A Nuclear Envelope-associated Kinase Phosphorylates Arginine-Serine Motifs and Modulates Interactions between the Lamin B Receptor and Other Nuclear Proteins

Nikolakaki, Eleni; Simos, George; Georgatos, Spyros D.; Giannakouŕos, Thomas

doi:10.1074/jbc.271.14.8365

Cited by 104 publications

(142 citation statements)

References 43 publications

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“…The positions of the 1000-and 300-bp markers are indicated. F, detection of Lys 14 -acetylated and Lys 9 -trimethylated histone H3 in LBR-precipitated chromatin, as documented by Western blotting. Equal proportions of the nonbound (lanes 1) and the LBR-bound (lanes 2) material are shown.…”

Section: Methodsmentioning

confidence: 99%

“…Likewise, another peak at 985.5 could be attributed either to a twice trimethylated or to a trimethylated/ acetylated peptide. Pull-down assays with recombinant LBR and Western blotting with anti-histone modification antibodies showed that H3 subspecies trimethylated at Lys 9 or acetylated Lys 14 were present in the LBR precipitate, albeit to a different extent (Fig. 2F).…”

Section: Lbr Associates With Peripheralmentioning

confidence: 99%

“…LBR is a polytopic inner nuclear membrane protein consisting of a long, N-terminal domain, seven or eight hydrophobic transmembrane regions, and a C-terminal tail (13). The N-terminal part of the molecule protrudes to the nucleoplasm and contains multiple serine-arginine motifs that are phosphorylated by the SRPK1 and the cdc2 kinases (14,15); the hydrophobic region represents, instead, a (functional) form of sterol reductase and is involved in cholesterol metabolism (16).…”

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The Inner Nuclear Membrane Protein Lamin B Receptor Forms Distinct Microdomains and Links Epigenetically Marked Chromatin to the Nuclear Envelope

Makatsori

Kourmouli

Polioudaki

et al. 2004

Journal of Biological Chemistry

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143

109

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Using heterochromatin-enriched fractions, we have detected specific binding of mononucleosomes to the N-terminal domain of the inner nuclear membrane protein lamin 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.A significant proportion of heterochromatin is localized in the periphery of the cell nucleus and maintains a close spatial association with the inner nuclear membrane (1-5). This spatial association reflects a multiplicity of interactions between chromatin components and integral or peripheral proteins of the nuclear envelope (NE) 1 (6, 7).Because chromatin is extensively and differentially modified (8, 9), it is tempting to think that certain epigenetic marks or factors associated with histone modifying enzymes provide binding sites for NE proteins. However, it is equally possible that transcriptionally active, noncondensed chromatin is subjected to silencing and "heterochromatinization" upon contact to the NE. Both of these hypotheses receive experimental support: chromatin that is silenced through binding to SIRs can tether itself to the NE (10), whereas targeting of marker genes to the inner nuclear membrane suppresses their expression (11).One of the factors that have been implicated in chromatin anchorage to the NE is the lamin B receptor (12). LBR is a polytopic inner nuclear membrane protein consisting of a long, N-terminal domain, seven or eight hydrophobic transmembrane regions, and a C-terminal tail (13). The N-terminal part of the molecule protrudes to the nucleoplasm and contains multiple serine-arginine motifs that are phosphorylated by the SRPK1 and the cdc2 kinases (14, 15); the hydrophobic region represents, instead, a (functional) form of sterol reductase and is involved in cholesterol metabolism (16).Immunodepletion of LBR from detergent-solubilized NE vesicles results in proteoliposomes with a diminished ability to bind chromatin. Furthermore, direct binding of electrophoretically purified LBR to metaphase chromosomes can be demonstrated by in vitro assays (17). Corroborating these observations, anti-LBR antibodies block nuclear assembly in sea urchin egg extracts (18), whereas direct (19) and indirect (20) interactions with heterochromatin protein 1 (HP1) have been claimed in the literature.Two critical parameters in LBR-chromatin interactions are the physical state of LBR and the molecular features of LBRassociated chromatin. To address these issues, we have isolated fragments of peripheral heterochromatin attached to the inner nuclear membrane. These subcellular fractions were utilized to affinity select mononucleosomes that associate with LBR and investigate L...

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Section: Methodsmentioning

confidence: 99%

Section: Lbr Associates With Peripheralmentioning

confidence: 99%

mentioning

confidence: 99%

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The Inner Nuclear Membrane Protein Lamin B Receptor Forms Distinct Microdomains and Links Epigenetically Marked Chromatin to the Nuclear Envelope

Makatsori

Kourmouli

Polioudaki

et al. 2004

Journal of Biological Chemistry

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143

109

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“…Plasmids, Expression of Proteins, and Antibodies-Human SRPK1, the wild-type NH 2 -terminal domain of LBR (GST-wtNt; amino acids 1-205), and a mutant form lacking the RS motifs (GST-⌬RS; lacks amino acids [75][76][77][78][79][80][81][82][83][84] 75 RSRSRSRSRS 84 ) were expressed as fusion proteins with GST using the pGEX-2T vector (Amersham Biosciences) (18,19). Fulllength SRPK1 was also subcloned into the p-FLAG-CMV-2 (Eastman Kodak) vector and expressed in 293T cells with a FLAG tag fused at its NH 2 terminus (20).…”

Section: Methodsmentioning

confidence: 99%

Temporal Association of Protamine 1 with the Inner Nuclear Membrane Protein Lamin B Receptor during Spermiogenesis

Mylonis

Drosou

Brancorsini

et al. 2004

Journal of Biological Chemistry

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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 lamin B receptor (LBR), an inner nuclear membrane protein that also possesses a stretch of RS dipeptides at its nucleoplasmic NH 2 -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.The development of spermatids into spermatozoa, termed spermiogenesis, is characterized by the replacement of histones by the highly basic, arginine-rich, protamines (1). As a result of this exchange, the nucleosomal-type chromatin is transformed into a smooth fiber and compacted in a volume of about 5% of that of a somatic cell nucleus (2, 3). Although the exchange of chromatin proteins during spermiogenesis has long been known, the molecular mechanisms and the signaling pathways governing the histone to protamine transition have remained obscure.The deposition of protamines on sperm chromatin and the subsequent chromatin condensation appear to be controlled by phosphorylation-dephosphorylation events. Protamines are highly phosphorylated, shortly after their synthesis and before binding to DNA, whereas they become largely dephosphorylated during sperm maturation (4 -8). Phosphorylation of P2 protamine has been shown to be essential, because deletion of the calmodulin-dependent protein kinase Camk4, which phosphorylates P2 protamine, impairs the replacement of transition protein-2 with P2 protamine, resulting in defective spermiogenesis and male sterility (9). On the other hand, all P1 protamines contain short arginine/serine-rich (RS) 1 domains that are efficiently phosphorylated by SRPK1 (SR protein kinase 1) (10), but the physiological significance of this modification is mostly unknown.In this respect, Biggiogera et al. (11) reported that protamines initially appear at the nuclear periphery, implying that the nuclear envelope might play a role in the replacement of transition proteins by protamines during spermiogenesis. Given that RS domains mediate protein-protein interactions (12), we sought to investigate the potential interaction of P1 protamine with the inner nuclear membrane protein lamin B receptor (LBR), which also possesses a repeat of RS dipeptides at its nucleoplasmic NH 2 -terminal domain. In the present study we demonstrate a direct a...

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“…A number of mammalian kinases have been shown to cause disassembly of nuclear speckles and relocalization of splicing factors, and to subsequently affect splicing factor activity. SRPK-1/2, Clk-1/2/3/4, cdc2-kinase, cyclin Ecdk2, topoisomerase I, U1 70-kDa associated kinase, cGMPdependent kinase, and lamin B-receptor kinase were all shown to phosphorylate SR proteins and other splicing factors in vitro (Woppmann et al, 1993;Gui et al, 1994;Colwill et al, 1996;Nikolakaki et al, 1996;Rossi et al, 1996;Nayler et al, 1997;Duncan et al, 1998;Kuroyanagi et al, 1998;Okamoto et al, 1998;Seghezzi et al, 1998;Wang et al, 1998;Koizumi et al, 1999;Wang et al, 1999). In addition, some of these kinases were shown to affect the splicing activity of such factors (Mermoud et al, 1994;Cao et al, 1997;Xiao and Manley, 1998;Prasad et al, 1999).…”

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confidence: 99%

Nuclear Relocalization of the Pre-mRNA Splicing Factor PSF during Apoptosis Involves Hyperphosphorylation, Masking of Antigenic Epitopes, and Changes in Protein Interactions

Shav‐Tal

Cohen

Lapter

et al. 2001

MBoC

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The spatial nuclear organization of regulatory proteins often reflects their functional state. PSF, a factor essential for pre-mRNA splicing, is visualized by the B92 mAb as discrete nuclear foci, which disappeared during apoptosis. Because this mode of cell death entails protein degradation, it was considered that PSF, which like other splicing factors is sensitive to proteolysis, might be degraded. Nonetheless, during the apoptotic process, PSF remained intact and was N-terminally hyperphosphorylated on serine and threonine residues. Retarded gel migration profiles suggested differential phosphorylation of the molecule in mitosis vs. apoptosis and under-phosphorylation during blockage of cells at G1/S. Experiments with the use of recombinant GFP-tagged PSF provided evidence that in the course of apoptosis the antigenic epitopes of PSF are masked and that PSF reorganizes into globular nuclear structures. In apoptotic cells, PSF dissociated from PTB and bound new partners, including the U1-70K and SR proteins and therefore may acquire new functions. INTRODUCTIONSplicing factors are found within the nucleus both in a diffuse form and in distinct domains termed interchromatin granules (IG) or "speckles" (Spector, 1993). These functional compartments are spatially dynamic with regard to movement and composition (Eils et al., 2000). Splicing factors within IGs are highly mobile and are constantly associating and dissociating from their compartments (Phair and Misteli, 2000). The mAb, B92, which recognizes the polypyrimidine tract binding protein (PTB)-associated splicing factor (PSF), produces typical specked nuclear pattern in immunostaining. These PSF speckles disappear during granulocytic differentiation (Lee et al., 1996;Shav-Tal et al., 2000). We recently showed that this disappearance is associated with partial degradation (Shav-Tal et al., 2000) and by the formation of alternative nuclear structures (Shav-Tal et al., 2001). The present study indicates that PSF speckles disappear also through a different mechanism, involving conformational changes that cause breakdown of PSF speckles and relocalization of the molecule in the nucleus. The functional nature of speckles is a matter of some controversy (for review, see Park and De Boni, 1999). These structures have been suggested to play a role in storage and recycling of splicing factors (Jimenez-Garcia and Spector, 1993), whereas a portion may act as reservoirs for the recruitment to active sites of transcription (Huang and Spector, 1996;Zeng et al., 1997). This view does not predict a direct involvement of speckles in pre-mRNA splicing. On the other hand, it has been shown that nascent mRNA transcripts and transcription sites are closely associated with speckles, that transcription occurs at the surface of speckles (Clemson and Lawrence, 1996), and that microinjected pre-mRNAs have high affinity to speckles (Wang et al., 1991). It has thus been proposed that speckles can act coordinately in transcription and splicing and that pre-mRNA splicing can take place bo...

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A Nuclear Envelope-associated Kinase Phosphorylates Arginine-Serine Motifs and Modulates Interactions between the Lamin B Receptor and Other Nuclear Proteins

Cited by 104 publications

References 43 publications

The Inner Nuclear Membrane Protein Lamin B Receptor Forms Distinct Microdomains and Links Epigenetically Marked Chromatin to the Nuclear Envelope

The Inner Nuclear Membrane Protein Lamin B Receptor Forms Distinct Microdomains and Links Epigenetically Marked Chromatin to the Nuclear Envelope

Temporal Association of Protamine 1 with the Inner Nuclear Membrane Protein Lamin B Receptor during Spermiogenesis

Nuclear Relocalization of the Pre-mRNA Splicing Factor PSF during Apoptosis Involves Hyperphosphorylation, Masking of Antigenic Epitopes, and Changes in Protein Interactions

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