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

Abstract

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, Ser49, Ser66, Thr67, Ser120, and Ser175, 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 Ser175 regulates the dissociation of emerin from BAF. 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, Ser49, Ser66, Thr67, Ser120, and Ser175, 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 Ser175 regulates the dissociation of emerin from BAF. The nuclear envelope (NE) 2The abbreviations used are: NEnuclear envelopeGSTglutathione S-transferaseΔTMGST-fused fragment comprising amino acid residues 1–213 of human emerinΔLTGST-fused fragment comprising amino acid residues 37–213 of human emerinBAFbarrier-to-autointegration factorLAPlamina-associated polypeptideLBRlamin B receptorMCM-phase Xenopus egg cytosol fractionSCS-phase Xenopus egg cytosol fractionMALDI-TOF MSmatrix-assisted laser desorption ionization-time of flight mass spectrometryERendoplasmic reticulumESI-IT MSelectrospray ionization-ion trap mass spectrometryCHCAα-cyano-4-hydroxycinnamic acidHFhydrofluoric acidCBBCoomassie Brilliant Blue. 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-2β (LAP2β), 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 (1Gant T.M. Wilson K.L. Annu. Rev. Cell. Dev. Biol. 1997; 13: 669-695Crossref PubMed Scopus (200) Google Scholar). 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 LAP2α and 2β are well understood (2Nikolakaki E. Meier J. Simos G. Georgatos S.D. Giannakouros T. J. Biol. Chem. 1997; 272: 6208-6213Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 3Takano M. Koyama Y. Ito H. Hoshino S. Onogi H. Hagiwara M. Furukawa K. Horigome T. J. Biol. Chem. 2004; 279: 13265-13271Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 4Foisner R. Gerace L. Cell. 1993; 73: 1267-1279Abstract Full Text PDF PubMed Scopus (451) Google Scholar, 5Dreger M. Otto H. Neubauer G. Mann M. Hucho F. Biochemistry. 1999; 38: 9426-9434Crossref PubMed Scopus (32) Google Scholar, 6Martins S.B. Marstad A. Collas P. Biochemistry. 2003; 42: 10456-10461Crossref PubMed Scopus (5) Google Scholar, 7Gajewski A. Csaszar E. Foisner R. J. Biol. Chem. 2004; 279: 35813-35821Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). LBR directly binds to DNA in vitro and dissociates on phosphorylation by cdc2 kinase and other kinase(s) in a mitotic egg extract (3Takano M. Koyama Y. Ito H. Hoshino S. Onogi H. Hagiwara M. Furukawa K. Horigome T. J. Biol. Chem. 2004; 279: 13265-13271Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). LAP2β binds to lamin B1 and chromatin, and cell cycle-dependent phosphorylation of LAP2β cancels this binding (4Foisner R. Gerace L. Cell. 1993; 73: 1267-1279Abstract Full Text PDF PubMed Scopus (451) Google Scholar). 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. nuclear envelope glutathione S-transferase GST-fused fragment comprising amino acid residues 1–213 of human emerin GST-fused fragment comprising amino acid residues 37–213 of human emerin barrier-to-autointegration factor lamina-associated polypeptide lamin B receptor M-phase Xenopus egg cytosol fraction S-phase Xenopus egg cytosol fraction matrix-assisted laser desorption ionization-time of flight mass spectrometry endoplasmic reticulum electrospray ionization-ion trap mass spectrometry α-cyano-4-hydroxycinnamic acid hydrofluoric acid Coomassie Brilliant Blue. Human emerin is a serine-rich protein exhibiting an apparent mass of 34 kDa on SDS-PAGE (8Bione S. Maestrini E. Rivella S. Mancini M. Regis S. Romeo G. Toniolo D. Nat. Genet. 1994; 8: 323-327Crossref PubMed Scopus (773) Google Scholar) and is phosphorylated in a cell cycle-dependent manner (9Ellis J.A. Craxton M. Yates J.R. Kendrick-Jones J. J. Cell Sci. 1998; 111: 781-792Crossref PubMed Google Scholar). Emerin belongs to the LEM (LAP2β, emerin, MAN1) protein family, whose members have approximately a 40-residue domain named the LEM (10Lin F. Blake D.L. Callebaut I. Skerjanc I.S. Holmer L. McBurney M.W. Paulin-Levasseur M. Worman H.J. J. Biol. Chem. 2000; 275: 4840-4847Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar). These proteins directly bind to barrier-to-autointegration factor (BAF) (11Furukawa K. J. Cell Sci. 1999; 112: 2485-2492Crossref PubMed Google Scholar, 12Lee K.K. Haraguchi T. Lee R.S. Koujin T. Hiraoka Y. Wilson K.L. J. Cell Sci. 2001; 114: 4567-4573Crossref PubMed Google Scholar, 13Liu J. Lee K.K. Segura-Totten M. Neufeld E. Wilson K.L. Gruenbaum Y. Proc. Natl. Sci. S. A. 2003; PubMed Scopus Google Scholar). is a protein with a mass of kDa and is highly in and the interactions with DNA and LEM proteins are critical for nuclear membrane targeting to chromatin and chromatin during nuclear assembly M. R. Wilson K.L. J. Cell Biol. PubMed Scopus Google Scholar). the onset of emerin from the NE to the endoplasmic and is to the surface of the of chromatin, the during telophase Y. A. M. H. K. J. 1999; PubMed Scopus Google Scholar, T. Koujin T. Segura-Totten M. Lee K.K. Y. Y. Wilson K.L. Hiraoka Y. J. Cell Sci. 2001; 114: PubMed Google Scholar). An LEM domain mutant of emerin be to this that the binding of emerin to through the LEM domain is for this T. Koujin T. Segura-Totten M. Lee K.K. Y. Y. Wilson K.L. Hiraoka Y. J. Cell Sci. 2001; 114: PubMed Google Scholar). It is that emerin binding and proteins L. Wilson K.L. Cell Biol. 2004; PubMed Scopus Google Scholar, Kendrick-Jones J. J.A. J. Cell Sci. 1999; 112: PubMed Google Scholar, L. S. 2000; PubMed Scopus Google Scholar, G. S. E. L. S. 2003; PubMed Scopus Google Scholar, Wilson K.L. Biol. 2004; PubMed Scopus Google Scholar, Lee K.K. Segura-Totten M. Wilson K.L. PubMed Scopus Google Scholar, Lee K.K. Wilson K.L. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, A. S. I. S. Wilson K.L. J. 2003; PubMed Scopus Google Scholar, T. M. Koujin T. Wilson K.L. Hiraoka Y. J. 2004; PubMed Scopus Google Scholar). In binding to lamin is for the of emerin in the NE in the a mutant of emerin residues which Emery-Dreifuss muscular and bind to lamin cell cycle-dependent phosphorylation (9Ellis J.A. Craxton M. Yates J.R. Kendrick-Jones J. J. Cell Sci. 1998; 111: 781-792Crossref PubMed Google Scholar). The suggested that the phosphorylation of emerin regulates the binding of emerin to lamin (9Ellis J.A. Craxton M. Yates J.R. Kendrick-Jones J. J. Cell Sci. 1998; 111: 781-792Crossref PubMed Google Scholar). we were in the cell cycle-dependent regulation of the binding of emerin to chromatin and BAF. We the binding of emerin to chromatin by means of a binding assay involving a GST-fused fragment of emerin and chromatin in a Xenopus egg cell-free system. We analyzed the cell cycle-dependent phosphorylation and sites of emerin. from emerin with a Xenopus egg mitotic cytosol were by means of a Titansphere column, and five phosphorylation sites were identified on mass an in vitro binding assay involving an emerin point mutant that Ser175 phosphorylation is for the dissociation of emerin from BAF. of GST-fused Emerin and of the of human emerin amino acid residues was by was with a human using the and The product was with and the at the at the of a point mutant with the at with a was used to the was with using the and by by DNA The was with and the fragment was the at the the was with and and the fragment was the using the in the The of human was from the of was using and The product was with and and the at the and The DNA of the in and were using an DNA of Xenopus were and to S-phase and M-phase cytosol as S. M. T. S. M. M. T. R. A. T. S. Horigome T. J. 1997; PubMed Scopus Google Scholar, T. Y. A. S. K. M. M. Furukawa K. S. Horigome T. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). the chromatin binding assay was as (3Takano M. Koyama Y. Ito H. Hoshino S. Onogi H. Hagiwara M. Furukawa K. Horigome T. J. Biol. Chem. 2004; 279: 13265-13271Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, M. M. Ito H. Furukawa K. K. S. Horigome T. J. PubMed Scopus Google Scholar) for the of Xenopus chromatin of the with an extract was was as E. were and at for of the was with in binding and at for The were with binding and used for the chromatin binding Xenopus chromatin, which was with and with Xenopus egg in of in was to the which were in of at for the binding was by with of a and The were by were for and of with was was used as an of the of emerin and chromatin. The in the are of a The was in using with of The of the of emerin with in the chromatin binding assay was by means of In a M. M. Ito H. Furukawa K. K. S. Horigome T. J. PubMed Scopus Google we this and an in vitro binding involving proteins and chromatin, that this the as the we used this to the of protein to chromatin in this In of Emerin and was as of with Xenopus cytosol were with binding and and with E. extract at for The were with binding The was to SDS-PAGE and to a to was with an and Emerin phosphorylated with a Xenopus egg cytosol fraction was by and the emerin was Phosphorylation of the protein was with to the and a M. Y. J.A. PubMed Scopus Google Scholar). In of Emerin and were with a as the were with cell cycle-dependent Xenopus egg cytosol fraction at for The were to and to a to the was as Phosphorylation of Emerin with a Xenopus of GST-fused emerin to was with S-phase and M-phase egg cytosol of at for with the were with and at for The proteins were by SDS-PAGE and by the phosphorylation was with The emerin were from the and the phosphorylated emerin was by of emerin was phosphorylated as by and to a The emerin was in in acid for at and with The protein was with in for at The peptides were in and a in the was at for at and in the was with a system of and acid in The was to of from Emerin with a Titansphere was to the of I. Y. M. S. M. Sci. 2004; PubMed Scopus Google Scholar) with some of GST-fused emerin were with a Xenopus egg M-phase cytosol fraction as that was The were by SDS-PAGE and by Emerin were from the and with in at for The peptides were in and to a Titansphere inner column, with at the of for on the Titansphere were with at the of for The were directly to a inner column, with and with a of B at a of The were analyzed with an using as a of of from M-phase emerin was to the of H. M. K. 2003; PubMed Scopus Google Scholar). The were in of hydrofluoric acid and at for The peptides were in of and analyzed with an which were with the Titansphere and were to an inner with and with a from to of at the of using a system The were directly to with a and a on the phosphopeptides, the mass was in the the was by the of the mass of the as The of Emerin to of emerin i.e. emerin and emerin to were in E. and used in this Emerin the and emerin the domain and of LEM GST-fused emerin and GST-fused emerin were from E. extract using a with of and which were by that protein in and by were of and in this were using with chromatin and its is regulated in a cell cycle-dependent for some other inner nuclear membrane proteins, i.e. LBR and we an in vitro chromatin binding We this assay to the binding of inner nuclear membrane proteins to chromatin M. M. Ito H. Furukawa K. K. S. Horigome T. J. PubMed Scopus Google Scholar). were with in the of the Xenopus egg cytosol to chromatin in However, were with a cytosol fraction the binding of chromatin to was and in with a mitotic cytosol fraction the binding in the chromatin binding of was on with and in the other the chromatin binding of was on with and in These that the emerin fragment bind to chromatin, and that the chromatin binding assay be used to the cell cycle-dependent binding of emerin to chromatin in The of the binding of to chromatin on to be of phosphorylation of the was on of with for a protein kinase i.e. and in the of the binding on of the and in These that the be by the binding of the in to which is to the binding of emerin to chromatin, the of the binding was by kinase the was for which the binding domain in and Xenopus egg cytosol contains M. R. Wilson K.L. J. Cell Biol. PubMed Scopus Google Scholar). the other of the binding of to chromatin on with be by the phosphorylation of emerin, the was by a kinase i.e. in 2The of the binding of emerin to chromatin is by mitotic phosphorylation of emerin. were and the for and were with a cytosol and a mitotic cytosol and at for The were with and and and and with of and and with of and at for The were with chromatin at for and by of DNA with The of with were as and of the for with were with chromatin in the as for the The are the means S.D. for a from the control of in the of Emerin to the mechanism for the binding of to chromatin on we the binding of emerin to chromatin is by in assay system. were with an E. extract BAF. The were used for the assay with the E. extract to chromatin, with the E. extract bind to chromatin. The binding of to the in this assay system was by with These that the binding of to chromatin is by and that the of the binding of to chromatin on with is by the binding of to emerin. Phosphorylation of Emerin the to the binding of emerin to is cell cycle-dependent were with E. extract to bind to The were with a cell cycle-dependent Xenopus egg extract In the of with the and to on and However, to by These that of dissociation of from the other with were with the E. extract proteins to the were by SDS-PAGE and to a membrane, and was with an The binding of was by the of with that with and by the binding of to These were with the The phosphorylation of emerin shown in were by which is as a means of with M. Y. J.A. PubMed Scopus Google Scholar). The for the of with is the be to be The on SDS-PAGE of emerin on with and was with in phosphorylated emerin in (9Ellis J.A. Craxton M. Yates J.R. Kendrick-Jones J. J. Cell Sci. 1998; 111: 781-792Crossref PubMed Google Scholar). However, the depending on the phosphorylation for cell were in this system. with and were strongly phosphorylated and the binding of was strongly the other of with to the mitotic phosphorylation of emerin this B and and of phosphorylation of by to be by cause and we that the binding of emerin to is by we focused on analysis of the cell cycle-dependent phosphorylation of emerin, mitotic phosphorylation sites of emerin. Cell Phosphorylation of the cell cycle-dependent phosphorylation of emerin in a Xenopus egg cell-free system on was with egg cytosol with binding and analyzed by The was with and to The was strongly phosphorylated with the phosphorylation by was (9Ellis J.A. Craxton M. Yates J.R. Kendrick-Jones J. J. Cell Sci. 1998; 111: 781-792Crossref PubMed Google Scholar). mitotic was for with These that one phosphorylation sites that are of the LEM domain the of the LEM domain a on with the of with in the of was and were from the and The phosphorylated in the M-phase was as as that in the These that the phosphorylation of emerin in the S-phase is the mitotic phosphorylation of emerin, and the in the phosphorylation between and we and were with in the of the and were to and proteins were to a Then, the emerin were from the membrane and with The peptides were to on a of phosphorylated peptides were for the from and with the with in were for and were with well focused were In these and and and were in and These suggested the at one phosphorylation in the LEM of the LEM domain of a phosphorylation in other the LEM and binds an emerin kinase and to These are The phosphorylation of emerin with were for with the phosphorylation of were to be to of the were we the mitotic phosphorylation sites in the of the Phosphorylation of the mitotic phosphorylation sites of emerin by means of mass spectrometry we used a system comprising a of a Titansphere column, which specifically binds phosphopeptides, and a and this was by and phosphorylated by and with We used a chymotryptic of emerin, a chymotryptic a a tryptic as be in The chymotryptic tryptic from phosphorylated were to the Titansphere and with The fraction was directly the column, with a and The of the peptides were by of the were with the and that these peptides were with to and the mass was analyzed by The mass of that the peptides were and that one sites in were phosphorylated and be in phosphorylation we by means of to which residues are The mass of the product of and and of the of the phosphorylation to Ser175 by and was we the a on product and the which a and the a on product In to the of we which Ser175 was phosphorylated in However, we focused on Ser175 Ser175, was identified as a phosphorylation by the analysis the for of the phosphorylated the phosphorylation sites of other Ser66, Thr67, and were However, we the that in the was that was In the of the we the phosphorylated In the of and to the phosphorylation be the other we by acid analysis of phosphorylated by in the of we identified five phosphorylation sites, Ser49, Ser66, Thr67, Ser120, and These phosphorylation sites the of well i.e. protein kinase kinase and kinase kinase and kinase by and the of acid as an during and the of acid as an during in a Ser175 Phosphorylation of Emerin by for the five mitotic phosphorylation sites of emerin were identified and one phosphorylation i.e. Ser175, was on we phosphorylation in the LEM these identified phosphorylation sites and of and were was suggested that the for the and and and in be to the LEM domain and that phosphorylation at these sites is regulated through an mechanism by the LEM Then, we the system to and phosphorylated with a mitotic extract to the phosphorylation to the in the in a to a phosphorylated Ser175 in to in for the was in other We the to in a to a fragment of the the in was These that phosphorylation at Ser175, which is of the LEM be through some mechanism by the LEM domain and suggested that the phosphorylation in Then, we a point mutant at Ser175 of with an this we that the from the of from is that of the we to that the Ser175 phosphorylation was by of the LEM be in and which from the from the in and in S175A with and in However, the to be a of the the from one to to and to These suggested that the LEM domain is for the Ser175 phosphorylation by Then, we an in vitro binding assay using and to the Ser175 phosphorylation regulates the dissociation of emerin and The S175A point mutant with binding with from BAF. that the Ser175 phosphorylation in the M-phase in the dissociation of emerin and BAF. Phosphorylation of that emerin is highly phosphorylated at the M-phase in human (9Ellis J.A. Craxton M. Yates J.R. Kendrick-Jones J. J. Cell Sci. 1998; 111: 781-792Crossref PubMed Google the role of phosphorylation of emerin poorly understood. G. S. E. L. S. 2003; PubMed Scopus Google Scholar) that the of emerin and is by of emerin, that phosphorylation of emerin regulates its binding to means of a system involving a Titansphere column, we that emerin is phosphorylated mitotic in vitro with at five and one Ser49, Ser66, Thr67, Ser120, and Ser175 These phosphorylation sites are at the binding for Ser49, Ser66, and for and Lee K.K. Wilson K.L. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, A. S. I. S. Wilson K.L. J. 2003; PubMed Scopus Google Scholar, T. M. Koujin T. Wilson K.L. Hiraoka Y. J. 2004; PubMed Scopus Google Scholar) for lamin and K.K. Haraguchi T. Lee R.S. Koujin T. Hiraoka Y. Wilson K.L. J. Cell Sci. 2001; 114: 4567-4573Crossref PubMed Google Scholar, Wilson K.L. Biol. 2004; PubMed Scopus Google and Ser175 for and Wilson K.L. Biol. 2004; PubMed Scopus Google Scholar, Lee K.K. Wilson K.L. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, A. S. I. S. Wilson K.L. J. 2003; PubMed Scopus Google Scholar, T. M. Koujin T. Wilson K.L. Hiraoka Y. J. 2004; PubMed Scopus Google phosphorylation at some of these sites regulates In of and/or Ser175 the binding of emerin to the phosphorylation sites are in the binding The for the of Emerin and In this study, was suggested that the dissociation of emerin from through mitotic phosphorylation of emerin in a Xenopus cell-free system. However, these mitotic of which its binding to emerin. point mutant suggested that the phosphorylation at Ser175, which is of the LEM in the dissociation of emerin from in the M-phase and Wilson L. Wilson K.L. Cell Biol. 2004; PubMed Scopus Google Scholar) and the by Lee K.K. Haraguchi T. Lee R.S. Koujin T. Hiraoka Y. Wilson K.L. J. Cell Sci. 2001; 114: 4567-4573Crossref PubMed Google Scholar) that at emerin with of residues and which of the LEM were with the of was by these suggested that the LEM domain other of emerin to participate in the binding of emerin to BAF. with this Then, we that the LEM domain and binding in emerin a binding and that the phosphorylation at Ser175 on a of the binding the Ser175 to be a that consists of a K. Callebaut I. Worman H.J. S. 2001; PubMed Scopus Google Scholar). cause the we Ser175 of was phosphorylated by the be of the LEM domain from a Ser175 and phosphorylation was a kinase to the LEM domain in the Ser175 phosphorylation of in this the LEM domain be phosphorylated at Ser175, the kinase be one that Ser175 of in this be the we the emerin phosphorylation assay using whose proteins were proteins were from by with and were phosphorylated in the of by was between and in phosphorylation of the and the be to the Ser175 phosphorylation Cell Phosphorylation and of inner nuclear membrane proteins, i.e. emerin, and MAN1, are to bind directly to chromatin and to participate in of the regulation of and other L. Wilson K.L. Cell Biol. 2004; PubMed Scopus Google Scholar, M. Lee K.K. Wilson K.L. Gruenbaum Y. Sci. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, M. Wilson K.L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). nuclear envelope in these proteins from chromatin and the nuclear lamina to to the membrane T. Koujin T. Segura-Totten M. Lee K.K. Y. Y. Wilson K.L. Hiraoka Y. J. Cell Sci. 2001; 114: PubMed Google Scholar, T. Koujin T. T. T. J. Y. Hiraoka Y. J. Cell Sci. 2000; PubMed Google Scholar). In the of we have that phosphorylation of LBR in the by cdc2 kinase and an kinase in an M-phase egg extract dissociation from chromatin (3Takano M. Koyama Y. Ito H. Hoshino S. Onogi H. Hagiwara M. Furukawa K. Horigome T. J. Biol. Chem. 2004; 279: 13265-13271Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). In this study, we analyzed the LEM domain focusing on emerin. suggested that the binding of emerin to chromatin by in a egg extract was by phosphorylation of emerin by one in a mitotic egg suggested mechanism for the binding of emerin to chromatin be to other LEM proteins, and these proteins are to bind to through a LEM domain (11Furukawa K. J. Cell Sci. 1999; 112: 2485-2492Crossref PubMed Google Scholar, 13Liu J. Lee K.K. Segura-Totten M. Neufeld E. Wilson K.L. Gruenbaum Y. Proc. Natl. Sci. S. A. 2003; PubMed Scopus Google Scholar). in the of dissociation from chromatin on with a mitotic cell extract (4Foisner R. Gerace L. Cell. 1993; 73: 1267-1279Abstract Full Text PDF PubMed Scopus (451) Google Scholar). of LBR and LEM proteins from chromatin through these phosphorylation mechanisms cause the of the nuclear membrane from chromatin at the onset of It shown that emerin to the membrane in in the of the in telophase in T. Koujin T. Segura-Totten M. Lee K.K. Y. Y. Wilson K.L. Hiraoka Y. J. Cell Sci. 2001; 114: PubMed Google Scholar, T. Koujin T. T. T. J. Y. Hiraoka Y. J. Cell Sci. 2000; PubMed Google Scholar). In this is to the emerin, and this is for the assembly of emerin T. Koujin T. Segura-Totten M. Lee K.K. Y. Y. Wilson K.L. Hiraoka Y. J. Cell Sci. 2001; 114: PubMed Google Scholar). and that the of emerin to telophase of emerin to the of chromatin. this the binding of emerin to chromatin, on with is by with and the of the binding with is by of with M. K. and T. which is as a protein the other is to of mechanism regulates the to of emerin and chromatin, Haraguchi T. Koujin T. Segura-Totten M. Lee K.K. Y. Y. Wilson K.L. Hiraoka Y. J. Cell Sci. 2001; 114: PubMed Google Scholar) have that emerin be to the chromatin surface in telophase