Dataset Information

Shaggy/glycogen synthase kinase 3? and phosphorylation of Sarah/regulator of calcineurin are essential for completion of Drosophila female meiosis.

ABSTRACT: The Ca(2+)/Calmodulin-dependent phosphatase calcineurin is essential for exit from meiotic arrest at metaphases I and II in Drosophila and Xenopus oocytes. We previously found that Sarah, the Drosophila homolog of regulator of calcineurin, acts as a positive regulator of calcineurin and is required to complete anaphase I of female meiosis. Here, we undertook biochemical approaches, including MS and posttranslational modification analyses, to better understand the mechanism by which Sarah regulates calcineurin. A search for phosphorylated residues revealed that Sarah is highly phosphorylated at Ser100, Thr102, and Ser219 in both ovaries and activated eggs and that Ser215 is phosphorylated only in activated eggs. Functional analyses using mutant forms of Sarah showed that phosphorylation at Ser215, a consensus phosphorylation site for glycogen synthase kinase 3? (GSK-3?) and its priming kinase site Ser219, are essential for Sarah function. Furthermore, germ-line clones homozygous for a null allele of shaggy (Drosophila GSK-3?) both fail to complete meiosis and lack phosphorylation of Sarah at Ser215, suggesting that the phosphorylation of Sarah by Shaggy/GSK-3? is required to complete meiosis. Our findings suggest a mechanism in which Shaggy/GSK-3? activates calcineurin through Sarah phosphorylation on egg activation in Drosophila.

SUBMITTER: Takeo S

PROVIDER: S-EPMC3340032 | biostudies-literature | 2012 Apr

REPOSITORIES: biostudies-literature

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Shaggy/glycogen synthase kinase 3β and phosphorylation of Sarah/regulator of calcineurin are essential for completion of Drosophila female meiosis.

Takeo Satomi S Swanson Selene K SK Nandanan Kavyasree K Nakai Yasuhiro Y Aigaki Toshiro T Washburn Michael P MP Florens Laurence L Hawley R Scott RS

Proceedings of the National Academy of Sciences of the United States of America 20120315 17

The Ca(2+)/Calmodulin-dependent phosphatase calcineurin is essential for exit from meiotic arrest at metaphases I and II in Drosophila and Xenopus oocytes. We previously found that Sarah, the Drosophila homolog of regulator of calcineurin, acts as a positive regulator of calcineurin and is required to complete anaphase I of female meiosis. Here, we undertook biochemical approaches, including MS and posttranslational modification analyses, to better understand the mechanism by which Sarah regulat ...[more]

PMID: 22421435

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Project description:BACKGROUND: Plant Glycogen Synthase Kinase 3/ SHAGGY-like kinases (GSKs) have been implicated in numerous biological processes ranging from embryonic, flower, stomata development to stress and wound responses. They are key regulators of brassinosteroid signaling and are also involved in the cross-talk between auxin and brassinosteroid pathways. In contrast to the human genome that contains two genes, plant GSKs are encoded by a multigene family. Little is known about Liliopsida resp. Poaceae in comparison to Brassicaceae GSKs. Here, we report the identification and structural characterization of two GSK homologs named TaSK1 and TaSK2 in the hexaploid wheat genome as well as a widespread phylogenetic analysis of land plant GSKs. RESULTS: Genomic and cDNA sequence alignments as well as chromosome localization using nullisomic-tetrasomic lines provided strong evidence for three expressed gene copies located on homoeolog chromosomes for TaSK1 as well as for TaSK2. Predicted proteins displayed a clear GSK signature. In vitro kinase assays showed that TaSK1 and TaSK2 possessed kinase activity. A phylogenetic analysis of land plant GSKs indicated that TaSK1 and TaSK2 belong to clade II of plant GSKs, the Arabidopsis members of which are all involved in Brassinosteroid signaling. Based on a single ancestral gene in the last common ancestor of all land plants, paralogs were acquired and retained through paleopolyploidization events, resulting in six to eight genes in angiosperms. More recent duplication events have increased the number up to ten in some lineages. CONCLUSIONS: To account for plant diversity in terms of functionality, morphology and development, attention has to be devoted to Liliopsida resp Poaceae GSKs in addition to Arabidopsis GSKs. In this study, molecular characterization, chromosome localization, kinase activity test and phylogenetic analysis (1) clarified the homologous/paralogous versus homoeologous status of TaSK sequences, (2) pointed out their affiliation to the GSK multigene family, (3) showed a functional kinase activity, (4) allowed a classification in clade II, members of which are involved in BR signaling and (5) allowed to gain information on acquisition and retention of GSK paralogs in angiosperms in the context of whole genome duplication events. Our results provide a framework to explore Liliopsida resp Poaceae GSKs functions in development.

Project description:BackgroundGlycogen Synthase Kinase 3/SHAGGY-like kinases (GSKs) are multifunctional non-receptor ser/thr kinases. Plant GSKs are involved in hormonal signaling networks and are required for growth, development, light as well as stress responses. So far, most studies have been carried out on Arabidopsis or on other eudicotyledon GSKs. Here, we evaluated the role of TaSK1 and TaSK2, two homolog wheat (Triticum aestivum) GSKs, in brassinosteroid signaling. We explored in addition the physiological effects of brassinosteroids on wheat growth and development.ResultsA bin2-1 like gain-of-function mutation has been inserted respectively in one of the homoeologous gene copies of TaSK1 (TaSK1-A.2-1) and in one of the homoeologous gene copies of TaSK2 (TaSK2-A.2-1). Arabidopsis plants were transformed with these mutated gene copies. Severe dwarf phenotypes were obtained closely resembling those of Arabidopsis bin2-1 lines and Arabidopsis BR-deficient or BR-signaling mutants. Expression of BR downstream genes, SAUR-AC1, CPD and BAS1 was deregulated in TaSK1.2-1 and TaSK2.2-1 transgenic lines. Severe dwarf lines were partially rescued by Bikinin beforehand shown to inhibit TaSK kinase activity. This rescue was accompanied with changes in BR downstream gene expression levels. Wheat embryos and seedlings were treated with compounds interfering with BR signaling or modifying BR levels to gain insight into the role of brassinosteroids in wheat development. Embryonic axis and scutellum differentiation were impaired, and seedling growth responses were affected when embryos were treated with Epibrassinolides, Propiconazole, and Bikinin.ConclusionsIn view of our findings, TaSKs are proposed to be involved in BR signaling and to be orthologous of Arabidopsis Clade II GSK3/SHAGGY-like kinases. Observed effects of Epibrassinolide, Propiconazole and Bikinin treatments on wheat embryos and seedlings indicate a role for BR signaling in embryonic patterning and seedling growth.

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Project description:Introduction: The ability of synapses to maintain physiological levels of evoked neurotransmission is essential for neuronal stability. A variety of perturbations can disrupt neurotransmission, but synapses often compensate for disruptions and work to stabilize activity levels, using forms of homeostatic synaptic plasticity. Presynaptic homeostatic potentiation (PHP) is one such mechanism. PHP is expressed at the Drosophila melanogaster larval neuromuscular junction (NMJ) synapse, as well as other NMJs. In PHP, presynaptic neurotransmitter release increases to offset the effects of impairing muscle transmitter receptors. Prior Drosophila work has studied PHP using different ways to perturb muscle receptor function-either acutely (using pharmacology) or chronically (using genetics). Some of our prior data suggested that cytoplasmic calcium signaling was important for expression of PHP after genetic impairment of glutamate receptors. Here we followed up on that observation. Methods: We used a combination of transgenic Drosophila RNA interference and overexpression lines, along with NMJ electrophysiology, synapse imaging, and pharmacology to test if regulators of the calcium/calmodulin-dependent protein phosphatase calcineurin are necessary for the normal expression of PHP. Results: We found that either pre- or postsynaptic dysregulation of a Drosophila gene regulating calcineurin, sarah (sra), blocks PHP. Tissue-specific manipulations showed that either increases or decreases in sra expression are detrimental to PHP. Additionally, pharmacologically and genetically induced forms of expression of PHP are functionally separable depending entirely upon which sra genetic manipulation is used. Surprisingly, dual-tissue pre- and postsynaptic sra knockdown or overexpression can ameliorate PHP blocks revealed in single-tissue experiments. Pharmacological and genetic inhibition of calcineurin corroborated this latter finding. Discussion: Our results suggest tight calcineurin regulation is needed across multiple tissue types to stabilize peripheral synaptic outputs.

Dataset Information

Shaggy/glycogen synthase kinase 3? and phosphorylation of Sarah/regulator of calcineurin are essential for completion of Drosophila female meiosis.

Publications

Shaggy/glycogen synthase kinase 3β and phosphorylation of Sarah/regulator of calcineurin are essential for completion of Drosophila female meiosis.

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