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S Che

Publications and source records attributed to S Che.

10 recordsLinked to original sources
PubMed1999

Identification and cloning of xp95, a putative signal transduction protein in Xenopus oocytes.

A 95-kDa protein in Xenopus oocytes, Xp95, was shown to be phosphorylated from the first through the second meiotic divisions during progesterone-induced oocyte maturation. Xp95 was purified and cloned. The Xp95 protein sequence exhibited homology to mouse Rhophilin, budding yeast Bro1, and Aspergillus PalA, all of which are implicated in signal transduction. It also contained three conserved features including seven conserved tyrosines, a phosphorylation consensus sequence for the Src family of tyrosine kinases, and a proline-rich domain near the C terminus that contains multiple SH3 domain-binding motifs. We showed the following: 1) that both Xp95 isolated from Xenopus oocytes and a synthetic peptide containing the Src phosphorylation consensus sequence of Xp95 were phosphorylated in vitro by Src kinase and to a lesser extent by Fyn kinase; 2) Xp95 from Xenopus oocytes or eggs was recognized by an anti-phosphotyrosine antibody, and the relative abundance of tyrosine-phosphorylated Xp95 increased during oocyte maturation; and 3) microinjection of deregulated Src mRNA into Xenopus oocytes increased the abundance of tyrosine-phosphorylated Xp95. These results suggest that Xp95 is an element in a tyrosine kinase signaling pathway that may be involved in progesterone-induced Xenopus oocyte maturation.

Amino Acid Sequence↗
PubMed1998

A phosphatase activity in Xenopus oocyte extracts preferentially dephosphorylates the MPM-2 epitope.

MPM-2 antigens are a large family of mitotic phosphoproteins that contain similar phosphoepitopes recognized by the anti-phosphoepitope antibody MPM-2 (MPM-2 epitopes). These proteins are phosphorylated during M phase induction and dephosphorylated from the onset of anaphase through interphase. Since biochemical characterization of the MPM-2 epitope phosphatase requires a specific assay for its activity, we tested different methods for measurement of the MPM-2 epitope phosphatase activity in crude cell lysates. First, an ELISA-based assay was designed that measured the phosphatase-induced reduction of the MPM-2 reactivity in crude M phase cell lysates. Using this assay to follow the phosphatase activity during sequential chromatography of Xenopus oocyte extracts, one predominant peak of phosphatase activity was detected which was separated from the majority of PP1 and PP2A activities. This phosphatase activity dephosphorylated the MPM-2 epitope on multiple MPM-2 antigens. The second method measured dephosphorylation of cdc25, a known MPM-2 antigen. Two major peaks of cdc25 dephosphorylating activities were detected during the sequential chromatography, one that copurified with the major peak of MPM-2 epitope phosphatase activity, and the other with the major peak of PP2A activity. Finally, we examined whether GST-MPM2, a fusion protein between glutathione S-transferase and a 19-residue peptide that contained two representative MPM-2 epitope sequences, could be dephosphorylated efficiently and specifically by the major MPM-2 epitope phosphatase activity in Xenopus oocyte extracts. Neither the crude extract nor the partially purified MPM-2 epitope phosphatase activity efficiently dephosphorylated the MPM-2 epitope on GST-MPM2. These results demonstrate that the ELISA-based assay preferentially detects the MPM-2 epitope phosphatase activity in crude cell lysates which may represent a physiological MPM-2 epitope phosphatase.

Animals↗
PubMed1997

Molecular cloning of a splice variant of Caenorhabditis elegans YNK1, a putative element in signal transduction.

YNK1 is a 98.3-kDa protein whose sequence was originally deduced from a genomic sequence in Caenorhabditis elegans. It was recently found that YNK1 is homologous to three different proteins implicated in signal transduction, suggesting that YNK1 is a signal transduction protein. In this report we describe the isolation of a full-length cDNA that encodes a splice variant of YNK1, designated YNK1a. We also present evidence that both YNK1 and YNK1a transcripts exist in vivo. Furthermore, using an antibody raised against a YNK1a recombinant protein, we demonstrate that the YNK1 protein is expressed in vivo throughout development.

Animals↗
PubMed1997

MPM-2 epitope sequence is not sufficient for recognition and phosphorylation by ME kinase-H.

Monoclonal antibody MPM-2 recognizes a large family of mitotic phosphoproteins in a phosphorylation-dependent manner. The antigenic phosphoepitope, designated the MPM-2 epitope, putatively consists of hydrophobic residue-Thr/Ser-Pro-hydrophobic residue-uncharged/basic residue. In this study, we addressed whether this sequence motif contains all the information necessary for recognition and phosphorylation by the kinase that phosphorylates most MPM-2 antigens. A fusion protein between glutathione S-transferase and a 19-residue peptide that contained two representative MPM-2 epitope sequences overlapping with two potential MAP kinase phosphorylation sites was constructed. Both the MPM-2 epitope sequences in the fusion protein (GST-MPM2) were phosphorylated by Xenopus egg extract, making the fusion protein MPM-2 reactive. However, while MAP kinase phosphorylated both the MPM-2 epitope sequences, neither ME kinase-H, a good candidate for a major MPM-2 epitope kinase, nor mitotic cdc2 kinase, which is known to phosphorylate certain MPM-2 antigens in vitro, phosphorylated GST-MPM2 to any significant extent. Furthermore, depletion of MAP kinase activity removed most, if not all, of the GST-MPM2 phosphorylating activity from crude Xenopus egg extracts. These results suggest that additional or different structural information than that provided by the deduced MPM-2 epitope sequence is required for recognition and phosphorylation by ME kinase-H or other major MPM-2 epitope kinases. They also offer a valid explanation for selective phosphorylation of certain MPM-2 antigens by MAP kinase as well as selective recognition of certain phosphorylated MAP kinase substrates by MPM-2.

Amino Acid Sequence↗
PubMed1997

Mitogen-activated protein kinase and cyclin B/Cdc2 phosphorylate Xenopus nuclear factor 7 (xnf7) in extracts from mature oocytes. Implications for regulation of xnf7 subcellular localization.

Xenopus nuclear factor 7 (xnf7) is a maternally expressed putative transcription factor that exhibits phosphorylation-dependent changes in subcellular localization during early Xenopus development. Xnf7 is localized to the germinal vesicle (nucleus) of immature oocytes in a hypophosphorylated state. Xnf7 is phosphorylated during oocyte maturation and released to the cytoplasm. The protein is retained in the cytoplasm during early embryonic cleavage stages but returns to nuclei at the mid-blastula transition. Xnf7 is phosphorylated at two sites during oocyte maturation, designated P1, consisting of one threonine at position 103, and P2, consisting of three clustered threonines at positions 209, 212, and 218. Phosphorylation of both sites is important in regulating xnf7 localization. The P1 site can be phosphorylated by cyclin B/Cdc2 in vitro. To further understand the mechanisms regulating subcellular localization of xnf7 during early development, kinases capable of catalyzing phosphorylation of the P2 site were purified from mature oocyte extracts. We found that mitogen-activated protein kinase phosphorylated Thr212 and cyclin B/Cdc2 phosphorylated Thr 209 and Thr212. No other kinase in mature oocyte extracts phosphorylated the xnf7 P2 site to a significant extent. These results implicate mitogen-activated protein kinase and cyclin B/Cdc2 in regulating xnf7 localization during oocyte maturation. This also suggests that localization of xnf7 may be regulated by multiple kinase activation pathways.

Animals↗
PubMed1997

Novel and frequent mutations of hepatitis B virus coincide with a major histocompatibility complex class I-restricted T-cell epitope of the surface antigen.

We examined the full-length hepatitis B virus (HBV) envelope (surface antigen or HBV small surface antigen [HBsAg]) sequences of 12 different liver samples from 10 different hepatoma-containing chronic carriers. Surprisingly, novel and frequent mutations occurred predominantly at amino acids 40 and 47 of HBsAg, in addition to within a known protective B-cell epitope (so-called group a determinant of HBsAg 124-148). Approximately 58% of chronic carriers contain mutations at the group a determinant. The mutation frequency at the hotspot codons 40 and 47 is approximately 83%, 1 order of magnitude higher than at the known polymorphic sites of subtype-specific determinants at codons 122 and 160, which is approximately 4%. This new mutational domain is found to coincide with a major histocompatibility complex class I-restricted T-cell epitope. The potential biological significance of this novel mutation in the immunopathogenesis of HBV chronic carriers is discussed.

Adult↗
PubMed1996

Finely tuned regulation of cytoplasmic retention of Xenopus nuclear factor 7 by phosphorylation of individual threonine residues.

Xenopus nuclear factor 7 (xnf7) is a maternal gene product that functi ons in dorsal/ventral patterning of the embryo. The xnf7 protein is stored in the oocyte nucleus germinal vesicle in a hypophosphorylated state. At oocyte maturation, xnf7 is hyperphosphorylated and released into the cytoplasm, where it is anchored until the midblastula stage, where it is dephosphorylated and enters the nucleus. We demonstrated that cytoplasmic anchoring of xnf7 was regulated by changes in the phosphorylation status of four threonines within two sites, site 1 (Thr-103) and site 2 (Thr-209, Thr-212, and Thr-218), which function in an additive manner. A mutant form of xnf7 (xnf7thr-glu) in which the threonines at sites 1 and 2 were mutated to glutamic acids to mimic a permanent state of phosphorylation was retained in the cytoplasm in oocytes and embryos through the gastrula stage. The cytoplasmic form of xnf7 was detected in a large 670-kDa protein complex probably consisting of xnf7 and several other unknown protein components. Anchoring of xnf7 was not dependent on association with either microtubule or microfilament components of the cytoskeleton, since treatment with cytochalasin B and nocodazole did not affect cytoplasmic retention. Both wild-type xnf7 and xnf7thr-glu form dimers in the yeast two-hybrid system; however, homodimerization was not required for cytoplasmic retention. We suggest that the cytoplasmic retention of xnf7 depends on the phosphorylation state of the protein whereas the cytoplasmic anchoring machinery appears to be constitutively present in oocytes and throughout development until the gastrula stage.

Amino Acid Sequence↗
PubMed1995

Body temperature effect on methylenedioxymethamphetamine-induced acute decrease in tryptophan hydroxylase activity.

Brain tryptophan hydroxylase activity decreases within 15 min after a single administration of 3,4-methylenedioxymethamphetamine. In the present study, the effect of body temperature on this acute decrease of tryptophan hydroxylase activity was examined. 2 h after a single dose of 3,4-methylenedioxymethamphetamine (20 mg/kg, s.c.), rats exhibited hyperthermia (38.7 degrees C) or hypothermia (35.8 degrees C) when maintained at 25 degrees C or 6 degrees C, respectively. The rectal temperature of control animals maintained at 6 degrees C was not altered. Tryptophan hydroxylase activity measured in the hippocampus, striatum and frontal cortex of hyperthermic rats treated with 3,4-methylenedioxymethamphetamine was decreased to 61%, 65%, and 71% of control levels, respectively, 2 h after drug treatment. However, in hypothermic rats, 3,4-methylenedioxymethamphetamine had no effect on tryptophan hydroxylase activity in the hippocampus, striatum or frontal cortex. Non-drug-induced hyperthermia or hypothermia did not affect tryptophan hydroxylase activity. Since hypothermia may prevent the 3,4-methylenedioxymethamphetamine-induced decrease in tryptophan hydroxylase activity by reducing the formation of free radicals, the effect of a free radical scavenging agent, N-tert-butyl-alpha-phenylnitrone, was examined. N-tert-butyl-alpha-phenylnitrone (200 mg/kg, i.p.) alone caused hypothermia but had no direct effect on tryptophan hydroxylase activity. Preadministration of N-tert-butyl-alpha-phenylnitrone prevented 3,4-methylenedioxymethamphetamine from raising the temperature above normal and attenuated the drug-induced decrease in tryptophan hydroxylase activity in hippocampus, striatum and frontal cortex. However, when the rats treated with a combination of N-tert-butyl-alpha-phenylnitrone and 3,4-methylenedioxymethamphetamine were maintained at hyperthermic conditions, N-tert-butyl-alpha-phenylnitrone had no protective effect. These results suggest that body temperature plays a prominent role in the 3,4-methylenedioxymethamphetamine-induced acute decrease in tryptophan hydroxylase activity.

Animals↗
PubMedDate not supplied

Natural killer cell suppression of IgM production.

The mechanisms by which natural killer (NK) cells regulate B cell function are not well understood. In this paper, the suppressive effects of NK cells on IgM production by lipopolysaccharide (LPS)-stimulated B cells were studied. We found that interleukin (IL)-2-activated NK (NKa) cells, but not unstimulated NK cells, suppressed IgM production by B cells stimulated with LPS. Suppression of antibody production required direct NKa-B cell contact, as demonstrated in cultures utilizing semiporous membranes for cell separation, and was the consequence of a reduction in the number of IgM-producing cells, as determined by enzyme-linked immunospot assays. Suppression could not be accounted for by cytotoxic mechanisms since the NKa cells caused neither cytolysis of 51Cr-labelled B cells or B cell apoptosis. While NKa-B cell contact was necessary for suppression, cell contact alone was not sufficient. Rather, both NKa-B cell contact and NKa production of interferon (IFN)-gamma were necessary. Since only IL-2-activated, but not unstimulated, NK cells suppressed IgM production, we investigated the potential for IL-4, which has been reported to downregulate IL-2-induced NK cell proliferation, to prevent NKa cell suppressive activity. While IL-4 antagonized IL-2-induced NK cell proliferation, it was completely ineffective in antagonizing NKa cell suppression of IgM production. The requirement for IL-2 activation of NK cells for suppression of IgM production suggests that NK cells may be part of a physiologic negative feedback mechanism to downregulate antibody production.

Animals↗