In Vitro Modulation of the Interaction between HA95 and LAP2β by cAMP Signaling

Martins, Sandra; Marstad, and Anne; Collas, Philippe

doi:10.1021/bi0350699

Cited by 7 publications

(6 citation statements)

References 35 publications

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“…Subsequent dephosphorylation of BAF Ser-4 would allow BAF to serve positive roles in recruiting both emerin and lamins to the reforming nuclear envelope, as shown in C. elegans embryos and human cells Dechat et al, 2004). Many of BAF's known binding partners at the nuclear envelope are also mitotically phosphorylated (Ellis et al, 1998;Foisner, 2003;Gajewski et al, 2004;Martins et al, 2003). Thus, our results collectively support models in which phosphorylation and dephosphorylation at Ser-4 regulates BAF activity during both interphase and mitosis.…”

Section: Discussionsupporting

confidence: 79%

Barrier-to-Autointegration Factor Phosphorylation on Ser-4 Regulates Emerin Binding to Lamin A In Vitro and Emerin Localization In Vivo

Bengtsson

Wilson

2006

MBoC

116

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Barrier-to-autointegration factor (BAF) is a conserved 10-kDa chromatin protein essential in proliferating cells. BAF dimers bind double-stranded DNA, histone H3, histone H1.1, lamin A, and transcription regulators, plus emerin and other LEM-domain nuclear proteins. Two-dimensional gel analysis showed that endogenous human and Xenopus BAF are posttranslationally modified by phosphorylation and potentially other modifications and that they are hyperphosphorylated during mitosis. The invariant Ser-4 residue on BAF is a major site of phosphorylation during both interphase and mitosis. In HeLa cells that overexpressed the phosphomimetic BAF missense mutant S4E, but not S4A, emerin mislocalized from the nuclear envelope, suggesting Ser-4-nonphosphorylated BAF normally promotes emerin localization at the nuclear envelope. Supporting this model, wild-type BAF but not mutant S4E enhanced emerin binding to lamin A in vitro. Thus, Ser-4-unphosphorylated BAF has a positive role in localizing emerin; this role may be disease relevant because loss or mislocalization of emerin causes Emery-Dreifuss muscular dystrophy. Our findings further suggest Ser-4 phosphorylation inhibits BAF binding to emerin and lamin A, and thereby weakens emerin-lamin interactions during both mitosis and interphase.

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Section: Discussionsupporting

confidence: 79%

Barrier-to-Autointegration Factor Phosphorylation on Ser-4 Regulates Emerin Binding to Lamin A In Vitro and Emerin Localization In Vivo

Bengtsson

Wilson

2006

MBoC

116

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“…AKAPA8L (HA95) contains two C2H2-type zinc finger motifs and also localizes to the nucleus (25). Like AKAP95, it co-localizes with condensed chromosomes at metaphase, suggesting that the two nuclear proteins have related cellular functions (25)(26)(27)(28). A recent integrative system analysis of protein-protein interaction has mapped AKAPA8L as one of the components of TNFα/NFκB signaling pathway (29).…”

Section: Discussionmentioning

confidence: 99%

Characterization of gene expression regulated by American ginseng and ginsenoside Rg3 in human colorectal cancer cells

Luο¹,

Wang²,

Jin³

et al. 2008

Int J Oncol

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American ginseng (Panax quinquefolius L., Araliaceae) possesses anti-cancer potential and is one of the most commonly used herbal medicines in the United States. Ginsenoside Rg3, one of the saponins in American ginseng, has been shown to inhibit tumor growth. In this study, we sought to characterize the downstream genes targeted by American ginseng extracts in HCT-116 human colorectal cancer cells. We first demonstrated that the content of Rg3 in American ginseng steamed at 120°C for 2 h (referred to as S2h) was significantly increased when compared with that of the unsteamed ginseng. Both S2h and Rg3 exhibited antiproliferative effects on HCT-116 cells. Using the Affymetrix high density genechips containing more than 40,000 genes and ESTs, the gene expression profiling of HCT-116 cells were assayed. Microarray data indicated that the expression levels of 76 genes were changed significantly after treatment with S2h or Rg3, whereby it was found that 52 of the 76 genes were up-regulated while the remaining 24 were down-regulated. Ingenuity Pathways Analysis of top functions affected by both S2h and Rg3 were carried out. The most effected pathway is the Ephrin receptor pathway. To validate the microarray data, quantitative real-time PCR of six candidate target genes was conducted, whereby it was found that three genes were up-regulated (AKAPA8L, PMPCB and PDE5A) and three were down-regulated (PITPNA, DUS2L and RIC8A). Although further studies are needed to elucidate the mechanisms of action, our findings should expand the understanding of the molecular framework of American ginseng as an anti-cancer agent.

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“…The interactions between some of the inner nuclear membrane proteins and chromatin are regulated through phosphorylation of these inner nuclear membrane proteins. The phosphorylation mechanisms for LBR and LAP2␣ and 2␤ are well understood (2)(3)(4)(5)(6)(7). LBR directly binds to DNA in vitro and dissociates on phosphorylation by cdc2 kinase and other kinase(s) in a mitotic egg extract (3).…”

mentioning

confidence: 99%

“…Furthermore, an in vitro binding assay involving an emerin point mutant revealed that Ser 175 phosphorylation is responsible for the dissociation of emerin from BAF. 6 Tag BAF-Cloning of the nucleoplasmic region of human emerin (⌬TM, amino acid residues 1-213) was performed by PCR. PCR was carried out with a human testis cDNA library using the following primers: 5Ј-CGG-GATCCCCATGGACAACTAGCAGAT-3Ј and 5Ј-CGGGATCCA-GAGCACGGTTTTCAGG-3Ј.…”

mentioning

confidence: 99%

Dissociation of Emerin from Barrier-to-autointegration Factor Is Regulated through Mitotic Phosphorylation of Emerin in a Xenopus Egg Cell-free System

Hirano

Segawa

Ouchi

et al. 2005

Journal of Biological Chemistry

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Emerin is the gene product of STA whose mutations cause Emery-Dreifuss muscular dystrophy. It is an inner nuclear membrane protein and phosphorylated in a cell cycle-dependent manner. However, the means of phosphorylation of emerin are poorly understood. We investigated the regulation mechanism for the binding of emerin to chromatin, focusing on its cell cycle-dependent phosphorylation in a Xenopus egg cell-free system. It was shown that emerin dissociates from chromatin depending on mitotic phosphorylation of the former, and this plays a critical role in the dissociation of emerin from barrier-to-autointegration factor (BAF). Then, we analyzed the mitotic phosphorylation sites of emerin. Emerin was strongly phosphorylated in an M-phase Xenopus egg cell-free system, and five phosphorylated sites, Ser 49 , Ser 66 , Thr 67 , Ser 120 , and Ser 175 , were identified on analysis of chymotryptic and tryptic emerin peptides using a phosphopeptide-concentrating system coupled with a Titansphere column, which specifically binds phosphopeptides, and tandem mass spectrometry sequencing. An in vitro binding assay involving an emerin S175A point mutant protein suggested that phosphorylation at Ser 175 regulates the dissociation of emerin from BAF. The nuclear envelope (NE)2 is a highly dynamic structure that disassembles at the onset of mitosis and reassembles on the surface of chromatin during telophase in vertebrates. These changes of NE are crucial for cell cycle progression. The NE consists of an outer nuclear membrane, inner nuclear membrane, nuclear pore complex, and nuclear lamina. The inner nuclear membrane contains integral membrane proteins, i.e. lamin B receptor (LBR), lamina-associated polypeptide-2␤ (LAP2␤), emerin, MAN1, and others, which interact with DNA and/or chromatin, and these proteins are proposed to participate in nuclear membrane targeting to chromatin at an early stage of nuclear assembly (1). The interactions between some of the inner nuclear membrane proteins and chromatin are regulated through phosphorylation of these inner nuclear membrane proteins. The phosphorylation mechanisms for LBR and LAP2␣ and 2␤ are well understood (2-7). LBR directly binds to DNA in vitro and dissociates on phosphorylation by cdc2 kinase and other kinase(s) in a mitotic egg extract (3). LAP2␤ binds to lamin B1 and chromatin, and cell cycle-dependent phosphorylation of LAP2␤ cancels this binding (4). Phosphorylation of these inner nuclear proteins, therefore, is likely to be one of the key mechanisms that control the interactions between the inner nuclear proteins and components of the nuclear lamina as well as chromatin. In this study, we focused on the mitotic phosphorylation of emerin, one of the inner nuclear membrane proteins.Human emerin is a serine-rich protein exhibiting an apparent mass of 34 kDa on SDS-PAGE (8) and is phosphorylated in a cell cycle-dependent manner (9). Emerin belongs to the LEM (LAP2␤, emerin, MAN1) protein family, whose members have approximately a 40-residue domain named the LEM (10). These ...

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In Vitro Modulation of the Interaction between HA95 and LAP2β by cAMP Signaling

Cited by 7 publications

References 35 publications

Barrier-to-Autointegration Factor Phosphorylation on Ser-4 Regulates Emerin Binding to Lamin A In Vitro and Emerin Localization In Vivo

Barrier-to-Autointegration Factor Phosphorylation on Ser-4 Regulates Emerin Binding to Lamin A In Vitro and Emerin Localization In Vivo

Characterization of gene expression regulated by American ginseng and ginsenoside Rg3 in human colorectal cancer cells

Dissociation of Emerin from Barrier-to-autointegration Factor Is Regulated through Mitotic Phosphorylation of Emerin in a Xenopus Egg Cell-free System

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