Vitamin B6 conjugation to nuclear corepressor
RIP140 and its role in gene regulation
M D Mostaqul Huq1, Nien-Pei Tsai1, Ya-Ping Lin1, LeeAnn Higgins2& Li-Na Wei1
Pyridoxal 5¢-phosphate (PLP), the biologically active form
of vitamin B6, is an important cofactor in amino acid
metabolism1, and supplementary vitamin B6 has protective
effects in many disorders2–5. Other than serving as a cofactor,
it can also modulate the activities of steroid hormone
receptors6–9and transcription factors10. However, the
molecular basis of this modulation is unclear. Here, we
report that mouse nuclear receptor interacting protein 140
(RIP140) can be modified by PLP conjugation. We mapped the
modification site to Lys613 by LC-ESI-MS/MS analysis. This
modification enhanced its transcriptional corepressive activity
and its physiological function in adipocyte differentiation. We
attribute this effect to increased interaction of RIP140 with
histone deacetylases and nuclear retention of RIP140. This
study uncovers a new physiological role of vitamin B6 in gene
regulation by PLP conjugation to a transcriptional coregulator,
which represents a new function of an old form11of
protein post-translational modification that has important
RIP140 is a ligand-dependent corepressor for many nuclear receptors.
Animal studies have revealed an important role for RIP140 in
reproductive biology and fat metabolism12–15. An LC-ESI-MS/MS
analysis of tryptic digests of the recombinant RIP140 expressed in
Sf21 insect ovary cells showed that the peptide spanning amino acid
residues 608–630 has a +228 Da mass shift (expected +229 Da)
relative to the unmodified peptide, which suggests a possible post-
translational modification of RIP140 by PLP (1) (Fig. 1a). A theore-
tical +229 Da mass shift (http://www.unimod.org/) was predicted for
PLP conjugation to lysine (2)16. Whereas the precursor ion mass of the
modified peptide showed a +228 Da shift for PLP-lysine Schiff base
(3) (Supplementary Fig. 1 online), the MS/MS analysis of the
precursor ion by collision-induced dissociation indeed showed two
protonated PLP fragment ions, at 230.12 m/z (229 Da +1H) and
229.12 m/z (228 Da +1H), which are attributable to 229 Da and
228 Da mass shifts, respectively (Supplementary Figs. 2 and 3 online).
Consistently, two PLP-conjugated immonium ions (PLP-lysine immo-
nium ion, 4), at 329.12 m/z and 330.12 m/z, also appeared as signature
ions for PLP-conjugated lysine for PLP moiety, with 228 Da and
229 Da mass, respectively (Supplementary Fig. 3). The 228 Da mass
shift in the first MS (LC-MS) may be due to either tautomerism of
PLP or a deprotonation of the 3-OH of PLP and subsequent forma-
tion of a quinoid17–20. Together, these data support the notion that
RIP140 expressed in insect cells is covalently modified by PLP.
The MS/MS spectrum covered the sequence of the modified
peptide, as determined by both y ions (y1–y9, y14, y15and y17) and
b ions (particularly b1–b5) (Supplementary Fig. 3). The spectrum
showed the y17
were also shown in the unmodified peptide spectrum. This suggested
modification on Lys613. In addition, the precursor mass of several b
ions and a ions, including b6-H2O at 883.32 m/z, [a8-H2O]+3at 341.13
m/z (1,023.38, M+3H), a10
741.80 m/z (1,483.60, M+2H), demonstrated a +229 Da mass shift for
To gather more evidence, we conducted in vitro PLP conjugation on
RIP140 purified from bacteria. We reduced the PLP-conjugated
RIP140 Schiff base by NaBH4to form a stable covalently conjugated
PLP-lysine adduct (reduced PLP-lysine Schiff base, 5) and to introduce
an extra 2U mass (total mass shift +2 Da). This provided further
evidence for PLP conjugation (+228 Da or + 229 Da) by a Schiff base.
As expected, a tryptic peptide spanning amino acid residues 606–630
showed a precursor ion at 685.305 m/z contributing a 231 Da (229 Da
+2H) mass shift relative to the unmodified peptide (Fig. 1b).
Although the MS/MS spectrum of the precursor ion (685.305 m/z)
of the modified peptide (Supplementary Figs. 4 and 5 online) had a
low signal-to-noise ratio (S/N), the presence of three peaks with
S/N 4 3, specifically b14
232.02 m/z (231 Da +1H) and the internal fragment 612-EKPAPSE
GAQNSTF-625-H2O (865.37 m/z), substantiated in vitro PLP con-
jugation of RIP140 at Lys613 (underlined residue). These peaks were
absent in the unmodified peptide spectra (Supplementary Figs. 4 and
5). This modification was further signified by an in vitro PLP
conjugation using a short synthetic peptide (amino acid residues
608–627), followed by ESI-MS/MS analysis (Supplementary Fig. 6
online). The MS/MS data of this reduced PLP-modified synthetic
peptide were similar to those of the in vitro–modified RIP140 full-
length protein, including d MS shift (231 Da), PLP fragment ion at
232.03 m/z and the immonium ion (reduced PLP-lysine immonium
ion, 6) at 332.12 m/z (Supplementary Fig. 6). Similarly, the MS/MS
data of the nonreduced PLP-modified syntheticpeptide were consistent
+2ion at 825.38 m/z and the b5ion at 545.22 m/z; both
+2at 613.25 m/z (1,227.50, M+2H) and a13
+2(814.34 m/z), a PLP fragment ion at
Received 5 September 2006; accepted 10 January 2007; published online 4 February 2007; doi:10.1038/nchembio861
1Department of Pharmacology and2Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota Medical School, 6-120 Jackson Hall,
321 Church Street SE, Minneapolis, Minnesota 55455, USA. Correspondence should be addressed to L.-N.W. (email@example.com).
NATURE CHEMICAL BIOLOGY
VOLUME 3NUMBER 3MARCH 2007161
with that for in vivo-modified RIP140 in ESI-MS/MS analyses (Sup-
plementary Fig. 6). It is notable that RIP140 can be modified by PLP
in vitro only at Lys613, given that it contains 88 lysine residues.
To determine whether RIP140 can be modified by PLP conjugation
in mammalian cells, and whether modification is limited to Lys613,
we developed a mouse antibody with antigen prepared from PLP-
conjugated (Lys613) RIP140 peptide (residues 608–627). We used this
antibody to monitor in vitro PLP-conjugated RIP140 on western blots,
which detected only PLP-conjugated RIP140 and not the unmodified
RIP140 (Supplementary Fig. 7 online), thereby attesting to its
specificity to PLP-conjugated RIP140 at Lys613.
We conducted a reciprocal immunoprecipitation (protein/antibody,
500 mg:5 mg) followed by western blot (1:1,000 dilution) using anti-
RIP140 or anti-PLP peptide to detect the PLP-conjugated endogenous
RIP140 in differentiated 3T3-L1 cells (adipocytes)13. The anti-PLP
peptide antibody detected PLP-conjugated endogenous RIP140 under
a normal culture condition (Fig. 2a). Under a vitamin B6–depleted
condition (by addition of the vitamin B6 antagonist 4-deoxypyridoxine
(7)8at 100 mM for 12 h), the concentration of PLP-conjugated RIP140
was reduced (Fig. 2a, left, compare lanes 2 and 3, lanes 5 and
6) but was substantially recovered upon PLP (100 mM, 12 h) loading
(Fig. 2a, lanes 7 and 8, top). Based on the western blot signal intensity,
it was estimated that approximately 20–30% of total endogenous
RIP140 was conjugated to PLP. A similar
experiment (immunoprecipitation followed
by western blot) was conducted to examine
exogenous RIP140 and a Lys613 mutant
(K613A) that cannot be modified by PLP in
COS-1 (African green monkey kidney) cells.
The anti-PLP peptide antibody detected PLP-
conjugated wild-type RIP140 but not the
K613A mutant, despite its comparable level
of expression (Fig. 2a, right). This confirmed
PLP conjugation at Lys613 of RIP140 in
We then examined the effects of PLP con-
jugation on the biological activity of RIP140.
To a Gal4 reporter, PLP rendered RIP140
more repressive (enhanced by B2.5-fold), and the vitamin B6
(Fig. 2b). We compared the wild type and its mutant (K613A)
(Fig. 2c), which showed that PLP (100 mM, 12 h) enhances the
repressive activity of the wild-type RIP140 by Bthree-fold; this
enhancement was abolished by the vitamin B6 antagonist. Although
(P o 0.05, Student’s t-test), it was not affected by the vitamin B6
antagonist (Fig. 2c). Together, these data support the conclusion that
RIP140 was modified by PLP in vivo at Lys613 and that this
modification enhanced its corepressive activity.
RIP140 exerts its repressive activity primarily by recruiting histone
deacetylases (HDACs) to the transcription complex21,22. We examined
whether vitamin B6 affects the interaction of RIP140 with HDAC3
using a mammalian two-hybrid test in COS-1 cells (Fig. 3).
PLP loading (100 mM, 12 h) enhanced RIP140 interaction with
HDAC3 by B2.5-fold; this enhancement was completely abolished
Mol. mass = 2,321.0886 Da
∆Mass = 228.1164 Da (calcd. 229.0140 Da)
∆Mass = 231.0113 Da (calcd. 231.0298 Da)
Mol. mass = 2,549.2050 DaMol. mass = 2,506.1677 DaMol. mass = 2,737.1790 Da
776.0 850.5 851.0 851.5 852.0627.5628.0 628.5629.0685.5 686.0 686.5
z = 3z = 3z = 4z = 4
Unmodified (SI: 124 c.p.s.)Unmodified (SI: 126 c.p.s.)Modified (SI: 36 c.p.s.)Modified (SI: 29 c.p.s.)
Figure 1 RIP140 is conjugated to PLP.
(a) Precursor ion of PLP-conjugated RIP140
peptide in vivo. (b) Precursor ion of PLP-
conjugated peptide from in vitro PLP-modified
RIP140. Approximately 20–25% of total RIP140
was conjugated to PLP, as estimated by TOF-MS
signal intensities (SI) of the precursor ions of
RIP140 peptides. Underlined lysine (Lys613)
within the peptide sequences indicates site of
modification by PLP. c.p.s., counts per second.
IP (endogenous RIP140)
Vit. B6 anta.
Vit. B6 anta.
IP: α-RIP140 (exogenous)
RLU (fold)RLU (fold)
Gal4Gal4Wild type K613A mutGal4-RIP140
Vit. B6 anta.
Figure 2 RIP140 is modified by PLP in mammalian cells. (a) A reciprocal
immunoprecipitation (IP)/western blot using antibody to PLP peptide and
antibody to RIP140 detected PLP-conjugated endogenous RIP140 in
differentiated 3T3-L1 cells (left). The wild-type RIP140, but not the K613A
mutant, was modified by PLP in COS-1 cells (right). IB, immunoblot; a,
antibodies for specific proteins. (b) trans-repressive activity of Gal4-RIP140
in COS-1 cells. (c) Effects of cellular vitamin B6 status on the repressive
activity of RIP140 and its mutant (K613A) in COS-1 cells. Error bars
indicate mean ± s.d. of three separate experiments. # versus *, P o 0.005;
# versus **, P o 0.05; * versus **, P o 0.05. PLP (100 mM, 12 h) and
vitamin B6 antagonist (100 mM, 12 h) were applied. RLU, relative light units.
162VOLUME 3NUMBER 3MARCH 2007NATURE CHEMICAL BIOLOGY
by depleting cells of vitamin B6 (vitamin B6 antagonist, 100 mM,
12 h). The repressive activity of RIP140 can also be regulated by its
interaction with C-terminal binding proteins (CtBPs)23,24. Notably,
vitamin B6 exerted no effect on the interaction of RIP140 with CtBP
(Supplementary Fig. 7), which supports the idea that vitamin B6
enhances the repressive activity of RIP140 by facilitating its interaction
We assessed the biological relevance of the effect of PLP conjugation
using 9-cis-retinoic acid (RA, 8) induction of the gene encoding the
RA receptor RAR-b2 as a model25. In reporter assays, the wild-type
RIP140 consistently repressed the activation of RARB reporter,
whereas the mutant (K613A), which was expressed at a similar level
(Supplementary Fig. 7), had only a partial activity (Fig. 4a). In
predifferentiated 3T3-L1 cells that lacked endogenous RIP140
(Fig. 4a), exogenously supplied RIP140 substantially repressed the
endogenous RAR-b2 gene in the presence of PLP (100 mM, 12 h)
(Fig. 4a, lanes 4 and 5). Without RIP140, PLP exerted no significant
effect on RARB expression (Fig. 4a, lanes 1 and 2). In loss-of-function
studies, two independent small interfering RNAs (siRNA1 and
siRNA2, see Methods) were used to knock down endogenous
RIP140 in differentiated 3T3-L1 cells that expressed abundant
RIP140 (Supplementary Fig. 7). As expected, PLP (100 mM, 12 h)
enhanced the repressive activity of endogenous RIP140 on the RARB
reporter (Fig. 4b, bars 2 and 4). In RIP140-silenced cells, this effect
was abrogated (Fig. 4b, bars 2 and 6), and excess PLP could no longer
significantly enhance the repression of this reporter (Fig. 4b, bars 4
and 8; Supplementary Fig. 7). The partial repression of the RARB
reporter by PLP in RIP140 knockdown cells might be due to the
modulation of supplemented RAR and retinoid X receptor (RXR) by
PLP in this overexpression system. To address the effect of PLP
conjugation on RIP140 in an entirely physiological condition, we
examined the expression of endogenous RARB in differentiated
3T3-L1 cells. It seemed that RARB was effectively repressed in cells
supplemented with vitamin B6 (Fig. 4b, lanes 1 and 2), but silencing
endogenous RIP140 abrogated vitamin B6–stimulated repression
(Fig. 4b, lanes 3 and 4).
We then evaluated the effect of PLP conjugation on the physiolo-
gical function of RIP140 in fat accumulation in adipocytes, because
vitamin B6 has been reported to facilitate adipocyte differentiation and
fat accumulation26. Endogenous RIP140 was silenced in differentiated
3T3-L1 cells using both siRNA1 (Supplementary Fig. 7) and siRNA2
(Fig. 4c). In differentiated control (scramble RNA) cells, triglyceride
(TG) content was increased relative to undifferentiated cells as
predicted, and this effect was further enhanced in cells incubated
with PLP (Fig. 4c, P o 0.005). In contrast, TG accumulation was
decreased in RIP140 knockdown cells (P o 0.05), in which vitamin B6
could no longer enhance TG accumulation (P o 0.005). These data
support a physiological role for vitamin B6 in fat accumulation,
mediated in part by modifying Lys613 of RIP140 (Fig. 4c).
Vitamin B6 may affect subcellular distribution of steroid hormone
receptors27. We examined whether vitamin B6 can affect the distribu-
tion of green fluorescent protein (GFP)-RIP140 in COS-1 cells
(Fig. 5a). RIP140 was located primarily in the nuclei (only 10–15%
of cells showed even distribution, Fig. 5a). Under a vitamin B6–
depleted (vitamin B6 antagonist, 100 mM, 12 h) condition, RIP140
was increasingly shuttled to the cytoplasm (approximately 40% of
cells, Fig. 5a). Importantly, the K613A mutant was more efficiently
exported (for approximately 65% of cells, Fig. 5a) and was affected by
disturbing vitamin B status (Fig. 5a).
We then sought to determine whether vitamin B6 can affect RIP140
transport, for example by modulating its interaction with exportin
(CRM1) or importin complexes. In coimmunoprecipitation experi-
ments, the mutant RIP140 was found to be preferentially associated
with CRM1 (Fig. 5b), which might contribute to its increased
cytoplasmic distribution. The interaction with importin-b1 was not
affected (Fig. 5b), which suggests that vitamin B6 facilitates nuclear
retention of RIP140 by reducing its interaction with the export
machinery. This was demonstrated in a direct in vitro protein-protein
interaction assay (Fig. 5c) in which direct interaction of RIP140 with
Vit. B6 anta.
RLU (× 10,000)
Figure 3 PLP conjugation enhances RIP140 interaction with HDAC3.
Shown is the mammalian two-hybrid test using Gal4-BD–RIP140 and
VP16-AD–HDAC3 in COS-1 cells. PLP and vitamin B6 antagonist
(100 mM) were added for 12 h. Error bars indicate mean ± s.d. of three
separate experiments. Gal4-BD, Gal4 DNA binding domain; VP16-AD,
Gal4 activation domain.
RLU (× 10,000)
1 2 3 4 5 6
Vit. B6 anta.
+ ++ +
TG level (fold)
Figure 4 PLP modulates the biological activities of RIP140. (a) Effects on RA induction of RAR-b2 reporter in COS-1 cells (left panel). PLP affects
endogenous RAR-b2 gene expression (real-time PCR) in undifferentiated 3T3-L1 cells (right, lanes 4 and 5). (b) PLP modulates RA induction of RAR-b2
reporter (left, bars 1–4) and endogenous RAR-b2 gene (real-time PCR) in differentiated 3T3-L1 cells (right, lanes 1 and 2). (c) PLP (100 mM, 16 h) effects
on TG accumulation in differentiated 3T3-L1 cells. All experiments were repeated at least twice. Error bars indicate mean ± s.d. * versus **, P o 0.005;
# versus ##, P o 0.05; * versus #, P o 0.05; ** versus ##, P o 0.005. scRNA, scramble RNA. Gray bars, control cells.
NATURE CHEMICAL BIOLOGY
VOLUME 3NUMBER 3MARCH 2007163
CRM1 was substantially disturbed by PLP conjugation of RIP140, as
detected by antibody to PLP-modified peptide.
This study uncovered a physiological role of vitamin B6 in gene
regulation by direct PLP conjugation to a transcription regulator,
which represents a new function of an old form of post-translational
modification. Modification of RIP140 by PLP enhances its corepres-
sive activity by increasing its interaction with HDAC3 and facilitating
its nuclear retention through disrupting interaction with the export
machinery. As a result, vitamin B6 might enhance the physiological
function of RIP140 and promote fat accumulation in adipocytes.
Materials. Commercial sources: pyridoxal 5¢-phosphate, Sigma; 4-deoxypy-
ridoxine, Sigma; 9-cis-RA, Sigma; NaBH4, Sigma; antibody to CRM1, Abcam,
Ltd; antibody to CtBP, Santa Cruz Biotechnology; antibody to karyopherin-b1,
Santa Cruz Biotechnology; and synthetic peptide spanning RIP140 amino acid
residues 608–627, EZbiolab. Mouse polyclonal antibody to RIP140 was against
the N-terminal domain (residues 1–495).
Plasmids. We described Gal4-BD and GFP fusions of full-length RIP140, GST-
RIP140, His6-RIP140 for baculovirus expression, Gal4-tk-Luc, and RAR-b2
reporter plasmids previously25,28,29, and we made the K613A mutation in GFP-
RIP140 and Gal4-BD–fused RIP140 using a QuickChange mutagenesis kit
(Stratagene). Mutagenic (bold letters) primers were as follows: sense, 5¢-GTC
CCA AGC CGA GGC ACC AGC CCC GAG TGA AG-3¢; antisense, 5¢-CT TCA
CTC GGG GCT GGT GCC TCG GCT TGG GAC-3¢.
Cell culture, transfection and reporter assays. We cultured COS-1 cells, HEK
293 cells and 3T3-L1 cells in DMEM supplemented with 10% FBS. We
conducted transfections using Lipofectamine-2000 (Invitrogen), and we carried
out mammalian two-hybrid tests in COS-1 cells.
Expression, purification and mass spectrometry of RIP140. See Supplemen-
tary Methods online.
RIP140 silencing, real-time PCR and fat accumulation in 3T3-L1 cells. We
differentiated 3T3-L1 cells to adipocytes using a differentiation mixture
containing insulin, 3-isobutyl-1-methylxanthine (IBMX), dexamethasone and
thyroid hormone as reported13. RIP140 siRNAs were siRNA1 (target sequence
5¢-GGAATGAGCTCGATTATAA-3¢, from Dharmacon)15and siRNA2 (target
sequence 5¢-AAGCTTCTTTCTTTAATCTAA-3¢, Qiagen). We harvested cells
72 h after transfection and prepared cell extracts in a coimmunoprecipitation
buffer (50 mM Tris-HCl, 100 mM NaCl, 0.1 mM EDTA, 10% glycerol, 0.1%
NP-40, pH 8.0) for estimating TG concentration by enzymatic endpoint assay
kit (Thermo DMA), and we normalized the TG concentration to the dried
weight of the total cell lysate.
In vitro PLP conjugation and generation of antibody. We conducted in vitro
PLP conjugation as reported30, separated RIP140 protein on SDS-PAGE
and followed by LC-ESI-MS/MS analysis as described29. We confirmed PLP
conjugation to the synthetic peptide by MALDI-TOF MS in a positive-ion
reflection mode (Qstar XL, Applied Biosystems) using a-cyano-4-hydroxycin-
namic acid as a matrix. We diluted the modified peptide in adjuvant for
injection. We tested the specificity of antibody to PLP-conjugated RIP140 on
Fluorescence microscopy. We obtained GFP images of cells grown in six-well
plate culture dishes using an inverted fluorescence microscope (Olympus IX70)
with ?20 objective lenses coupled to a DVC camera (Model 1412, DVC
Company). We captured each image at a fixed frame rate of 10.2 s–1, with a
resolution of 1,380 ? 1,025 (height ? width) pixels, using the acquisition
software DVC view, version 2.2.8 (DVC Company). We cropped typical fields
containing multiple cells of the unprocessed images with a similar dimension
for each image using a crop tool in Adobe Photoshop.
Statistical data analysis. We present data as mean ± s.d., and we determined
statistical significance with the Student’s t-test (two-tailed) at a confidence level
of P o 0.05.
Note: Supplementary information and chemical compound information is available on
the Nature Chemical Biology website.
This work was supported by US National Institutes of Health grants DA11190,
DA11806, DK54733, DK60521 and K02-DA13926 to L.-N.W. We also thank
the staffs of the Mass Spectrometry Consortium for the Life Sciences, Department
of Biochemistry, Molecular Biology and Biophysics at the University of
Minnesota, St. Paul for recording the mass spectra, and the staff in L.-N.W.’s lab
for their help.
M.D.M.H. conceived of and directed the project, conducted overall experiments,
interpreted data and prepared the manuscript. N.P.T. and Y.P.L. prepared
antibodies and conducted site-directed mutagenesis. L.A.H. recorded mass and
assisted in interpretation of mass data and text editing. L.-N.W. provided overall
guidance and direction in experimentation, interpretation of data and manuscript
preparation and provided all financial support.
COMPETING INTERESTS STATEMENT
The authors declare that they have no competing financial interests.
Published online at http://www.nature.com/naturechemicalbiology
Reprints and permissions information is available online at http://npg.nature.com/
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