The homeobox gene Pitx2: mediator of asymmetric left-right signaling in vertebrate heart and gut looping.
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Left-right asymmetry in vertebrates is controlled by activities emanating from the left lateral plate. How these signals get transmitted to the forming organs is not known. A candidate mediator in mouse, frog and zebrafish embryos is the homeobox gene Pitx2. It is asymmetrically expressed in the left lateral plate mesoderm, tubular heart and early gut tube. Localized Pitx2 expression continues when these organs undergo asymmetric looping morphogenesis. Ectopic expression of Xnr1 in the right lateral plate induces Pitx2 transcription in Xenopus. Misexpression of Pitx2 affects situs and morphology of organs. These experiments suggest a role for Pitx2 in promoting looping of the linear heart and gut. (+info)
The mitogen-activated protein kinase signaling pathway stimulates mos mRNA cytoplasmic polyadenylation during Xenopus oocyte maturation.
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The Mos protein kinase is a key regulator of vertebrate oocyte maturation. Oocyte-specific Mos protein expression is subject to translational control. In the frog Xenopus, the translation of Mos protein requires the progesterone-induced polyadenylation of the maternal Mos mRNA, which is present in the oocyte cytoplasm. Both the Xenopus p42 mitogen-activated protein kinase (MAPK) and maturation-promoting factor (MPF) signaling pathways have been proposed to mediate progesterone-stimulated oocyte maturation. In this study, we have determined the relative contributions of the MAPK and MPF signaling pathways to Mos mRNA polyadenylation. We report that progesterone-induced Mos mRNA polyadenylation was attenuated in oocytes expressing the MAPK phosphatase rVH6. Moreover, inhibition of MAPK signaling blocked progesterone-induced Mos protein accumulation. Activation of the MAPK pathway by injection of RNA encoding Mos was sufficient to induce both the polyadenylation of synthetic Mos mRNA substrates and the accumulation of endogenous Mos protein in the absence of MPF signaling. Activation of MPF, by injection of cyclin B1 RNA or purified cyclin B1 protein, also induced both Mos protein accumulation and Mos mRNA polyadenylation. However, this action of MPF required MAPK activity. By contrast, the cytoplasmic polyadenylation of maternal cyclin B1 mRNA was stimulated by MPF in a MAPK-independent manner, thus revealing a differential regulation of maternal mRNA polyadenylation by the MAPK and MPF signaling pathways. We propose that MAPK-stimulated Mos mRNA cytoplasmic polyadenylation is a key component of the positive-feedback loop, which contributes to the all-or-none process of oocyte maturation. (+info)
Identification and cloning of xp95, a putative signal transduction protein in Xenopus oocytes.
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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. (+info)
Voltage sensors in domains III and IV, but not I and II, are immobilized by Na+ channel fast inactivation.
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Using site-directed fluorescent labeling, we examined conformational changes in the S4 segment of each domain of the human skeletal muscle sodium channel (hSkM1). The fluorescence signals from S4 segments in domains I and II follow activation and are unaffected as fast inactivation settles. In contrast, the fluorescence signals from S4 segments in domains III and IV show kinetic components during activation and deactivation that correlate with fast inactivation and charge immobilization. These results indicate that in hSkM1, the S4 segments in domains III and IV are responsible for voltage-sensitive conformational changes linked to fast inactivation and are immobilized by fast inactivation, while the S4 segments in domains I and II are unaffected by fast inactivation. (+info)
Acute effects of ethanol on kainate receptors with different subunit compositions.
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Previous studies showed that recombinant homomeric GluR6 receptors are acutely inhibited by ethanol. This study examined the acute actions of ethanol on recombinant homomeric and heteromeric kainate (KA) receptors with different subunit configurations. Application of 25 to 100 mM ethanol produced inhibition of a similar magnitude of both GluR5-Q and GluR6-R KA receptor-dependent currents in Xenopus oocytes. Ethanol decreased the KA Emax without affecting the EC50 and its effect was independent of the membrane holding potential for both of these receptors subtypes. Ethanol also inhibited homomeric and heteromeric receptors transiently expressed in human embryonic kidney (HEK) 293 cells. In these cells, the expression of heteromeric GluR6-R subunit-containing receptors was confirmed by testing their sensitivity to 1 mM alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid. Ethanol inhibited to a similar extent KA-gated currents mediated by receptors composed of either GluR6 or GluR6 + KA1 subunits, and to a slightly lesser extent receptors composed of GluR6 + KA2 subunits. Acute ethanol's effects were tested on GluR5 KA receptors that are expressed as homomers (GluR5-Q) or heteromers (GluR5-R + KA1 and GluR5-R + KA2). Homomeric and heteromeric GluR5 KA receptors were all inhibited to a similar extent by ethanol; however, there was slightly more inhibition of GluR5-R + KA2 receptors. Thus, recombinant KA receptors with different subunit compositions are all acutely inhibited to a similar extent by ethanol. In light of recent reports that KA receptors regulate neurotransmitter release and mediate synaptic currents, we postulate that these receptors may play a role in acute ethanol intoxication. (+info)
Modulation of the channel activity of the epsilon2/zeta1-subtype N-methyl D-aspartate receptor by PSD-95.
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A channel-associated protein PSD-95 has been shown to induce clustering of N-methyl D-aspartate (NMDA) receptors, interacting with the COOH terminus of the epsilon subunit of the receptors. The effects of PSD-95 on the channel activity of the epsilon2/zeta1 heteromeric NMDA receptor were examined by injection of PSD-95 cRNA into Xenopus oocytes expressing the NMDA receptors. Expression of PSD-95 decreased the sensitivity of the NMDA receptor channels to L-glutamate. Mutational studies showed that the interaction between the COOH terminus of the epsilon2 subunit of the NMDA receptor and the second PSD-95/Dlg/Z0-1 domain of PSD-95 is critical for the decrease in glutamate sensitivity. It is known that protein kinase C markedly potentiates the channel activity of the NMDA receptor expressed in oocytes. PSD-95 inhibited the protein kinase C-mediated potentiation of the channels. Thus, we demonstrated that PSD-95 functionally modulates the channel activity of the epsilon2/zeta1 NMDA receptor. PSD-95 makes signal transmission more efficient by clustering the channels at postsynaptic sites. In addition to this, our results suggest that PSD-95 plays a protective role against neuronal excitotoxicity by decreasing the glutamate sensitivity of the channels and by inhibiting the protein kinase C-mediated potentiation of the channels. (+info)
Characterization of two related Drosophila gamma-tubulin complexes that differ in their ability to nucleate microtubules.
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gamma-tubulin exists in two related complexes in Drosophila embryo extracts (Moritz, M., Y. Zheng, B.M. Alberts, and K. Oegema. 1998. J. Cell Biol. 142:1- 12). Here, we report the purification and characterization of both complexes that we name gamma-tubulin small complex (gammaTuSC; approximately 280,000 D) and Drosophila gammaTuRC ( approximately 2,200,000 D). In addition to gamma-tubulin, the gammaTuSC contains Dgrip84 and Dgrip91, two proteins homologous to the Spc97/98p protein family. The gammaTuSC is a structural subunit of the gammaTuRC, a larger complex containing about six additional polypeptides. Like the gammaTuRC isolated from Xenopus egg extracts (Zheng, Y., M.L. Wong, B. Alberts, and T. Mitchison. 1995. Nature. 378:578-583), the Drosophila gammaTuRC can nucleate microtubules in vitro and has an open ring structure with a diameter of 25 nm. Cryo-electron microscopy reveals a modular structure with approximately 13 radially arranged structural repeats. The gammaTuSC also nucleates microtubules, but much less efficiently than the gammaTuRC, suggesting that assembly into a larger complex enhances nucleating activity. Analysis of the nucleotide content of the gammaTuSC reveals that gamma-tubulin binds preferentially to GDP over GTP, rendering gamma-tubulin an unusual member of the tubulin superfamily. (+info)
Identification of a novel domain shared by putative components of the endocytic and cytoskeletal machinery.
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We have identified a approximately 140 amino acid domain that is shared by a variety of proteins in budding and fission yeast, nematode, rat, mouse, frog, oat, and man. Typically, this domain is located within 20 residues of the N-terminus of the various proteins. The percent identity among the domains in the 12 proteins ranges from 42 to 93%, with 16 absolutely conserved residues: N-x(11-13)-V-x2-A-T-x(34-36)-R-x(7-8)-W-R-x3-K-x12-G-x-E-x15 -L-x11-12-D-x-G-R-x11-D-x7-R. Even though these proteins share little beyond their segment of homology, data are emerging that several of the proteins are involved in endocytosis and or regulation of cytoskeletal organization. We have named this protein segment the ENTH domain, for Epsin N-terminal Homology domain, and hypothesize that it is a candidate for binding specific ligands and/or enzymatic activity in the cell. (+info)