xyk plays an important role in the early events ofXenopusegg activation in a manner independent or upstream of calcium signaling.

The adaptor protein Shc exists in three isoforms; p46, p52, and p66, and is a key regulator of a variety of biological processes. Our previous studies have shown that the tyrosine kinase c-Src phosphorylates Shc in a phosphatidylinositol (PtdIns) 4,5-bisphosphate-dependent manner. Here we demonstrate that PtdIns 3,4,5-trisphosphate stimulates phosphorylation of Shc by c-Src. The phosphorylation is blocked by a glutathione S-transferase fusion protein containing Shc phosphotyrosine binding (PTB) domain or a phosphotyrosine-containing Shc PTB domain-binding peptide. In rat pheochromocytoma cell line PC12, nerve growth factor (NGF) stimulates tyrosine phosphorylation of both Triton-soluble and -insoluble Shc which was maximal at 2-5 min after NGF treatment. We find that pretreatment of PC12 cells with the PtdIns 3-kinase inhibitor wortmannin or LY294002 results in almost half inhibition of the NGF-dependent tyrosine phosphorylation of only Triton-insoluble Shc. Similar inhibitory effect is observed with tyrosine kinase inhibitors genistein and PP1. Upon NGF stimulation, c-Src also becomes tyrosine-phosphorylated and accumulates in the Triton-insoluble fraction. The c-Src events are insensitive to wortmannin but sensitive to genistein. These results suggest that coordinate action of PtdIns 3-kinase and/or PtdIns 3,4,5-trisphosphate and c-Src can function as positive regulator in tyrosine phosphorylation of Shc in vitro and in vivo.

Mitogen-activated protein kinase (MAPK) is activated by phosphorylation within its activation segment. Upon phosphorylation, the activation segment refolds to provide the active conformation of the enzyme. We reported previously that a phosphorylation-sensitive secondary structure could be formed in a 26-amino-acid long synthetic peptide corresponding to the activation segment ofXenopusMAPK, termed IDA (Inter-DFG-APE) MAPK peptide (Tokmakov, A. A.,et al.1997,Biochem. Biophys. Res. Commun.236, 243–247). Here, we show that unphosphorylated IDA MAPK peptide can inhibitin vitroboth MAPK and MAPK kinase activities with the inhibition constants of 82 and 18 μM, respectively. Phosphorylated forms of the peptide were of little effect. IDA MAPK peptide did not inhibit significantly the activity of some other protein kinases, including MAPK homologue p38 kinase, suggesting the specificity for MAPK and MAPK kinase. Microinjection of unphosphorylated IDA MAPK peptide into immatureXenopusoocytes significantly suppressed progesterone-induced oocyte maturation by inhibiting activation of both MAPK and maturation promoting factor. Similar inhibition of maturation was registered upon oocyte treatment with another specific inhibitor of MAPK pathway, PD098059. These results depict IDA MAPK peptide as a selective inhibitor of the MAPK pathway that can be used for the investigations of MAPK-mediated signaling.

The adaptor protein Shc was prepared as glutathione S‐transferase fusion proteins (GST–Shc) and used as in vitro substrate for c‐Src. Since phosphotyrosine‐binding domain of Shc has been shown to bind phosphatidyl‐inositol 4,5‐bisphosphate (PtdIns(4,5)P2) [Zhou et al. (1995) Nature 378, 584–592], effect of PtdIns(4,5)P2 on the phosphorylation of GST–Shc by c‐Src was examined. PtdIns(4,5)P2 stimulated the phosphorylation of GST–Shc without any effect on the c‐Src activity as judged by both its autophosphorylation and phosphorylation of exogenous substrate, Cdc2 peptide. On the other hand, phosphatidylserine, phosphatidic acid, phosphatidylinositol, and phosphatidylinositol 4‐phosphate but not phosphatidylcholine stimulated the c‐Src activity itself. K m for GST–Shc in the presence of 1 μM PtdIns(4,5)P2 was calculated to be 90 nM. The PtdIns(4,5)P2‐dependent phosphorylation of GST–Shc was inhibited by a GST–fusion protein containing the phosphotyrosine‐binding domain of Shc. These results suggest that PtdIns(4,5)P2 can act as a regulator of phosphorylation of Shc by c‐Src through its binding to Shc.

A 26-amino acid long synthetic peptide corresponding to the activation loop ofXenopusMAP kinase (MAPK), termed IDA (Inter-DFG-APE) MAPK peptide, was found to efficiently inhibit the immunoprecipitation of the enzyme with anti-IDA MAPK serum. The value of half-inhibition concentration (100 nM) indicates that the IDA peptide and native MAPK activation loop are virtually indistinguishable in terms of antibody recognition. On the other hand, the Tyr-phosphorylated form of the peptide exerted its inhibitory action at around one order higher concentration. Shorter nonapeptides covering the epitope sequence of anti-IDA MAPK antibody could also affect the immunoprecipitation but at much higher concentrations (half-inhibition concentration ∼ 100 μM) and independently of their phosphorylation state. Circular dichroic study revealed that a secondary structure could be readily induced with the aid of trifluoroethanol in the unphosphorylated and, to a less extent, in the Tyr-phosphorylated IDA MAPK peptide but not in the shorter nonapeptides. These results suggest that the secondary structure similar to that of the unphosphorylated activation loop of MAPK can be formed in the IDA MAPK peptide and may be lost upon its Tyr-phosphorylation.