MINIREVIEW
What 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 differentiation, 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-

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.
1374 FEBS Journal 274 (2007) 1374–1382 ª 2007 The Author Journal compilation ª 2007 FEBS
through 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

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

xMAN1) comprise an RR-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].

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
What MAN1 does to the Smads L. Bengtsson
1376 FEBS Journal 274 (2007) 1374–1382 ª 2007 The Author Journal compilation ª 2007 FEBS

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

MAN1 lowers the cellular pool of phosphorylated R-
Smads [15,16] and prevents accumulation of 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
L. Bengtsson What MAN1 does to the Smads
FEBS Journal 274 (2007) 1374–1382 ª 2007 The Author Journal compilation ª 2007 FEBS 1377
of 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-

ing as a ‘molecular filter’, catching 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 phosphatase 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

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

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