MAN1 at the nuclear
envelope blocks TGF-
β
signaling by stimulating
dephosphorylation of the
Smad transcription factors
Stopping the signal at the inner nuclear
envelope. Activation of cell-surface receptors
for transforming growth factor
β
(TGF-
β
)
results in changes in gene expression
mediated by the transcription factors Smad2
and Smad3. Bourgeois et al. examined how
the nuclear membrane protein MAN1 inhibits
TGF-
β
signaling. Modeling from NMR and
SAXS data, biochemical assays, and cellular
assays revealed that MAN1 prevented
Smad2 and Smad3 from binding to purified
and cellular transcription factor FAST1
and to cellular Smad4, the latter of which
promotes transcription regulation by Smad2
and Smad3. In addition, in vitro assays
indicated that MAN1 could facilitate
the dephosphorylation and inactivation
of Smad2 and Smad3 by the phosphatase
PPM1A. These results contribute to explain
why individuals deficient in the gene
encoding MAN1 have developmental defects
suggestive of aberrant TGF-
β
signaling.
In the metazoan cell, the genome is
protected by a double membrane rich
in proteins called the nuclear envelope.
Increasing evidences indicate that the
inner nuclear envelope modulates gene
expression by recruiting histone modifying
enzymes and sequestering transcription
factors (1). This essential functional role
is supported by the large number of
mutations causing diseases found in inner
nuclear membrane proteins that impact
cell differentiation, tissue development
and aging (2,3).
The Laboratory of Structural Biology
and Radiobiology at CEA Saclay has
used Nuclear Magnetic Resonance (NMR),
Small Angle X-ray Scattering (SAXS) and
Molecular Modeling to describe the 3D
structure of several inner nuclear envelope
protein fragments, often composed
of globular domains linked by poorly
structured polypeptide chains (see for
example (4,5,6)). However, very few 3D
structures of complexes involving inner
nuclear protein regions are yet available.
Here, we report a model of the complex
between the C-terminal region of the
inner nuclear envelope protein MAN1
and the MH2 domain of the Smad2
transcription regulator. In order to calculate
this model, the 3D structure of the MH2
domain of Smad2 was obtained by X-ray
crystallography, the 3D structure of the
Smad2 binding domain from MAN1 was
calculated from NMR and SAXS data, and
the relative position of the two domains in
the complex was derived from biochemical
identification of the residues at the
interface and SAXS data on the global
shape of the complex. From this model,
we proposed mechanisms for the inhibition
of the TGF-
b
signaling pathway by MAN1.
In particular, our model suggested that
when Smad2 is bound to MAN1, it can still
bind to its essential coactivator Smad4.
Interaction of MAN1 with the Smad2/
Smad4 complex was confirmed in vitro.
However, overexpression of MAN1 clearly
inhibited the formation of the Smad2/
Smad4 complex in cell.
We showed that MAN1 overexpression led
to dephosphorylation of Smad2 in cell, and
it is known that dephosphorylated Smad2
cannot bind to Smad4. Finally, we showed
in vitro that MAN1 binds to PPM1A,
a phosphatase that dephosphorylates
Smad2. Thus we propose that in cell,
MAN1 recruits PPM1A, favors Smad2
dephosphorylation and inhibits the TGF-
b
signaling pathway. These results explain
why patients showing genetic bone
diseases due to a deletion of the Smad2
binding domain of MAN1 present aberrant
TGF-
β
signaling.
BIOLOGY AND HEALTH SCIENCES
58
SYNCHROTRON
HIGHLIGHTS
2013
