Mutations in several of the integral membrane proteins of the inner nuclear membrane emerin, MAN1, lamin B receptor and their common binding partners lamins cause distinct diseases, the
Trang 1What MAN1 does to the Smads
TGFb/BMP signaling and the nuclear envelope
Luiza Bengtsson
Institute for Chemistry and Biochemistry, Free University Berlin, Germany
Introduction
Our knowledge about the nuclear membrane has
advanced dramatically in the recent years We now
know that protein residents of the nuclear membrane
regulate processes as diverse as DNA replication and
transcription, control of the shape and stability of the
nucleus, cell cycle progression, chromatin
organiza-tion, cell development and differentiaorganiza-tion, nuclear
anchoring and migration, and apoptosis (reviewed in
[1,2]) Mutations in several of the integral membrane
proteins of the inner nuclear membrane (emerin,
MAN1, lamin B receptor) and their common binding
partners (lamins) cause distinct diseases, the molecular
mechanisms of which are not yet understood [1,3,4] One of the current hypotheses suggests that the diseases result from altered gene expression in affec-ted tissues and that integral membrane proteins of the inner nuclear membrane (INM) regulate gene expression either directly, or as components of tran-scription regulating protein complexes [3,5,6] Indeed, both emerin and MAN1 bind the transcriptional repressors germ cell-less (GCL) and Bcl-2-associated transcription factor (Btf) [7,8] In addition, loss of emerin leads to up-regulation of expression of 28 genes, which can be rescued by reintroducing emerin [9] LAP2b, another INM protein, can repress trans-cription by recruiting histone deacetylase [10], or
Keywords
BMP; laminopathy; MAN1; nuclear
envelope; phosphatase; signal transduction;
Smad; TGFb
Correspondence
L Bengtsson, Institute for Chemistry and
Biochemistry, Free University Berlin,
Thielallee 63 14195 Berlin, Germany
Tel: +49 30 838 54789
E-mail: lbengts@chemie.fu-berlin.de
Previous address
Department of Cell Biology, Johns Hopkins
University School of Medicine, 725 N Wolfe
St, Baltimore, MD 21205, USA
(Received 8 March 2006, accepted 8
Janu-ary 2007)
doi:10.1111/j.1742-4658.2007.05696.x
The inner nuclear membrane protein MAN1 has been identified as an important factor in transforming growth factor b⁄ bone morphogenic pro-tein (TGFb⁄ BMP) signaling Loss of MAN1 results in three autosomal dominant diseases in humans; all three characterized by increased bone density Xenopus embryos lacking MAN1 develop severe morphological defects Both in humans and in Xenopus embryos the defects originate from deregulation of TGFb⁄ BMP signaling Several independent studies have shown that MAN1 is antagonizing TGFb⁄ BMP signaling through binding
to regulatory Smads Here, recent progress in understanding MAN1 func-tions is summarized and a model for MAN1-dependent regulation of TGFb⁄ BMP signaling is proposed
Abbreviations
BAF, barrier-to-autointegration factor; BMP, bone morphogenic protein; Btf, Bcl-2-associated transcription factor; GCL, germ cell-less; INM, inner nuclear membrane; LAP, lamina associated polypeptide; MH-domain, Mad homology domain; pRb, retinoblastoma protein;
PP, protein phosphatase; RR-motif, RNA recognition motif; R-Smads, regulatory Smads; SANE, Smad1 antagonistic effector; TGFb, transforming growth factor b; UHM, U2AF homology motif; WH, winged-helix.
Trang 2through binding to GCL [11] Lamin A binds the
transcription repressors retinoblastoma protein (pRb)
and MOK2 (reviewed in [1,12]) Finally, the nuclear
envelope protein MAN1, the subject of this review,
has been shown to bind regulatory Smads (R-Smads)
and antagonize the transforming growth factor
b⁄ bone morphogenic protein (TGFb ⁄ BMP)-induced
signal transduction pathway [13–17]
Who is MAN1?
MAN1 was first discovered as one of the autoantigens
for the autoantibodies from a patient with collagen
vascular disease [18] MAN1 is an integral membrane
protein of the INM and belongs to the LEM
(Lap2-emerin-MAN1)-domain family of proteins [18,19] The
LEM domain is a structural motif [20–22] also found
in emerin, lamina associated polypeptide (LAP)2,
Lem2 [23,24], the Drosophila specific proteins otefin
[25] and Bocksbeutel [26], and other as yet
uncharac-terized proteins named Lem3–5 [23] LEM domains
bind barrier-to-autointegration factor (BAF [8,27–29]),
an essential DNA-binding protein that has been
impli-cated in the organization of chromatin structure [30–
32] and recruitment of nuclear envelope proteins to the
chromosomes during nuclear assembly [33] The LEM
domain in MAN1, located at the very N-terminus of
this 100 kDa protein ([18], Fig 1), is highly conserved
with 82% identity between human and Xenopus MAN1
(xMAN1 [14]) In contrast, the N-terminus outside the
LEM domain is only 30% identical between human
and Xenopus MAN1 [14]
The functions of a MAN1 homolog, the Lem2
pro-tein, might be representative for the functions of the
MAN1 N-terminus Lem2 is 19% homologous to MAN1, has an N-terminal LEM domain, two trans-membrane domains and a conserved C-terminal nucleo-plasmic domain [24], but is lacking the C-terminal RNA recognition motif (RR-motif) found in MAN1 (Fig 1) Thus, structurally, Lem2 appears as a shorter version of MAN1 Overexpression of Lem2 in mamma-lian cells does not affect cell viability, but disturbs nuc-lear organization, which is manifested by protein bridges containing lamins and BAF connecting nuclei
of cells that have otherwise completed mitosis [24] In contrast, knockdown of the Caenorhabditis elegans ortholog, the Ce-Lem2 (the gene product of C elegans lem-2 gene, also known as ‘Ce-MAN1’ [8,24]), is lethal
in 15% of embryos [34] Interestingly, simultaneous down-regulation of Ce-Lem2 and Ce-emerin was lethal
in 100% of embryos by the 100-cell stage [34], while reduction of Ce-emerin had no noticeable effect [35], suggesting that Ce-Lem2 and Ce-emerin can substitute for each other to some extent It is not yet known whe-ther MAN1 and emerin are redundant, however, func-tional overlap is likely, because mammalian MAN1 and mammalian emerin do have many common part-ners (see below)
MAN1 needs lamins in order to localize to the INM [34,36,37] The N-terminus and the first transmem-brane domain of MAN1 are necessary and sufficient for MAN1 INM localization [13,38] The N-terminus
of human MAN1 (up to the first transmembrane domain) binds prelamin A and B1 [8] in vitro, while the LEM domain alone is sufficient to bind BAF (Fig 1; [8]) Prelamin A and BAF are also binding partners of emerin [39] Interestingly, the N-terminus
of human MAN1 binds the human emerin itself (Fig 1; [8]) Emerin is an integral membrane protein and localizes to the nuclear envelope [40] Mutations
in emerin cause Emery–Dreifuss muscular dystrophy [41] Although most disease causing mutations result in loss of emerin, in some cases the mutated emerin is present at normal levels and is also correctly localized (reviewed in [39]) Two of such mutations, the deletion
of residues 95–99 and the substitution Q133H, do affect MAN1 N-terminus binding to emerin: the bind-ing was abolished when tested in vitro [8] Given the possibility that MAN1 overlaps functionally with emerin, one might assume that MAN1 stabilizes⁄ regu-lates emerin’s functions Thus, loss of emerin binding
to MAN1 N-terminus and⁄ or loss of the
MAN1–emer-in complex functions could directly contribute to the Emery–Dreifuss muscular dystrophy disease mechanism
The C-terminus of MAN1 (human MAN1 residues 649–911; Fig 1) is 87% identical between human and
Fig 1 Map of binding sites on MAN1 Human and Xenopus MAN1
and C elegans lem2 sequences were retrieved from NCBI data
bank and pairwise aligned to human MAN1 using CLUSTALW
[8,13,14,16,17,34,69,70] Gaps between the boxed areas represent
gaps in the alignment that were larger than 10 amino acids.
Domains were either predicted using SMART [71] or taken from the
NMR structure [44] Numbers above the sequence mark the first
and last amino acid of each functional domain WH, Winged helix
domain; UHM, U2AF homology motif; L, LEM domain; 1, first
transmembrane domain; 2, second transmembrane domain; R,
RR-motif Black thick lines depict the smallest part of MAN1
required to bind each partner [8,13,14,16,44,69].
Trang 3Xenopus [14] and 55% identical between human and
Ciona intestinalis (a simple eukaryote of the chordate
lineage from which all vertebrates originate), implying
an evolutionarily conserved function The C-terminus
does not localize to the nuclear envelope by itself
[13,14,38], suggesting it has roles other than targeting
This part of MAN1 indeed binds several regulators of
gene expression, including transcriptional repressors
GCL and Btf and, surprisingly, also binds BAF [8,34]
There is no LEM domain in the MAN1 C-terminus,
however, a different BAF binding motif common to
MAN1 C-terminus, Histone H1 and the transcription
factor cone-rod homeobox (Crx) has been proposed [8]
The residues 801–857 in human MAN1 (655–734 in
xMAN1) comprise an motif (Fig 1 [8,13–15])
RR-motifs in other proteins are known to mediate
associ-ation with RNA [42], but can also function as protein–
protein interaction domains [43] Several studies have
identified the RR-motif in MAN1 as a binding site for
transcription regulators, the R-Smads [14] A detailed
NMR analysis of human MAN1 C-terminus revealed
the existence of two globular domains: the
experiment-ally confirmed winged-helix (WH) domain comprising
the residues 655–750 and a putative U2 auxillary factor
homology motif (UHM) consisting of residues 782–911
and including the RR-motif [15,44] Both the WH
domain and the UHM domain adopt a stable a⁄ b-fold
found in several DNA-interacting transcription factors
[45] Indeed, a MAN1 fragment consisting of the WH
domain binds DNA with nanomolar affinity and the
binding is further increased by the presence of the
UHM domain [44] Because the DNA binding site on
MAN1 does not overlap with the Smad binding site, it
seems possible for MAN1 to bind DNA and Smads
simultaneously [44]
MAN1 is essential for early
development and later tissue-specific
functions
MAN1 mRNA is maternally expressed in Xenopus
embryos [14] By the tailbud stage, the expression of
xMAN1 is restricted to anterior central nervous
sys-tem, eyes, otic vesicles and bronchial arches [14]
Strik-ingly, xMAN1 expression starts to diminish at stage 34
and is completely down-regulated by stage 45 [14,46]
It is not known whether xMAN1 is expressed in adult
frogs, however, various human cell lines do contain
endogenous MAN1 [13,15], which implies that MAN1
is reactivated in somatic cells Interestingly, as the
expression of xMAN1 is turned off, expression of both
Xenopusemerin genes is turned on [46], which suggests
yet another link between MAN1 and emerin functions
Xenopusembryos injected with antisense morpholino oligos against xMAN1 gastrulated normally [14] Like-wise, down-regulation of Drosophila MAN1 by RNAi does not affect the early development of the embryos [37] At later stages however, the Xenopus embryos showed severe morphological anomalies: their right eyes were absent or poorly formed [14] The eye defects correlated with several target genes of BMP signaling being up-regulated in the xMAN1 morphants, implica-ting xMAN1 in BMP signaling [14] It is not clear whether treatment with antisense morpholino oligos against xMAN1 resulted in a true null-phenotype, because, due to partial tetraploidy there might be another xMAN1 gene in Xenopus
In mammalian cells, MAN1 siRNA enhanced TGFb, activin and BMP signaling, because several gene targets of these pathways were up-regulated com-pared to controls [15] Reduced MAN1 expression also made the cells more sensitive to TGFb-induced growth inhibition [15]
Mutations in human MAN1 result in osteopoikilo-sis, Buschke–Ollendorff syndrome and melorheostosis [17] All three disorders are autosomal dominant and are characterized by increased bone density [47] In Buschke–Ollendorff syndrome, the osteopoikilosis is associated with disseminated connective tissue nevi In melorheostosis, the bone hyperostosis is accompanied
by abnormalities of adjacent soft tissues, such as joint contractures, sclerodermatous skin lesions, muscle atrophy, hemangiomas and lymphoedema [17] The disease causing mutations result in haploinsufficiency with respect to full-length MAN1 [17] There are two possibilities for how the mutations in MAN1 could cause disease: (a) the mutated protein is specifically interfering with remaining wildtype MAN1 functions, and⁄ or (b) half the amount of MAN1 in cells is not enough to keep up MAN1 functions The latter alter-native is more likely, because overexpression of mutated proteins in tissue culture cells expressing nor-mal levels of full-length endogenous MAN1 did not resemble the MAN1 siRNA phenotype, e.g., TGFb signaling was not enhanced [17]
TGFb/BMP signaling: the basics BMP, TGFb and activin belong to a family of pleio-tropic cytokines Each cytokine has many different iso-forms with highly specific functions These functions include the context-specific inhibition or stimulation
of cell proliferation, control of extracellular matrix synthesis and degradation, and the control of epi-thelial⁄ mesenchymal interactions during embryogene-sis Other functions include wound healing and the
Trang 4modulation of immune functions Misregulation of
these specific pathways results in developmental
disor-ders, cancer, fibrosis and autoimmune disorders
Signa-ling is initiated by binding of the cytokine to a
homodimeric complex of cytokine receptor type II,
which recruits type I receptor and activates it by
phos-phorylation Phosphorylated and thereby activated
type I receptor phosphorylates Smads, which then
form oligomeric complexes and enter the nucleus to
either induce or suppress gene expression by
interact-ing with cell type and signal-specific transcription
acti-vators or repressors There are three classes of Smads:
regulatory Smads (BMP-responsive R-Smads 1, 5 and
8 and TGFb-responsive R-Smads 2 and 3), the
co-Smad co-Smad4 and the inhibitory co-Smads 6 and 7 All
R-Smads and the co-Smad consist of three domains:
the N-terminal MH1 domain, the variable proline-rich
linker, and the C-terminal Mad homology (MH)2
domain The MH2 domain is highly conserved in
all Smads and is primarily responsible for binding to
different partners in a series of mutually exclusive
protein–protein interactions The specificity of the
BMP⁄ TGFb ⁄ activin signal is conferred by mixing and
matching of receptor subtypes in the oligomeric
recep-tor complexes as well as by regulation of Smad
interac-tions in the cytoplasm and in the nucleus Smads can
be either activated or inhibited by phosphorylation,
sumoylation and ubiquitination (reviewed in [48–54])
MAN1 antagonizes TGFb/BMP signaling
by binding R-Smads
Xenopus MAN1 was identified as a gene involved in
neuralization and neural patterning during Xenopus
development [14] The RR-motif in MAN1 was
neces-sary but not sufficient for the neuralizing activity,
while neither the LEM domain nor the whole
N-termi-nus of MAN1 showed any activity [14] Furthermore,
both full-length MAN1 [16] and the C-terminus alone
[14,16] could induce a partial secondary axis formation
in Xenopus embryos [14] Both the neuralizing activity
and the secondary axis induction indicate inhibited
BMP signaling An independent study also discovered
xMAN1 as a negative regulator of the BMP signaling,
but named the protein ‘SANE’ (Smad1 antagonistic
effector) [13] The cDNA sequences of SANE and
xMAN1 in the NCBI gene database are identical
(gi|56849616 and gi|29335751, respectively) and are
orthologous to human MAN1
The C-terminus of human MAN1 interacted with
Smads 2 and 3 in a yeast two-hybrid skeletal muscle
library [15] Additionally, in an affinity-purification of
Smad3 interacting proteins from TGFb-responsive
Hep3B (human liver carcinoma) and RIE-1 (rat intest-inal epithelial) cells, MAN1 was among the proteins that bound specifically [15] Various independent meth-ods ranging from in vivo coimmunoprecipitation to direct in vitro binding assays confirmed the direct inter-action between MAN1 and all regulatory Smads (BMP and TGFb-responsive) but not the co-Smad or the inhibitory Smads [13–17] The interaction was mapped to the RR-motif in MAN1 and the MH2 domain of R-Smads (Fig 1; [14,16]) RNAse treatment had no effect on the MAN1⁄ Smad binding suggesting that the RR-motif in MAN1 is a protein–protein inter-action domain [13–17]
Several independent experiments suggest that the antagonizing activity of MAN1 in TGFb⁄ BMP signa-ling depends on its ability to bind R-Smads When tes-ted using luciferase reporters containing response elements from the BMP-responsive gene Xvent2, both full-length xMAN1 and the C-terminus alone inhibited luciferase gene expression after BMP4 stimulation, while the N-terminus alone had no effect [16] Although TGFb and activin signaling were unaffected
by MAN1 overexpression in Xenopus embryos [13,15,17], in mammalian cell lines both the full-length MAN1 [13] and its C-terminus alone [15,17] were cap-able of antagonizing TGFb-, BMP- and activin-signa-ling Similarly, human MAN1 with mutated RR-motif was defective in antagonizing both BMP and TGFb signaling in tissue culture cells [15]
MAN1 does not bind inhibitory Smads or the co-Smad [15] Moreover, MAN1 does not bind R-Smad–co-Smad complexes [15] The association of MAN1 with R-Smads is not regulated by the signaling pathway, because neither stimulation with TGFb or BMP, nor overexpression of constitutively active type I receptor for TGFb, BMP or activin increases the amount of R-Smad bound to MAN1 [15] MAN1 binds both phosphorylated and unphosphorylated R-Smads [15] At the same time, overexpression of MAN1 lowers the cellular pool of phosphorylated R-Smads [15,16] and prevents accumulation of R-R-Smads
in the nucleus after cytokine-induced activation [15] Importantly, the R-Smads are not being degraded as a result of MAN1 overexpression (shown for Smad3 [13], Smad2 [16] and xSmad1 [16])
The model: MAN1 disrupts the R-Smad–co-Smad complexes and promotes dephosphorylation of R-Smads
How can MAN1 attenuate TGFb⁄ BMP signaling by binding R-Smads? As an INM protein and not a part
Trang 5of the nuclear pore complexes, MAN1 is unlikely to
block Smad entry into the nucleus It is also unlikely
that MAN1 simply sequesters R-Smads at the nuclear
envelope and thus prevents transcription from their
target genes [15,36,55] – this would result in an
accu-mulation of the R-Smads at the nuclear periphery and
not in the observed cytoplasmic accumulation [15]
MAN1 is predicted to be able to bind DNA and
R-Smads simultaneously [44], thus it may assist in
acti-vation or repression of TGFb⁄ BMP target genes at the
nuclear envelope It is formally possible that such
genes code for antagonists of TGFb⁄ BMP signaling
and their expression results in overall signal
attenu-ation However, effects on Smad phosphorylation and
Smad nuclear localization were studied after 1 h of
TGFb1 stimulation [15] implicating that the
antagon-izing mechanism is more direct
Smad-mediated signaling has two important
proper-ties: (a) only phosphorylated complexed R-Smads are
retained in the nucleus, and (b) only phosphorylated
R-Smads in complex with the co-Smad can initiate or
inhibit transcription of TGFb⁄ BMP target genes
[48–54,56] Thus MAN1 has to either disrupt R-Smad–
co-Smad complexes and⁄ or induce dephosphorylation
of R-Smads in order to attenuate the Smad-mediated
signal This hypothesis is supported by several
experi-mental data: (a) it has been shown that MAN1 bound
Smad3 is not associated with the co-Smad, in contrast
to ‘free’ Smad3 [15]; (b) overabundance of MAN1
cor-relates with lower cellular pool of phosphorylated
Smads [16]; (c) upon overexpression of MAN1
R-Smads do not accumulate in the nucleus, indicating
lost retention in the nucleus and accelerated nuclear
export [14–16], and (d) full-length MAN1 antagonizes
TGFb⁄ BMP signaling more effectively than the
C-ter-minus alone, implying that the correct nuclear
envel-ope localization of MAN1 is beneficial, but not
necessary for MAN1 functions in TGFb⁄ BMP
signa-ling [14] Taken together the data suggests a role for
MAN1 similar to that of the inhibitory Smad 6 Smad
6 inhibits TGFb⁄ BMP signaling not only by binding
the respective type I receptors and interfering with
phosphorylation of Smads, but also by binding
R-Smads and preventing them from
heterooligomeriz-ing with co-Smad and formheterooligomeriz-ing active complexes
(reviewed in [57]) Hypothetically, MAN1 may be
act-ing as a ‘molecular filter’, catchact-ing a portion of the
Smad complexes that enter the nucleus and forcing the
complexes apart by binding the R-Smad and displacing
the co-Smad Monomeric Smads would become
rap-idly dephosphorylated and exported out of the nucleus
MAN1 may also recruit a nuclear phosphatase to
dephosphorylate Smads and reinforce Smad complex
disassembly Two nuclear Smad phosphatases have recently been identified: pyruvate dehydrogenase phos-phatase (PDP) for BMP responsive R-Smads [58] and PPM1A for TGFb responsive R-Smads [59]; both are members of the metal-ion-dependent protein phospha-tase family and both are distributed throughout the nucleus Two further phosphatases, the protein phos-phatase 1 (PP1) and the protein phosphos-phatase 2 A (PP2A) are anchored at the nuclear periphery [60–62] Overexpression of the catalytic domains of PP1 and PP2A did not have any effect on Smad phosphoryla-tion [58,59]; however, both phosphatases need a regu-latory subunit in order to find their targets [63] PP1 is responsible for dephosphorylating lamins throughout the interphase, while PP2A dephosphorylates pRb in a cell cycle and lamin dependent manner [60–62] More-over, inhibition of PP2A increases the phospho-Smad pool in the cells only when lamins are present Thus, both PP1 and PP2A are potentially in the right place
to dephosphorylate MAN1-bound Smads The pro-posed model is summarized in Fig 2
Why MAN1?
Any inhibition of BMP⁄ TGFb signaling by MAN1 has to be a strictly local process restricted to the nuc-lear envelope Why is it important to have a signaling antagonist posted there? MAN1 can potentially bind both DNA and R-Smads and is therefore able to influ-ence gene expression directly [44] It is not yet known which exact genes are under transcriptional control by MAN1, but the fact that haploinsufficiency of MAN1 causes severe bone disorders [17] suggests that the genes in question are central for cell functions and have to be tightly regulated Thus MAN1 would hypo-thetically both transduce the Smad-mediated signal and attenuate it at the same time Alternatively, MAN1 might be safeguarding the nuclear periphery against concentration of active Smad complexes which could potentially interact with other INM proteins and the lamina and negatively influence regulation of gene expression [2]
Could emerin be involved?
At least in C elegans embryos, Ce-emerin seems to provide a backup mechanism for functions of the MAN1 homolog Ce-lem2 [34] In Xenopus embryos emerin gene expression begins as MAN1 expression diminishes [46] In somatic human cells, both emerin and MAN1 are expressed [17,18,39] Human emerin and human MAN1 have many common binding part-ners [8,39], but it is not yet known if emerin also binds
Trang 6Smads Emerin binds the N-terminus of MAN1 [8] and
has thus the potential to regulate the TGFb⁄ BMP
sign-aling antagonizing activity of MAN1 Emerin is
retained at the nuclear membrane by lamins (reviewed
in [39]) and Nesprin 2 [64,65] Interestingly, the
expres-sion of synaptic nuclear envelope-2, a short isoform of
the giant Nesprin 2 [64–66] also located at the nuclear
membrane, is specifically up-regulated in response to
TGFb signal [67,68] If nesprins serve as scaffolds for
protein complexes containing MAN1, emerin, lamins,
protein phosphatases and other components, then the
up-regulation of nesprin expression might function as a
feedback mechanism In such a feedback mechanism,
the cytokine signal results in translocation of
phos-phorylated Smads into the nucleus, leading to higher
expression of nesprins More nesprins could then
hypo-thetically link more emerin⁄ phosphatases ⁄ MAN1
pro-tein complexes which would eventually lead to
enhanced dephosphorylation of Smads and
attenu-ated⁄ terminated signal
The discovery that the INM protein MAN1 binds
Smads and antagonizes cytokine signaling also raises
the question what roles other nuclear envelope proteins
might have in cellular signal transduction We know
that several of them (LAP2b, emerin, lamin A) can
regulate gene expression [1,9,11,12]; future studies will
have to tell whether they do it on orders coming from the plasma membrane
Acknowledgements The first version of this review was written while I was
a postdoctoral fellow in Katherine L Wilson’s lab (spring 2005) Warmest thanks to Katherine L Wilson and members of the Wilson lab, especially K E Tifft,
M Mansharamani and M S Zastrow for comments
on the manuscript, to R Schwappacher for fruitful discussions and to Petra Knaus for her support LB was funded by a postdoctoral fellowship from the Deutsche Forschungsgemeinschaft
References
1 Gruenbaum Y, Margalit A, Goldman RD, Shumaker
DK & Wilson KL (2005) The nuclear lamina comes of age Nat Rev Mol Cell Biol 6, 21–31
2 D’Angelo MA & Hetzer MW (2006) The role of the nuclear envelope in cellular organization Cell Mol Life Sci 63, 316–332
3 Broers JL, Hutchison CJ & Ramaekers FC (2004) Laminopathies J Pathol 204, 478–488
Fig 2 Proposed model for TGFb⁄ BMP signaling regulation by MAN1 (1) MAN1 binds through its C-terminal RR-motif to the MH2-domain
of incoming R-Smads–co-Smad complexes MAN1 at the nuclear envelope is in a complex with emerin, other proteins and a putative phos-phatase (2) The recruitment of R-Smads to a MAN1 complex causes disassembly of the R-Smad–co-Smad complex, dephosphorylation of Smads and increased nuclear export (3) This results in fewer active Smad complexes capable of recruiting coactivators⁄ corepressors to DNA in the nuclear interior and overall less activation of gene expression Thus, MAN1 function is to fine tune the TGFb ⁄ BMP signaling NPC, nuclear pore complex; PP, protein phosphatase.
Trang 74 Broers JL, Ramaekers FC, Bonne G, Yaou RB &
Hutchison CJ (2006) Nuclear lamins: laminopathies and
their role in premature ageing Physiol Rev 86, 9671008
5 Wilson KL (2000) The nuclear envelope, muscular
dys-trophy and gene expression Trends Cell Biol 10, 125–129
6 Somech R, Shaklai S, Amariglio N, Rechavi G &
Simon AJ (2005) Nuclear envelopathies – raising the
nuclear veil Pediatr Res 57, 8R–15R
7 Holaska JM, Lee KK, Kowalski AK & Wilson KL
(2003) Transcriptional repressor germ cell-less (GCL)
and barrier to autointegration factor (BAF) compete for
binding to emerin in vitro J Biol Chem 278, 6969–6975
8 Mansharamani M & Wilson KL (2005) Nuclear
mem-brane protein MAN1: Direct binding to emerin in vitro
and two modes of binding to BAF J Biol Chem 280,
13863–13870
9 Tsukahara T, Tsujino S & Arahata K (2002) CDNA
microarray analysis of gene expression in fibroblasts of
patients with X-linked Emery-Dreifuss muscular
dystro-phy Muscle Nerve 25, 898–901
10 Somech R, Shaklai S, Geller O, Amariglio N, Simon AJ,
Rechavi G & Gal-Yam EN (2005) The nuclear-envelope
protein and transcriptional repressor LAP2beta interacts
with HDAC3 at the nuclear periphery, and induces
histone H4 deacetylation J Cell Sci 118, 4017–4025
11 Nili E, Cojocaru GS, Kalma Y, Ginsberg D, Copeland
NG, Gilbert DJ, Jenkins NA, Berger R, Shaklai S,
Amariglio N, et al (2001) Nuclear membrane protein
LAP2beta mediates transcriptional repression alone and
together with its binding partner GCL (germ-cell-less)
J Cell Sci 114, 3297–3307
12 Zastrow MS, Vlcek S & Wilson KL (2004) Proteins that
bind A-type lamins: integrating isolated clues J Cell Sci
117, 979–987
13 Lin F, Morrison JM, Wu W & Worman HJ (2005)
MAN1, an integral protein of the inner nuclear
mem-brane, binds Smad2 and Smad3 and antagonizes
trans-forming growth factor-beta signalling Hum Mol Genet
14, 437–445
14 Osada S, Ohmori SY & Taira M (2003) XMAN1, an
inner nuclear membrane protein, antagonizes BMP
sig-naling by interacting with Smad1 in Xenopus embryos
Development 130, 1783–1794
15 Pan D, Estevez-Salmeron LD, Stroschein SL, Zhu X,
He J, Zhou S & Luo K (2005) The integral inner
nuclear membrane protein MAN1 physically interacts
with the R-Smad proteins to repress signaling by the
transforming growth factor-b superfamily of cytokines
J Biol Chem 280, 15992–16001
16 Raju GP, Dimova N, Klein PS & Huang HC (2003)
SANE, a novel LEM domain protein, regulates bone
morphogenetic protein signaling through interaction
with Smad1 J Biol Chem 278, 428–437
17 Hellemans J, Preobrazhenska O, Willaert A, Debeer P,
Verdonk PC, Costa T, Janssens K, Menten B, Van Roy
N, Vermeulen SJ, et al (2004) Loss-offunction mutations in LEMD3 result in osteopoikilosis, Buschke–Ollendorff syndrome and melorheostosis Nat Genet 36, 1213–1218
18 Lin F, Blake DL, Callebaut I, Skerjanc IS, Holmer L, McBurney MW, Paulin-Levasseur M & Worman HJ (2000) MAN1, an inner nuclear membrane protein that shares the LEM domain with lamina-associated poly-peptide 2 and emerin J Biol Chem 275, 4840–4847
19 Lee KK, Gruenbaum Y, Spann P, Liu J & Wilson KL (2000) C elegans nuclear envelope proteins emerin, MAN1, lamin, and nucleoporins reveal unique timing
of nuclear envelope breakdown during mitosis Mol Biol Cell 11, 3089–3099
20 Wolff N, Gilquin B, Courchay K, Callebaut I, Worman
HJ & Zinn-Justin S (2001) Structural analysis of emerin,
an inner nuclear membrane protein mutated in X-linked Emery-Dreifuss muscular dystrophy FEBS Lett 501, 171–176
21 Laguri C, Gilquin B, Wolff N, Romi-Lebrun R, Courc-hay K, Callebaut I, Worman HJ & Zinn-Justin S (2001) Structural characterization of the LEM motif common
to three human inner nuclear membrane proteins Struc-ture 9, 503–511
22 Cai M, Huang Y, Ghirlando R, Wilson KL, Craigie R
& Clore GM (2001) Solution structure of the constant region of nuclear envelope protein LAP2 reveals two LEM-domain structures: one binds BAF and the other binds DNA EMBO J 20, 4399–4407
23 Lee KK & Wilson KL (2004) All in the family: evidence for four new LEM-domain proteins Lem2 (NET-25), Lem3, Lem4 and Lem5 in the human genome Symp Soc Exp Biol 56, 329–339
24 Brachner A, Reipert S, Foisner R & Gotzmann J (2005) LEM2 is a novel MAN1-related inner nuclear mem-brane protein associated with A-type lamins J Cell Sci
118, 5797–5810
25 Ashery-Padan R, Ulitzur N, Arbel A, Goldberg M, Weiss AM, Maus N, Fisher PA & Gruenbaum Y (1997) Localization and posttranslational modifications of otefin, a protein required for vesicle attachment to chro-matin, during Drosophila melanogaster development Mol Cell Biol 17, 4114–4123
26 Wagner N, Schmitt J & Krohne G (2004) Two novel LEM-domain proteins are splice products of the anno-tated Drosophila melanogaster gene CG9424 (Bocksbeu-tel) Eur J Cell Biol 82, 605–616
27 Shumaker DK, Lee KK, Tanhehco YC, Craigie R & Wilson KL (2001) LAP2 binds to BAF-DNA com-plexes: requirement for the LEM domain and modula-tion by variable regions EMBO J 20, 1754–1764
28 Lee KK, Haraguchi T, Lee RS, Koujin T, Hiraoka Y & Wilson KL (2001) Distinct functional domains in emerin bind lamin A and DNA-bridging protein BAF
J Cell Sci 114, 4567–4573
Trang 829 Segura-Totten M & Wilson KL (2004) BAF: roles in
chromatin, nuclear structure and retrovirus integration
Trends Cell Biol 14, 261–266
30 Furukawa K, Sugiyama S, Osouda S, Goto H,
Inag-aki M, Horigome T, Omata S, McConnell M, Fisher
PA & Nishida Y (2003) Barrier-to-autointegration
fac-tor plays crucial roles in cell cycle progression and
nuclear organization in Drosophila J Cell Sci 116,
3811–3823
31 Segura-Totten M, Kowalski AK, Craigie R & Wilson
KL (2002) Barrier-toautointegration factor: major roles
in chromatin decondensation and nuclear assembly
J Cell Biol 158, 475–485
32 Margalit A, Segura-Totten M, Gruenbaum Y & Wilson
KL (2005) Barrier-toautointegration factor is required
to segregate and enclose chromosomes within the
nuclear envelope and assemble the nuclear lamina Proc
Natl Acad Sci USA 102, 3290–3295
33 Haraguchi T, Koujin T, Segura-Totten M, Lee KK,
Matsuoka Y, Yoneda Y, Wilson KL & Hiraoka Y
(2001) BAF is required for emerin assembly into the
reforming nuclear envelope J Cell Sci 114, 4575–4585
34 Liu J, Lee KK, Segura-Totten M, Neufeld E, Wilson
KL & Gruenbaum Y (2003) MAN1 and emerin have
overlapping function(s) essential for chromosome
segre-gation and cell division in Caenorhabditis elegans Proc
Natl Acad Sci USA 100, 4598–4603
35 Gruenbaum Y, Lee KK, Liu J, Cohen M & Wilson KL
(2002) The expression, lamin-dependent localization and
RNAi depletion phenotype for emerin in C elegans
J Cell Sci 115, 923–929
36 Ostlund C, Sullivan T, Stewart CL & Worman HJ
(2006) Dependence of diffusional mobility of integral
inner nuclear membrane proteins on A-type lamins
Bio-chemistry 45, 1374–1382
37 Wagner N, Kagermeier B, Loserth S & Krohne G
(2006) The Drosophila melanogaster LEM-domain
pro-tein MAN1 Eur J Cell Biol 85, 91–105
38 Wu W, Lin F & Worman HJ (2002) Intracellular
traf-ficking of MAN1, an integral protein of the nuclear
envelope inner membrane J Cell Sci 115, 1361–1371
39 Bengtsson L & Wilson KL (2004) Multiple and
surpris-ing new functions for emerin, a nuclear membrane
pro-tein Curr Opin Cell Biol 16, 73–79
40 Yorifuji H, Tadano Y, Tsuchiya Y, Ogawa M, Goto K,
Umetani A, Asaka Y & Arahata K (1997) Emerin,
defi-ciency of which causes Emery-Dreifuss muscular
dystro-phy, is localized at the inner nuclear membrane
Neurogenetics 1, 135–140
41 Bione S, Maestrini E, Rivella S, Mancini M, Regis S,
Romeo G & Toniolo D (1994) Identification of a novel
X-linked gene responsible for Emery-Dreifuss muscular
dystrophy Nat Genet 8, 323–327
42 Birney E, Kumar S & Krainer AR (1993) Analysis of
the RNA-recognition motif and RS and RGG domains:
conservation in metazoan pre-mRNA splicing factors Nucleic Acids Res 21, 5803–5816
43 Dye BT & Patton JG (2001) An RNA recognition motif (RRM) is required for the localization of PTB-asso-ciated splicing factor (PSF) to subnuclear speckles Exp Cell Res 263, 131–144
44 Caputo S, Couprie J, Duband-Goulet I, Konde E, Lin
F, Braud S, Gondry M, Gilquin B, Worman HJ & Zinn-Justin S (2006) The Carboxyl-terminal Nucleoplasmic Region of MAN1 Exhibits a DNA Bind-ing WBind-inged Helix Domain J Biol Chem 281, 18208– 18215
45 Gajiwala KS & Burley SK (2000) Winged helix proteins Curr Opin Struct Biol 10, 110–116
46 Gareiss M, Eberhardt K, Kruger E, Kandert S, Bohm
C, Zentgraf H, Muller CR & Dabauvalle MC (2005) Emerin expression in early development of Xenopus laevis Eur J Cell Biol 84, 295–309
47 Hall CM (2002) International nosology and classifica-tion of constituclassifica-tional disorders of bone Am J Med Genet 113, 65–77
48 Schiller M, Javelaud D & Mauviel A (2004) TGF-[beta]-induced SMAD signaling and gene regulation: consequences for extracellular matrix remodeling and wound healing J Dermatol Sci 35, 83–92
49 Javelaud D & Mauviel A (2004) Mammalian trans-forming growth factor-[beta]s: Smad signaling and physio-pathological roles Int J Biochem Cell Biol 36, 1161–1165
50 Dijke PT & Hill CS (2004) New insights into TGF-[beta]-Smad signalling Trends Biochem Sci 29, 265– 273
51 Wan M & Cao X (2005) BMP signaling in skeletal development Biochem Biophys Res Commun 328, 651–657
52 Shi Y & Massague J (2003) Mechanisms of TGF-[beta] Signaling from Cell Membrane to the Nucleus Cell 113, 685–700
53 DaCosta Byfield S & Roberts AB (2004) Lateral signal-ing enhances TGF-[beta] response complexity Trends Cell Biol 14, 107–111
54 Nohe A, Keating E, Knaus P & Petersen NO (2004) Signal transduction of bone morphogenetic protein receptors Cellular Signalling 16, 291–299
55 Worman HJ (2006) Inner nuclear membrane and regula-tion of Smad-mediated signaling Biochim Biophys Acta
1761, 626–631
56 Schmierer B & Hill CS (2005) Kinetic analysis of Smad nucleocytoplasmic shuttling reveals a mechanism for transforming growth factor beta-dependent nuclear accumulation of Smads Mol Cell Biol 25, 9845– 9858
57 Feng XH & Derynck R (2005) Specificity and versatility
in tgf-beta signaling through Smads Annu Rev Cell Dev Biol 21, 659–693
Trang 958 Chen HB, Shen J, Ip YT & Xu L (2006) Identification
of phosphatases for Smad in the BMP⁄ DPP pathway
Genes Dev 20, 648–653
59 Lin X, Duan X, Liang YY, Su Y, Wrighton KH, Long
J, Hu M, Davis CM, Wang J, Brunicardi FC et al
(2006) PPM1A functions as a Smad phosphatase to
ter-minate TGFbeta signaling Cell 125, 915–928
60 Steen RL, Beullens M, Landsverk HB, Bollen M &
Col-las P (2003) AKAP149 is a novel PP1 specifier required
to maintain nuclear envelope integrity in G1 phase
J Cell Sci 116, 2237–2246
61 Kuntziger T, Rogne M, Folstad RL & Collas P (2006)
Association of PP1 with its regulatory subunit
AKAP149 is regulated by serine phosphorylation
flank-ing the RVXF motif of AKAP149 Biochemistry 45,
5868–5877
62 Van Berlo JH, Voncken JW, Kubben N, Broers JL,
Duisters R, van Leeuwen RE, Crijns HJ, Ramaekers FC,
Hutchison CJ & Pinto YM (2005) A-type lamins are
essential for TGF-beta1 induced PP2A to
dephosphory-late transcription factors Hum Mol Genet 14, 2839–2849
63 Gallego M & Virshup DM (2005) Protein serine⁄
threon-ine phosphatases: life, death, and sleeping Curr Opin
Cell Biol 17, 197–202
64 Zhang Q, Ragnauth CD, Skepper JN, Worth NF,
War-ren DT, Roberts RG, Weissberg PL, Ellis JA &
Shana-han CM (2005) Nesprin-2 is a multi-isomeric protein
that binds lamin and emerin at the nuclear envelope
and forms a subcellular network in skeletal muscle
J Cell Sci 118, 673–687
65 Libotte T, Zaim H, Abraham S, Padmakumar VC,
Schneider M, Lu W, Munck M, Hutchison C, Wehnert
M, Fahrenkrog B, et al (2005) Lamin A⁄ C Dependent
Localization of Nesprin-2, a Giant Scaffolder at the Nuclear Envelope Mol Biol Cell 16, 3411–3424
66 Apel ED, Lewis RM, Grady RM & Sanes JR (2000) Syne-1, a dystrophinand Klarsicht-related protein associated with synaptic nuclei at the neuromuscular junction J Biol Chem 275, 31986–31995
67 Karlsson G, Liu Y, Larsson J, Goumans M-J, Lee J-S, Thorgeirsson SS, Ringner M & Karlsson S (2005) Gene expression profiling demonstrates that TGF{beta}1 sig-nals exclusively through receptor complexes involving Alk5 and identifies targets of TGF-{beta} signalling Physiol Genomics 21, 396–403
68 Yang Y-C, Piek E, Zavadil J, Liang D, Xie D, Heyer J, Pavlidis P, Kucherlapati R, Roberts AB & Bottinger EP (2003) Hierarchical model of gene regulation by trans-forming growth factor {beta} Proc Natl Acad Sci USA
100, 10269–10274
69 Pan D, Estevez-Salmeron LD, Stroschein SL, Zhu X,
He J, Zhou S & Luo K (2005) The integral inner nuclear membrane protein MAN1 physically interacts with the R-Smad proteins to repress signaling by the TGFbeta superfamily of cytokines J Biol Chem 280, 15992–16001
70 Higgins D, Thompson J, Gibson T, Thompson JD, Hig-gins DG & Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence align-ment through sequence weighting,position-specific gap penalties and weight matrix choice Nucleic Acids Res
22, 4673–4680
71 Letunic I, Copley RR, Schmidt S, Ciccarelli FD, Doerks T, Schultz J, Ponting CP & Bork P (2004) SMART 4.0: towards genomic data integration Nucleic Acids Res 32, D142–D144