The Experts below are selected from a list of 1122 Experts worldwide ranked by ideXlab platform
Thomas Giannakouros - One of the best experts on this subject based on the ideXlab platform.
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the o β linked n acetylglucosaminylation of the <B>LaminB> B Receptor and its impact on dna Binding and phosphorylation
Biochimica et Biophysica Acta, 2018Co-Authors: Caroline Smetnocca, Eleni Nikolakaki, Adeline Page, Francoisxavier Cantrelle, Isabelle Landrieu, Thomas GiannakourosAbstract:ABstract <B>LaminB> B Receptor (LBR) is an integral protein of the interphase inner nuclear memBrane that is implicated in chromatin anchorage to the nuclear envelope. Phosphorylation of a stretch of arginine-serine (RS) dipeptides in the amino-terminal nucleoplasmic domain of LBR regulates the interactions of the Receptor with other nuclear proteins, DNA and RNA and thus modulates tethering of heterochromatin to the nuclear envelope. While phosphorylation has Been extensively studied, very little is known aBout other post-translational modifications of the protein. There is only one report on the O-β-linked N-acetyl-glucosaminylation (O-GlcNAcylation) of a serine residue downstream of the RS domain of rat LBR. In the present study we identify additional O-GlcNAcylation sites By using as suBstrates of O-β-N-acetylglucosaminyltransferase (OGT) a set of peptides containing the entire LBR RS domain or parts of it as well as flanking sequences. The in vitro activity of OGT was assessed By tandem mass spectrometry and NMR spectroscopy. Furthermore, we provide evidence that O-GlcNAcylation hampers DNA Binding while it marginally affects RS domain phosphorylation mediated By SRPK1, Akt2 and cdk1 kinases. General significance Our methodology providing a quantitative description of O-GlcNAc patterns Based on a comBination of mass spectrometry and high resolution NMR spectroscopy on short peptide suBstrates allows suBsequent functional analyses. Hence, our approach is of general interest to a wide audience of Biologists aiming at deciphering the functional role of O-GlcNAc glycosylation and its crosstalk with phosphorylation.
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<B>LaminB> B Receptor: Interplay Between Structure, Function and Localization.
Cells, 2017Co-Authors: Eleni Nikolakaki, Ilias Mylonis, Thomas GiannakourosAbstract:<B>LaminB> B Receptor (LBR) is an integral protein of the inner nuclear memBrane, containing a hydrophilic N-terminal end protruding into the nucleoplasm, eight hydrophoBic segments that span the memBrane and a short, nucleoplasmic C-terminal tail. Two seemingly unrelated functions have Been attriButed to LBR. Its N-terminal domain tethers heterochromatin to the nuclear periphery, thus contriButing to the shape of interphase nuclear architecture, while its transmemBrane domains exhiBit sterol reductase activity. Mutations within the transmemBrane segments result in defects in cholesterol synthesis and are associated with diseases such as the Pelger–Huet anomaly and GreenBerg skeletal dysplasia, whereas no such harmful mutations related to the anchoring properties of LBR have Been reported so far. Recent evidence suggests a dynamic regulation of LBR expression levels, structural organization, localization and function, in response to various signals. The molecular mechanisms underlying this dynamic Behavior have not yet Been fully unraveled. Here, we provide an overview of the current knowledge of the interplay Between the structure, function and localization of LBR, and hint at the interconnection of the two distinct functions of LBR.
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<B>LaminB> B Receptor: Interplay Between Structure, Function and Localization
MDPI AG, 2017Co-Authors: Eleni Nikolakaki, Ilias Mylonis, Thomas GiannakourosAbstract:<B>LaminB> B Receptor (LBR) is an integral protein of the inner nuclear memBrane, containing a hydrophilic N-terminal end protruding into the nucleoplasm, eight hydrophoBic segments that span the memBrane and a short, nucleoplasmic C-terminal tail. Two seemingly unrelated functions have Been attriButed to LBR. Its N-terminal domain tethers heterochromatin to the nuclear periphery, thus contriButing to the shape of interphase nuclear architecture, while its transmemBrane domains exhiBit sterol reductase activity. Mutations within the transmemBrane segments result in defects in cholesterol synthesis and are associated with diseases such as the Pelger–Huët anomaly and GreenBerg skeletal dysplasia, whereas no such harmful mutations related to the anchoring properties of LBR have Been reported so far. Recent evidence suggests a dynamic regulation of LBR expression levels, structural organization, localization and function, in response to various signals. The molecular mechanisms underlying this dynamic Behavior have not yet Been fully unraveled. Here, we provide an overview of the current knowledge of the interplay Between the structure, function and localization of LBR, and hint at the interconnection of the two distinct functions of LBR
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srpk1 and akt protein kinases phosphorylate the rs domain of <B>LaminB> B Receptor with distinct specificity a comBined Biochemical and in silico approach
PLOS ONE, 2016Co-Authors: Nikolaos Voukkalis, Eleni Nikolakaki, Metaxia Vlassi, Maria Koutroumani, Christoforos Zarkadas, Thomas GiannakourosAbstract:Activated Akt has Been previously implicated in acting on RS domain-containing proteins. However, it has Been questioned whether its action is direct or it is mediated By co-existing SR kinase activity. To address this issue we studied in detail the phosphorylation of <B>LaminB> B Receptor (LBR) By Akt. Using synthetic peptides and a set of recomBinant proteins expressing mutants of the LBR RS domain we now demonstrate that while all serines of the RS domain represent more or less equal phosphoacceptor sites for SRPK1, Ser80 and Ser82 are mainly targeted By Akt. 3D-modeling comBined with molecular dynamics (MD) simulations show that amongst short, overlapping LBR RS-containing peptides complying with the minimum Akt recognition consensus sequence, only those Bearing phosphosites either at Ser80 or Ser82 are aBle to fit into the active site of Akt, at least as effectively as its known suBstrate, GSK3-β. ComBined our results provide evidence that Akt kinases directly phosphorylate an RS domain-containing protein and that Both the residues N-terminal the phosphosite and at position +1 are essential for Akt specificity, with the latter suBstrate position Being compatiBle with the arginine residue of RS-repeats.
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phosphorylation of the arginine serine repeats of <B>LaminB> B Receptor By srpk1 insights from molecular dynamics simulations
Biochimica et Biophysica Acta, 2012Co-Authors: Diamantis Sellis, Thomas Giannakouros, Victoria Drosou, Dimitrios Vlachakis, Nikolas Voukkalis, Metaxia VlassiAbstract:ABstract Background Arginine/serine (RS) repeats are found in several proteins in metazoans with a wide variety of functions, many of which are regulated By SR protein kinase 1 (SRPK1)-mediated phosphorylation. <B>LaminB> B Receptor (LBR) is such a protein implicated in chromatin anchorage to the nuclear envelope. Methods Molecular dynamics simulations were used to investigate the conformation of two LBR peptides containing four (human-) and five (turkey-orthologue) consecutive RS dipeptides, in their unphosphorylated and phosphorylated forms and of a conserved peptide, in isolation and in complex with SRPK1. GST pull-down assays were employed to study LBR interactions. Results Unphosphorylated RS repeats adopt short, transient helical conformations, whereas serine phosphorylation induces Arginine-claw-like structures. The SRSRSRSPGR peptide, overlapping with the LBR RS repeats, docks into the known, acidic docking groove of SRPK1, in an extended conformation. Phosphorylation By SRPK1 is necessary for the association of LBR with histone H3. Conclusions The C-terminal region of the LBR RS domain constitutes a recognition platform for SRPK1, which uses the same recognition mechanism for LBR as for suBstrates with long RS domains. This docking may promote unfolding of the RS repeats destined to Be phosphorylated. Phosphorylation induces Arginine-claw-like conformations, irrespective of the RS-repeat length, that may facilitate interactions with Basic partners. General significance Our results shed light on the conformational preferences of an important class of repeats Before and after their phosphorylation and support the idea that even short RS domains may Be constituents of recognition platforms for SRPK1, thus adding to knowledge towards a full understanding of their phosphorylation mechanism.
Spyros D. Georgatos - One of the best experts on this subject based on the ideXlab platform.
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Dynamics and Structure-Function Relationships of the <B>LaminB> B Receptor (LBR).
PloS one, 2017Co-Authors: Ioannis Giannios, Eleftheria Chatzantonaki, Spyros D. GeorgatosAbstract:The <B>LaminB> B Receptor (LBR) is a multi-spanning memBrane protein of the inner nuclear memBrane that is often employed as a "reporter" of nuclear envelope dynamics. We show here that the diffusional moBility of full-length LBR exhiBits significant regional variation along the nuclear envelope, consistent with the existence of discrete LBR microdomains and the occurrence of multiple, asymmetrically-spaced anastomoses along the nuclear envelope-endoplasmic reticulum interface. Interestingly, a commonly used fusion protein that contains the amino-terminal region and the first transmemBrane domain of LBR exhiBits reduced moBility at the nuclear envelope, But Behaves similarly to full-length LBR in the endoplasmic reticulum. On the other hand, carBoxy-terminally truncated mutants that retain the first four transmemBrane domains and a part or the whole of the amino-terminal region of LBR are generally hyper-moBile. These results suggest that LBR dynamics is structure and compartment specific. They also indicate that native LBR is proBaBly "configured" By long-range interactions that involve the loops Between adjacent transmemBrane domains and parts of the amino-terminal region.
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<B>LaminB> A, <B>LaminB> B, and <B>LaminB> B Receptor Analogues in Yeast
2013Co-Authors: Spyros D. Georgatos, I Maroulakou, G BlobelAbstract:ABstract. Previous studies have shown that turkey erythrocyte <B>LaminB> B is anchored to the nuclear envelope via a 58-kD integral memBrane protein termed p58 or <B>LaminB> B Receptor (Worman H. J., J. Yuan, G. BloBel, and S. D. Georgatos. 1988. Proc. Natl. Acad. Sci. USA. 85:8531-8534). We now identify a p58 analogue in the yeast Saccharomyces cerevisiae. Turkey erythrocyte <B>LaminB> B Binds to yeast ureaextracted nuclear envelopes with high affinity, associating predominantly with a 58-kD polypeptide. This yeast polypeptide is recognized By polyclonal antiBodies against turkey p58, partitions entirely with the nuclear fraction, remains memBrane Bound after urea extraction of the nuclear envelopes, and is structurally similar to turkey p58 By peptide mapping criteria
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solution structure and molecular interactions of <B>LaminB> B Receptor tudor domain
Journal of Biological Chemistry, 2012Co-Authors: Stamatis Liokatis, Spyros D. Georgatos, Ioannis Giannios, Christian Edlich, Katerina Soupsana, Parthena Panagiotidou, Konstantinos Tripsianes, Michael Sattler, Anastasia S PolitouAbstract:<B>LaminB> B Receptor (LBR) is a polytopic protein of the nuclear envelope thought to connect the inner nuclear memBrane with the underlying nuclear <B>LaminB>a and peripheral heterochromatin. To Better understand the function of this protein, we have examined in detail its nucleoplasmic region, which is predicted to harBor a Tudor domain (LBR-TD). Structural analysis By multidimensional NMR spectroscopy estaBlishes that LBR-TD indeed adopts a classical β-Barrel Tudor fold in solution, which, however, features an incomplete aromatic cage. Removal of LBR-TD renders LBR more moBile at the plane of the nuclear envelope, But the isolated module does not Bind to nuclear <B>LaminB>s, heterochromatin proteins (MeCP2), and nucleosomes, nor does it associate with methylated Arg/Lys residues through its aromatic cage. Instead, LBR-TD exhiBits tight and stoichiometric Binding to the “histone-fold” region of unassemBled, free histone H3, suggesting an interesting role in histone assemBly. Consistent with such a role, roBust Binding to native nucleosomes is oBserved when LBR-TD is extended toward its carBoxyl terminus, to include an area rich in Ser-Arg residues. The Ser-Arg region, alone or in comBination with LBR-TD, Binds Both unassemBled and assemBled H3/H4 histones, suggesting that the TD/RS interface may operate as a “histone chaperone-like platform.”
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The Inner Nuclear MemBrane Protein <B>LaminB> B Receptor Forms Distinct Microdomains and Links Epigenetically Marked Chromatin to the Nuclear Envelope
The Journal of biological chemistry, 2004Co-Authors: Dimitra Makatsori, Leonard D. Shultz, Niki Kourmouli, Hara Polioudaki, Kelvin Mclean, Panayiotis A. Theodoropoulos, Prim B. Singh, Spyros D. GeorgatosAbstract:Using heterochromatin-enriched fractions, we have detected specific Binding of mononucleosomes to the N-terminal domain of the inner nuclear memBrane protein <B>LaminB> B Receptor. Mass spectrometric analysis reveals that LBR-associated particles contain complex patterns of methylated/acetylated histones and are devoid of "euchromatic" epigenetic marks. LBR Binds heterochromatin as a higher oligomer and forms distinct nuclear envelope microdomains in vivo. The organization of these memBrane assemBlies is affected significantly in heterozygous ic (ichthyosis) mutants, resulting in a variety of structural aBnormalities and nuclear defects.
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Mitotic Phosphorylation of the <B>LaminB> B Receptor By a Serine/Arginine Kinase and p34cdc2
The Journal of biological chemistry, 1997Co-Authors: Eleni Nikolakaki, Spyros D. Georgatos, Juergen Meier, George Simos, Thomas GiannakourosAbstract:ABstract The <B>LaminB> B Receptor (LBR) is an integral protein of the inner nuclear memBrane that is modified at interphase By a nuclear envelope-Bound protein kinase. This enzyme (RS kinase) specifically phosphorylates arginine-serine dipeptide motifs located at the NH2-terminal domain of LBR and regulates its interactions with other nuclear envelope proteins. To compare the phosphorylation state of LBR during interphase and mitosis, we performed phosphopeptide mapping of in vitro and in vivo 32P-laBeled LBR and analyzed a series of recomBinant proteins and synthetic peptides. Our results show that LBR undergoes two types of mitotic phosphorylation mediated By the RS and the p34cdc2 protein kinases, respectively. The RS kinase modifies similar sites at interphase and mitosis (i.e. Ser76, Ser78, Ser80, Ser82, Ser84), whereas p34cdc2 mainly phosphorylates Ser71. These findings clarify the phosphorylation state of LBR during the cell cycle and provide new information for understanding the mechanisms responsiBle for nuclear envelope assemBly and disassemBly.
Richard M Pauli - One of the best experts on this subject based on the ideXlab platform.
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an anadysplasia like spontaneously remitting spondylometaphyseal dysplasia secondary to <B>LaminB> B Receptor lBr gene mutations further definition of the phenotypic heterogeneity of lBr Bone dysplasias
American Journal of Medical Genetics Part A, 2015Co-Authors: Nara Sobreira, Peggy Modaff, Gary Steel, Jing You, Sonia Nanda, David Valle, Julie Hooverfong, Richard M PauliAbstract:We descriBe a Boy who has an anadysplasia-like spondylometaphyseal dysplasia. By whole exome sequencing he was shown to have compound heterozygous mutations of LBR that codes for the <B>LaminB> B Receptor. He shares many similarities with a case previously descriBed, But in whom the early natural history could not Be estaBlished [Borovik et al., 2013]. Thus, in addition to GreenBerg dysplasia (a perinatal lethal disorder), homozygosity or compound heterozygosity of mutations in LBR can result in a mild, spontaneously regressing Bone dysplasia. © 2014 Wiley Periodicals, Inc.
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An anadysplasia-like, spontaneously remitting spondylometaphyseal dysplasia secondary to <B>LaminB> B Receptor (LBR) gene mutations: further definition of the phenotypic heterogeneity of LBR-Bone dysplasias.
American journal of medical genetics. Part A, 2014Co-Authors: Nara Sobreira, Peggy Modaff, Gary Steel, Jing You, Sonia Nanda, Julie Hoover-fong, David Valle, Richard M PauliAbstract:We descriBe a Boy who has an anadysplasia-like spondylometaphyseal dysplasia. By whole exome sequencing he was shown to have compound heterozygous mutations of LBR that codes for the <B>LaminB> B Receptor. He shares many similarities with a case previously descriBed, But in whom the early natural history could not Be estaBlished [Borovik et al., 2013]. Thus, in addition to GreenBerg dysplasia (a perinatal lethal disorder), homozygosity or compound heterozygosity of mutations in LBR can result in a mild, spontaneously regressing Bone dysplasia.
Eleni Nikolakaki - One of the best experts on this subject based on the ideXlab platform.
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the o β linked n acetylglucosaminylation of the <B>LaminB> B Receptor and its impact on dna Binding and phosphorylation
Biochimica et Biophysica Acta, 2018Co-Authors: Caroline Smetnocca, Eleni Nikolakaki, Adeline Page, Francoisxavier Cantrelle, Isabelle Landrieu, Thomas GiannakourosAbstract:ABstract <B>LaminB> B Receptor (LBR) is an integral protein of the interphase inner nuclear memBrane that is implicated in chromatin anchorage to the nuclear envelope. Phosphorylation of a stretch of arginine-serine (RS) dipeptides in the amino-terminal nucleoplasmic domain of LBR regulates the interactions of the Receptor with other nuclear proteins, DNA and RNA and thus modulates tethering of heterochromatin to the nuclear envelope. While phosphorylation has Been extensively studied, very little is known aBout other post-translational modifications of the protein. There is only one report on the O-β-linked N-acetyl-glucosaminylation (O-GlcNAcylation) of a serine residue downstream of the RS domain of rat LBR. In the present study we identify additional O-GlcNAcylation sites By using as suBstrates of O-β-N-acetylglucosaminyltransferase (OGT) a set of peptides containing the entire LBR RS domain or parts of it as well as flanking sequences. The in vitro activity of OGT was assessed By tandem mass spectrometry and NMR spectroscopy. Furthermore, we provide evidence that O-GlcNAcylation hampers DNA Binding while it marginally affects RS domain phosphorylation mediated By SRPK1, Akt2 and cdk1 kinases. General significance Our methodology providing a quantitative description of O-GlcNAc patterns Based on a comBination of mass spectrometry and high resolution NMR spectroscopy on short peptide suBstrates allows suBsequent functional analyses. Hence, our approach is of general interest to a wide audience of Biologists aiming at deciphering the functional role of O-GlcNAc glycosylation and its crosstalk with phosphorylation.
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<B>LaminB> B Receptor: Interplay Between Structure, Function and Localization.
Cells, 2017Co-Authors: Eleni Nikolakaki, Ilias Mylonis, Thomas GiannakourosAbstract:<B>LaminB> B Receptor (LBR) is an integral protein of the inner nuclear memBrane, containing a hydrophilic N-terminal end protruding into the nucleoplasm, eight hydrophoBic segments that span the memBrane and a short, nucleoplasmic C-terminal tail. Two seemingly unrelated functions have Been attriButed to LBR. Its N-terminal domain tethers heterochromatin to the nuclear periphery, thus contriButing to the shape of interphase nuclear architecture, while its transmemBrane domains exhiBit sterol reductase activity. Mutations within the transmemBrane segments result in defects in cholesterol synthesis and are associated with diseases such as the Pelger–Huet anomaly and GreenBerg skeletal dysplasia, whereas no such harmful mutations related to the anchoring properties of LBR have Been reported so far. Recent evidence suggests a dynamic regulation of LBR expression levels, structural organization, localization and function, in response to various signals. The molecular mechanisms underlying this dynamic Behavior have not yet Been fully unraveled. Here, we provide an overview of the current knowledge of the interplay Between the structure, function and localization of LBR, and hint at the interconnection of the two distinct functions of LBR.
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<B>LaminB> B Receptor: Interplay Between Structure, Function and Localization
MDPI AG, 2017Co-Authors: Eleni Nikolakaki, Ilias Mylonis, Thomas GiannakourosAbstract:<B>LaminB> B Receptor (LBR) is an integral protein of the inner nuclear memBrane, containing a hydrophilic N-terminal end protruding into the nucleoplasm, eight hydrophoBic segments that span the memBrane and a short, nucleoplasmic C-terminal tail. Two seemingly unrelated functions have Been attriButed to LBR. Its N-terminal domain tethers heterochromatin to the nuclear periphery, thus contriButing to the shape of interphase nuclear architecture, while its transmemBrane domains exhiBit sterol reductase activity. Mutations within the transmemBrane segments result in defects in cholesterol synthesis and are associated with diseases such as the Pelger–Huët anomaly and GreenBerg skeletal dysplasia, whereas no such harmful mutations related to the anchoring properties of LBR have Been reported so far. Recent evidence suggests a dynamic regulation of LBR expression levels, structural organization, localization and function, in response to various signals. The molecular mechanisms underlying this dynamic Behavior have not yet Been fully unraveled. Here, we provide an overview of the current knowledge of the interplay Between the structure, function and localization of LBR, and hint at the interconnection of the two distinct functions of LBR
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srpk1 and akt protein kinases phosphorylate the rs domain of <B>LaminB> B Receptor with distinct specificity a comBined Biochemical and in silico approach
PLOS ONE, 2016Co-Authors: Nikolaos Voukkalis, Eleni Nikolakaki, Metaxia Vlassi, Maria Koutroumani, Christoforos Zarkadas, Thomas GiannakourosAbstract:Activated Akt has Been previously implicated in acting on RS domain-containing proteins. However, it has Been questioned whether its action is direct or it is mediated By co-existing SR kinase activity. To address this issue we studied in detail the phosphorylation of <B>LaminB> B Receptor (LBR) By Akt. Using synthetic peptides and a set of recomBinant proteins expressing mutants of the LBR RS domain we now demonstrate that while all serines of the RS domain represent more or less equal phosphoacceptor sites for SRPK1, Ser80 and Ser82 are mainly targeted By Akt. 3D-modeling comBined with molecular dynamics (MD) simulations show that amongst short, overlapping LBR RS-containing peptides complying with the minimum Akt recognition consensus sequence, only those Bearing phosphosites either at Ser80 or Ser82 are aBle to fit into the active site of Akt, at least as effectively as its known suBstrate, GSK3-β. ComBined our results provide evidence that Akt kinases directly phosphorylate an RS domain-containing protein and that Both the residues N-terminal the phosphosite and at position +1 are essential for Akt specificity, with the latter suBstrate position Being compatiBle with the arginine residue of RS-repeats.
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temporal association of protamine 1 with the inner nuclear memBrane protein <B>LaminB> B Receptor during spermiogenesis
Journal of Biological Chemistry, 2004Co-Authors: Ilias Mylonis, Eleni Nikolakaki, Victoria Drosou, Stefano Brancorsini, Paolo Sassonecorsi, Thomas GiannakourosAbstract:During mammalian spermiogenesis, histones are replaced By transition proteins, which are in turn replaced By protamines P1 and P2. P1 protamine contains a short arginine/serine-rich (RS) domain that is highly phosphorylated Before Being deposited into sperm chromatin and almost completely dephosphorylated during sperm maturation. We now demonstrate that, in elongating spermatids, this phosphorylation is required for the temporal association of P1 protamine with <B>LaminB> B Receptor (LBR), an inner nuclear memBrane protein that also possesses a stretch of RS dipeptides at its nucleoplasmic NH2-terminal domain. Previous studies have shown that the cellular protein p32 also Binds tightly to the unmodified RS domain of LBR. Extending those findings, we now present evidence that p32 prevents phosphorylation of LBR and furthermore that dissociation of this protein precedes P1 protamine association. Our data suggest that docking of protamine 1 to the nuclear envelope is an important intermediate step in spermiogenesis and reveal a novel role for SR protein kinases and p32.
George Simos - One of the best experts on this subject based on the ideXlab platform.
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Mitotic Phosphorylation of the <B>LaminB> B Receptor By a Serine/Arginine Kinase and p34cdc2
The Journal of biological chemistry, 1997Co-Authors: Eleni Nikolakaki, Spyros D. Georgatos, Juergen Meier, George Simos, Thomas GiannakourosAbstract:ABstract The <B>LaminB> B Receptor (LBR) is an integral protein of the inner nuclear memBrane that is modified at interphase By a nuclear envelope-Bound protein kinase. This enzyme (RS kinase) specifically phosphorylates arginine-serine dipeptide motifs located at the NH2-terminal domain of LBR and regulates its interactions with other nuclear envelope proteins. To compare the phosphorylation state of LBR during interphase and mitosis, we performed phosphopeptide mapping of in vitro and in vivo 32P-laBeled LBR and analyzed a series of recomBinant proteins and synthetic peptides. Our results show that LBR undergoes two types of mitotic phosphorylation mediated By the RS and the p34cdc2 protein kinases, respectively. The RS kinase modifies similar sites at interphase and mitosis (i.e. Ser76, Ser78, Ser80, Ser82, Ser84), whereas p34cdc2 mainly phosphorylates Ser71. These findings clarify the phosphorylation state of LBR during the cell cycle and provide new information for understanding the mechanisms responsiBle for nuclear envelope assemBly and disassemBly.
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mitotic phosphorylation of the <B>LaminB> B Receptor By a serine arginine kinase and p34cdc2
Journal of Biological Chemistry, 1997Co-Authors: Eleni Nikolakaki, Spyros D. Georgatos, Juergen Meier, George Simos, Thomas GiannakourosAbstract:ABstract The <B>LaminB> B Receptor (LBR) is an integral protein of the inner nuclear memBrane that is modified at interphase By a nuclear envelope-Bound protein kinase. This enzyme (RS kinase) specifically phosphorylates arginine-serine dipeptide motifs located at the NH2-terminal domain of LBR and regulates its interactions with other nuclear envelope proteins. To compare the phosphorylation state of LBR during interphase and mitosis, we performed phosphopeptide mapping of in vitro and in vivo 32P-laBeled LBR and analyzed a series of recomBinant proteins and synthetic peptides. Our results show that LBR undergoes two types of mitotic phosphorylation mediated By the RS and the p34cdc2 protein kinases, respectively. The RS kinase modifies similar sites at interphase and mitosis (i.e. Ser76, Ser78, Ser80, Ser82, Ser84), whereas p34cdc2 mainly phosphorylates Ser71. These findings clarify the phosphorylation state of LBR during the cell cycle and provide new information for understanding the mechanisms responsiBle for nuclear envelope assemBly and disassemBly.
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The <B>LaminB> B Receptor (LBR) provides essential chromatin docking sites at the nuclear envelope.
The EMBO journal, 1996Co-Authors: Athina Pyrpasopoulou, Juergen Meier, Christèle Maison, George Simos, Spyros D. GeorgatosAbstract:Morphological studies have estaBlished that peripheral heterochromatin is closely associated with the nuclear envelope. The tight coupling of the two structures has Been attriButed to nuclear <B>LaminB>s and <B>LaminB>-associated proteins; however, it remains to Be determined which of these elements are essential and which play an auxiliary role in nuclear envelope-chromatin interactions. To address this question, we have used as a model system in vitro reconstituted vesicles assemBled from octyl glucoside-soluBilized nuclear envelopes. Comparing the chromosome Binding properties of normal, immunodepleted and chemically extracted vesicles, we have arrived at the conclusion that the principal chromatin anchorage site at the nuclear envelope is the <B>LaminB> B Receptor (LBR), a uBiquitous integral protein of the inner nuclear memBrane. Consistent with this interpretation, purified LBR Binds directly to chromatin fragments and decorates the surface of chromosomes in a distinctive Banding pattern.
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characterization of p18 a component of the <B>LaminB> B Receptor complex and a new integral memBrane protein of the avian erythrocyte nuclear envelope
Journal of Biological Chemistry, 1996Co-Authors: George Simos, Christèle Maison, Spyros D. GeorgatosAbstract:Employing avian erythrocytes, we have previously isolated a multimeric complex consisting of the <B>LaminB> B Receptor (LBR, or p58), the nuclear <B>LaminB>s, an LBR-specific kinase, a 34-kDa protein, and an 18-kDa polypeptide termed p18. As the LBR kinase and the 34-kDa component have Been recently characterized, we now proceed in the characterization of p18. We show here that p18 is an integral memBrane protein specific to the erythrocyte nuclear envelope which Binds to LBR and B-type <B>LaminB>s. NH2-terminal sequencing indicates that p18 is distinct from other nuclear envelope components, But has similarity to the mitochondrial isoquinoline-Binding protein. In situ analysis By immunoelectron microscopy and examination of digitonin-permeaBilized cells By indirect immunofluorescence show that p18, unlike LBR and other <B>LaminB>-Binding proteins, is equally distriButed Between the inner and outer nuclear memBrane. Furthermore, cycloheximide inhiBition experiments reveal that the fraction of p18 that resides in the outer nuclear memBrane does not represent nascent chains en route to the inner nuclear memBrane, But rather material in equiliBrium with the p18 that partitions with the inner nuclear memBrane. The paradigm of p18 suggests that transmemBrane complexes formed By the nuclear <B>LaminB>s and LBR provide potential docking sites for integral memBrane proteins of the nuclear envelope that equiliBrate Between the rough endoplasmic reticulum and the inner nuclear memBrane.
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a nuclear envelope associated kinase phosphorylates arginine serine motifs and modulates interactions Between the <B>LaminB> B Receptor and other nuclear proteins
Journal of Biological Chemistry, 1996Co-Authors: Eleni Nikolakaki, Spyros D. Georgatos, George Simos, Thomas GiannakourosAbstract:ABstract Previous studies have identified a suBassemBly of nuclear envelope proteins, termed “the LBR complex.” This complex includes the <B>LaminB> B Receptor protein (LBR or p58), a kinase which phosphorylates LBR in a constitutive fashion (LBR kinase), the nuclear <B>LaminB>s A and B, an 18-kDa polypeptide (p18), and a 34-kDa protein (p34/p32). The latter polypeptide has Been shown to interact with the HIV-1 proteins Rev and Tat and with the splicing factor 2 (SF2). Using recomBinant proteins produced in Bacteria and synthetic peptides representing different regions of LBR, we now show that the LBR kinase modifies specifically arginine-serine (RS) dipeptide motifs located at the nucleoplasmic, NH-terminal domain of LBR and in memBers of the SR family of splicing factors. Furthermore, we show that the NH-terminal domain of LBR Binds to p34/p32, whereas a mutated domain lacking the RS region does not. Phosphorylation of LBR By the RS kinase completely aBolishes Binding of p34/p32, suggesting that this enzyme regulates interactions among the components of the LBR complex.
