Functional Analysis of Centrosomal Kinase Substrates in <i>Drosophila melanogaster</i> Reveals a New Function of the Nuclear Envelope Component Otefin in Cell Cycle Progression

Habermann, Karin; Mirgorodskaya, Ekaterina; Gobom, Johan; Lehmann, Verena; Müller, Hannah; Blümlein, Katharina; Deery, Michael J.; Czogiel, Irina; Erdmann, Christoph; Ralser, Markus; Kries, Jens Peter von; Lange, Bodo

doi:10.1128/mcb.00814-12

Cited by 13 publications

(14 citation statements)

References 70 publications

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“…In the INM, it partly colocalized (Fig. 1B) with the INM marker Otefin [24,25]. Both clonal and immunostaining analyses using different Schlank antibodies convincingly showed the presence of nuclear Schlank in the fat body.…”

Section: Discussionmentioning

confidence: 65%

“…1A). When we costained with anti-Schlank and either anti-Lamin Dm0 (Lam Dm0), which stains Lamin at the INM [23] or anti-Otefin, a LEM (LAP2, Emerin, and MAN1) domain protein [24,25], we detected Schlank protein in the nucleus and at the INM. Surprisingly, Schlank was mostly found underneath Lamin, in Otefin positive compartments in the nuclear periphery and in the nucleoplasmic structures in the fat body ( Fig.…”

Section: Detection Of Endogenous Schlank Protein In the Nucleus Of Famentioning

confidence: 99%

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Nuclear Drosophila CerS Schlank regulates lipid homeostasis via the homeodomain, independent of the lag1p motif

et al. 2016

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Edited by Laszlo NagyDrosophila Ceramide Synthase (CerS) Schlank regulates both ceramide synthesis and fat metabolism. Schlank contains a catalytic lag1p motif and, like many CerS in other species, a homeodomain of unknown function. Here, we show that the Drosophila CerS Schlank is imported into the nucleus and requires two nuclear localization signals (NLSs) within its homeodomain and functional Importin-b import machinery. Expression of Schlank variants containing the homeodomain without functional lag1p motif rescued the fat metabolism phenotype of schlank mutants whereas a variant with a mutated NLS site did not rescue. Thus, the homeodomain of Schlank is involved in the regulation of lipid metabolism independent of the catalytic lag1p motif.Keywords: ceramide synthase; Drosophila; homeodomain; importin; lipid homeostasis; nuclear function Ceramides and sphingolipids are key intermediates in diverse cellular processes for growth, stress resistance, apoptosis, neurodegeneration, insulin function, and lipid homeostasis [1][2][3][4][5].In synthesis of ceramide, Ceramide Synthases (CerSs) acylate a sphingoid long-chain base (LCB) with a fatty acyl-CoA of distinct length to generate (dihydro) ceramide, the backbone of all complex sphAbbreviations aa, amino acid; ASAH1, acid ceramidase; ATF2, activating transcription factor 2; cDNA, complementary DNA; CerSs, ceramide synthases; dsRNA, double-stranded RNA; ER, endoplasmic reticulum; Gal4, galactose-responsive transcription factor; GFP, green fluorescent protein; GlcT-1, glucosylceramide synthase; H215D, change in a histidine at position 215 of schlank into glutamate; INM, inner nuclear membrane;

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Section: Discussionmentioning

confidence: 65%

Section: Detection Of Endogenous Schlank Protein In the Nucleus Of Famentioning

confidence: 99%

Nuclear Drosophila CerS Schlank regulates lipid homeostasis via the homeodomain, independent of the lag1p motif

et al. 2016

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“…The dynamic pattern of Polo kinase in D. melanogaster embryos is consistent with many target substrates (Moutinho-Santos et al 1999). Several Polo kinase substrates have been identified in D. melanogaster (Bodenmiller et al 2007;Zhai et al 2008;Santamaria et al 2011;Habermann et al 2012) but most targets remain unknown. In mammals, two consensus phosphorylation sequences have been described for the orthologous Polo-like Kinase1 (Plk1).…”

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confidence: 71%

An Amino-Terminal Polo Kinase Interaction Motif Acts in the Regulation of Centrosome Formation and Reveals a Novel Function for centrosomin (cnn) in Drosophila

Eisman¹,

Phelps²

2015

Genetics

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The formation of the pericentriolar matrix (PCM) and a fully functional centrosome in syncytial Drosophila melanogaster embryos requires the rapid transport of Cnn during initiation of the centrosome replication cycle. We show a Cnn and Polo kinase interaction is apparently required during embryogenesis and involves the exon 1A-initiating coding exon, suggesting a subset of Cnn splice variants is regulated by Polo kinase. During PCM formation exon 1A Cnn-Long Form proteins likely bind Polo kinase before phosphorylation by Polo for Cnn transport to the centrosome. Loss of either of these interactions in a portion of the total Cnn protein pool is sufficient to remove native Cnn from the pool, thereby altering the normal localization dynamics of Cnn to the PCM. Additionally, Cnn-Short Form proteins are required for polar body formation, a process known to require Polo kinase after the completion of meiosis. Exon 1A Cnn-LF and Cnn-SF proteins, in conjunction with Polo kinase, are required at the completion of meiosis and for the formation of functional centrosomes during early embryogenesis. KEYWORDS Drosophila; centrosomin (cnn); Polo kinase; meiosis; cleavage mitosis T HE animal centrosome is the dominant microtubuleorganizing center (MTOC) in cells and is composed of a centrally located pair of centrioles surrounded by the pericentriolar matrix (PCM). In Drosophila melanogaster, Centrosomin (Cnn) is the most abundant protein in the PCM (Lange et al. 2000) and is required for the localization of many other PCM components during mitosis (Megraw et al. 1999; VaizelOhayon and Schejter 1999). The cnn gene is transcriptionally complex, using three promoters with unique initiating coding exons associated with multiple splice variants making up two protein families (Eisman et al. 2009), contrary to its frequent depiction as a single-protein gene. Much of the work on Cnn has been done on the Cnn-Long Form (Cnn-LF) protein family, containing a highly conserved KFC eukaryotic motif required for g-tubulin localization (Fu and Glover 2012) and three insect-specific conserved motifs (Eisman and Kaufman 2013). The gene also utilizes two of its promoters to produce several splice variants of the Cnn-Short Form (Cnn-SF) protein family, which contain the conserved KFC motif (Eisman et al. 2009) and a rapidly evolving coiled-coil carboxy terminus (Eisman and Kaufman 2013). While all members of either protein family are similar, their amino termini vary with respect to the promoter utilized during transcription.Many studies have shown Cnn-LF proteins are the predominant component of the PCM at mitotic centrosomes, based on immunostaining and the live localization of ectopically expressed GFP::Cnn-PA fusion proteins. In syncytial embryos Cnn-LF protein is always present at centrosomes, whereas in cells the protein is detectable only during mitosis. Two studies have shown Cnn-LF localizes to the centrosome during the peak of flare formation, which occurs at the onset of centrosome replication and continues during S phase and ...

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“…This prediction is suggested by our findings that ote −/− gonads contain fewer PGCs compared to ote +/+ gonads (Figure 2, data not shown) and that bam tumors in the absence of Ote are small compared to bam tumors with Ote (Figure 5A). A role of Ote in the cell cycle may be linked to its requirement in nuclear envelope reformation following mitosis (Ashery-Padan et al, 1997) or its localization to the centrosome (Habermann et al, 2012). Second, Ote may be required for germ cell differentiation.…”

Section: Discussionmentioning

confidence: 99%

The Drosophila Nuclear Lamina Protein Otefin Is Required for Germline Stem Cell Survival

et al. 2013

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Summary LEM domain (LEM-D) proteins are components of an extensive protein network that assembles beneath the inner nuclear envelope. Defects in LEM-D proteins cause tissue-restricted human diseases associated with altered stem cell homeostasis. Otefin (Ote) is a Drosophila LEM-D protein that is intrinsically required for female germline stem cells (GSCs) maintenance. Previous studies linked Ote loss with transcriptional activation of the key differentiation gene, bag-of-marbles (bam), leading to the model that Ote tethers the bam gene to the nuclear periphery for gene silencing. Using genetic and phenotypic analyses of multiple ote−/− backgrounds, we obtained evidence that is inconsistent with this model. We show that bam repression is maintained in ote−/− GSCs and that germ cell loss persists in ote−/−, bam−/− mutants, together demonstrating that GSC loss is independent of bam transcription. We show the primary defect in ote−/− GSCs is a block of differentiation, which ultimately leads to germ cell death.

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Functional Analysis of Centrosomal Kinase Substrates in Drosophila melanogaster Reveals a New Function of the Nuclear Envelope Component Otefin in Cell Cycle Progression

Cited by 13 publications

References 70 publications

Nuclear Drosophila CerS Schlank regulates lipid homeostasis via the homeodomain, independent of the lag1p motif

Nuclear Drosophila CerS Schlank regulates lipid homeostasis via the homeodomain, independent of the lag1p motif

An Amino-Terminal Polo Kinase Interaction Motif Acts in the Regulation of Centrosome Formation and Reveals a Novel Function for centrosomin (cnn) in Drosophila

The Drosophila Nuclear Lamina Protein Otefin Is Required for Germline Stem Cell Survival

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