Nucleic Acids Research Advance Access originally published online on January 27, 2009
Nucleic Acids Research 2009 37(4):e30; doi:10.1093/nar/gkp020
Nucleic Acids Research, 2009, Vol. 37, No. 4 e30
© 2009 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Mutually exclusive STAT1 modifications identified by Ubc9/substrate dimerization-dependent SUMOylation
Susan Zimnik,
Matthias Gaestel and
Rainer Niedenthal*
Institute for Physiological Chemistry/Biochemistry, Medical School Hannover, Carl-Neuberg Strasse 1, 30625 Hannover, Germany
*To whom correspondence should be addressed. Tel: +49 511 532 2826; Fax: +49 511 532 2827; Email: Niedenthal.Rainer{at}MH-Hannover.de
Received December 5, 2008. Revised January 6, 2009. Accepted January 7, 2009.
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ABSTRACT
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Post-translational modifications control the physiological activity
of the signal transducer and activator of transcription STAT1.
While phosphorylation at tyrosine Y701 is a prerequisite for
STAT1 dimerization, its SUMOylation represses the transcriptional
activity. Recently, we have demonstrated that SUMOylation at
lysine K703 inhibits the phosphorylation of nearby localized
Y701 of STAT1. Here, we analysed the influence of phosphorylation
of Y701 on SUMOylation of K703
in vivo. For that reason, an
Ubc9/substrate dimerization-dependent SUMOylation (USDDS) system
was developed, which consists of fusions of the SUMOylation
substrate and of the SUMO-conjugating enzyme Ubc9 to the chemically
activatable heterodimerization domains FKBP and FRB, respectively.
When FKBP fusion proteins of STAT1, p53, CRSP9, FOS, CSNK2B,
HES1, TCF21 and MYF6 are coexpressed with Ubc9-FRB, treatment
of HEK293 cells with the rapamycin-related dimerizer compound
AP21967 induces SUMOylation of these proteins
in vivo. For STAT1-FKBP
and p53-FKBP we show that this SUMOylation takes place at their
specific SUMOylation sites
in vivo. Using USDDS, we then demonstrate
that STAT1 phosphorylation at Y701 induced by interferon-β
treatment inhibits SUMOylation of K703
in vivo. Thus, pY701
and SUMO-K703 of STAT1 represent mutually exclusive modifications,
which prevent signal integration at this molecule and probably
ensure the existence of differentially modified subpopulations
of STAT1 necessary for its regulated nuclear cytoplasmic activation/inactivation
cycle.
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INTRODUCTION
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Functions of proteins are often controlled by post-translational
modifications, such as phosphorylation, myristoylation, acetylation,
ubiquitination and SUMOylation. These modifications can be constitutively
or regulated and often prime or hinder further modifications
(
1). Protein SUMOylation is a reversible conjugation process
with strong similarity to ubiquitination where the SUMO protein
is attached in a process of three enzymatic steps via an isopeptide
bond to the
-amino group of a lysine residue of the substrate
protein. In a fourth step, SUMOylation can be released by SUMO-specific
proteases (
2). SUMOylation is involved in the regulation of
several proteins and, consequently, potentially interferes with
other regulatory protein modifications. Interestingly, some
transcription factors, such as HSF1, GATA-1 and MEF2A, are regulated
by phosphorylation-dependent SUMOylation (
3–5), while
MEF2D, HIC1, NF-IL-6 and SP-3 show an interplay between SUMOylation
and acetylation (
6–9). Furthermore, SUMOylation competes
with IkB
ubiquitination (
10) and, as we have demonstrated recently
using Ubc9 fusion-directed SUMOylation (UFDS), SUMOylation inhibits
STAT1 phosphorylation at Y701 (
11).
The in vivo analysis of the interplay between different protein modifications mentioned above is often hampered by the low level of the specific modifications in the cell and by the lacking possibilities to manipulate a specific protein modification independently of other modifications. To increase the low level of SUMOylation in vivo, we have developed Ubc9 fusion-directed SUMOylation (UFDS) (11). However, because of the static fusion of Ubc9 to the substrate protein of interest, this method is not suited to study the kinetics of SUMOylation or the sequential SUMOylation after different preceding modification events. To overcome these limitations, we now introduce the Ubc9/substrate dimerization-dependent SUMOylation (USDDS) system. Instead of a static fusion, this system makes use of a chemically inducible interaction between Ubc9 and the substrate of interest allowing substrate-directed SUMOylation in vivo at a controlled time point. USDDS now enables us to study induced SUMOylation in dependence on other pre-existing modifications. Here, we demonstrate USDDS with eight substrate proteins and used USDDS to analyse the effect of STAT1 phosphorylation at Y701 on its SUMOylation at K703.
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MATERIALS AND METHODS
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Plasmids
We amplified the cDNA encoding for the FKBP domain from pC4EN-F1E
and the FRB (T2098L) domain from pC4-RHE (ARGENT Regulated Heterodimerization
Kit) by PCR using the primers FKBP-EcoRI (5'-GCGCGAATTCTCCAGAGGAGTGCAGGTGGAAACCATC-3')
and FKBP-XbaI (5'-GCGCTCTAGATTAACTAGTTTCCAGTTTTAGAAGCTC-3')
or the primers FRB-EcoRI (5'-GCGCGAATTCTCCAGAATCCTCTGGCATGAGATGTGG-3')
and FRB-XbaI (5'-GCGCTCTAGATTAACTAGTCTTTGAGATTCGTCGGAACACATGATA-3')
and cloned it into the EcoRI and XbaI sites of pcDNA3 (Invitrogen)
to obtain the pcDNA3-MCS-FKBP/FRB expression vectors. We have
taken the cDNA-encoding human STAT1a from the pcDNA3-STAT1-Ubc9
plasmid (
11) by BamHI/EcoRI digestion, and cloned it into the
BamHI and EcoRI sites of pcDNA3-MCS-FKBP/FRB to generate the
mammalian STAT1-FKBP/FRB expression vectors. Dependent on an
EcoRI site in the coding sequence, seven C-terminal amino acids
of the human STAT1a in the STAT1-FKBP/FRB fusion proteins are
missing. We then have taken the cDNA coding for human p53 from
the plasmid pcDNA3-p53-Ubc9 by BamHI/EcoRI digestion, and cloned
it into the BamHI and EcoRI sites of pcDNA3-MCS-FKBP/FRB to
generate the mammalian p53-FKBP/FRB expression vectors. For
generation of the destination vector (pcDNA3-RfB-FKBP) for fusion
of open reading frames to the N-terminus of FKBP, we amplified
the cDNA encoding the FKBP domain from pC4EN-F1E by PCR using
the primers FKBP-EcoRV (5'-GCGCGATATCTCCAGAGGAGTGCAGGTGGAAACCATC-3')
and FKBP-XhoI (5'-GCGCCTCGAGTTAACTAGTTTCCAGTTTTAGAAGCTC-3')
and cloned it into the EcoRV and XhoI sites of pcDNA3 (Invitrogen)
to obtain the pcDNA3-MCS-FKBP2 expression vector. We then inserted
the Gateway RfB recombination cassette (Invitrogen) into the
EcoRV site of the pcDNA3-MCS-FKBP2. The ORF-FKBP fusion protein
expression vectors were obtained by recombination of the above
described destination vector with the ORF (
Table 1) harbouring
entry plasmids using the Gateway recombination system (Invitrogen).
Transfection, cell lysis and western blotting
HEK293 cells were cultured in Dulbecco's modified Eagle's medium
with high glucose, complemented with 10% fetal calf serum, 2
mM
L-glutamine, 100 U/ml penicillin and 100 mg/ml streptomycin.
We performed transfection of 50–80% confluent HEK293 cells
in 12-well plates using the polyethylenimine transfection reagent
according to Christina Ehrhardt
et al. (
13). We grew the transfectants
for 24 h, then lysed them in 150 µl of gel loading buffer
[160 mM Tris–HCL, pH 6.8, 4% (w/v) sodium dodecyl sulphate
(SDS), 20% (v/v) glycerol, 0.5% β-mercaptoethanol (v/v),
0.008% (w/v) bromophenol blue] and incubated them for 10 min
at 95°C. For western blot analysis, we separated the proteins
by SDS–PAGE, blotted the proteins on a PVDF membrane and
detected them with specific primary antibodies [
-Ubc9 (H81,
Santa Cruz),
-pY701-STAT1 (Tyr701, Cell Signaling),
-STAT1 (Cell
Signaling),
-p53 (1C12, Cell Signaling),
-SUMO1 (Cell Signaling),
-FKBP (1-026A, Affinity Bioreagents)], a horseradish peroxidase-conjugated
secondary antibody, the Immobilon
TM Western (Millipore) and
the LAS-3000 imaging system (Fuji). For the interferon-stimulation
experiments, we grew the transfectants for 24 h and then either
stimulated them with 1000 U/ml of interferon-β or left
them unstimulated for 1 h. Then the cells were incubated further
0, 1, 2 or 4 h in the medium with the interferon-β without
or after adding AP21967 to a final concentration of 1 µM.
Then we lysed the cells and analysed the proteins by western
blotting.
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RESULTS
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AP21967-dependent binding of Ubc9 to various substrate proteins induces their SUMOylation
Post-translational protein modifications can act separately,
together or even counteract each other to integrate extracellular
signals and to ensure a specific function of a protein (
1).
To characterize the interplay of SUMOylation with other covalent
modifications in a direct and controlled way, we generated an
inducible USDDS system (
Figure 1). USDDS replaces the static
protein fusion-immanent properties of UFDS (
11) with the ‘protein
matchmaker’ approach utilizing rapamycin-induced heterodimer
formation between the 12 kDa-FK506-binding protein (FKBP12)
and the 12 kDa-FKBP12-rapamycin-associated protein (FRB) which
together form a relatively stable ternary complex (
14). Accordingly,
we fused the appropriate protein domains of FKBP and FRB, which
can be heterodimerized by the synthetic rapamycin derivative
AP21967 (
15), to Ubc9 and the SUMOylation substrate of interest,
respectively.
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Figure 1. Schematic representation of the USDDS. The SUMOylation substrate of choice is fused to one of the heterodimerization domains (FRB) and Ubc9 to the other (FKBP). When the fusion proteins are coexpressed in HEK293 cells, incubation with the membrane permeable compound AP21967 induces heterodimerization of the two fusion proteins. As a result, the SUMO-loaded conjugating enzyme Ubc9 is brought in close proximity to the substrate of interest and effective SUMO conjugation of the substrate occurs.
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As proteins of interest we have chosen STAT1 and, the tumour
suppressor protein p53, which displays significant SUMOylation
in vivo (
16–19). To generalize the approach, we also analysed
three SUMOylation substrates identified previously by UFDS and
verified without Ubc9 fusion, CRSP9, FOS and CSNK2B (
12), as
well as further potential nuclear proteins and SUMOylation substrates
in USDDS (summarized in
Table 1). All fusion proteins were expressed
in HEK293 cells to detectable levels (
Figure 2A–J and
data not shown). When p53-FRB or STAT1-FRB were coexpressed
with Ubc9-FKBP together with EGFP-SUMO1, no significant EGFP-SUMOylation
of STAT1-FRB and of p53-FRB could be detected (
Figure 2A and
B). In contrast, incubation of the transfected cells with the
dimerizer AP21967 leads to a strongly enhanced SUMOylation of
STAT1-FRB and p53-FRB already after 1 h, which reaches saturation
after 2–4 h (
Figure 2A and B). The estimated stoichiometry
of the SUMOylation of STAT1 and p53 in USDDS is similar to that
of UFDS. However, USDDS clearly functions in an inducible manner.
We also tested STAT1-FKBP, p53-FKBP and FKBP fusions of the
proteins listed in
Table 1 in combination with Ubc9-FRB. Again,
we found an even slightly stronger, AP21967-induced SUMOylation
of the STAT1- and p53-FKBP fusion proteins (
Figure 2C and D).
Furthermore, we found AP21967-induced SUMOylation of the CRSP9-FKBP,
FOS-FKBP, TCF21-FKBP, CSNK2B-FKBP, MYF6-FKBP and HES1-FKBP (
Figure 2E–J).
Overall, the results by the USDDS system summarized in
Table 1 resemble the data obtained using the UFDS system. However, there
are clear differences in SUMOylatability at least for HES1 or
MYF6, which are SUMOylated in USDDS only. These differences
could result from structural constrains of the static UFDS system,
which are not present in the more flexible USDDS approach.
USDDS targets the specific SUMOylation sites
To prove that USDDS displays specificity for the
in vivo SUMOylation
sites of p53 and STAT1, we coexpressed the mutant proteins p53K386R-FRB
and STAT1K703R-FRB with Ubc9-FKBP and EGFP-SUMO1. In HEK293
cells, we could not identify any SUMOylation of STAT1K703R-FRB
(
Figure 3A) but only weak second site SUMOylation of p53K386R-FRB
(
Figure 3B) which was also detected with the UFDS system (
11).
Obviously, the induced heterodimerization of the Ubc9 fusion
protein with the substrate fusion proteins leads preferentially
to a modification at their specific SUMOylation sites. This
let us suppose that USDDS is a useful tool to analyse the dynamic
interplay between SUMOylation and other protein modifications.
USDDS demonstrates that pY701 excludes K703 SUMOylation of STAT1
It has been shown that interferon-
stimulation of STAT1-transfected
COS7 cells, a treatment which also increases STAT1 phosphorylation
(
20), leads to an enhanced SUMOylation of STAT1 (
21,
22). This
let us postulate that STAT1 phosphorylation at Y701 might directly
enhance the SUMOylatability of STAT1 at K703. Using UFDS, we
have already demonstrated that STAT1 SUMOylation at K703 inhibits
Y701 phosphorylation (
11), but it was not possible to study
the opposite effect by UFDS (data not shown), possibly due to
the static SUMOylation of STAT1 in this experimental setting.
Because of that, we now used USDDS to study the effect of STAT1
phosphorylation at Y701 on K703 SUMOylation
in vivo (
Figure 4).
Therefore, we first coexpressed STAT1-FRB, Ubc9-FKBP and EGFP-SUMO1
in HEK293 cells. After 24 h, we stimulated the cells with interferon-β
for 1 h to induce STAT1 phosphorylation and subsequently incubated
the cells with 1 µM AP21967 to induce STAT1 SUMOylation
by the heterodimerization of STAT1-FRB with Ubc9-FKBP. The transfectants
were lysed after different incubation times and the proteins
were analysed by a pY701-STAT1 specific antibody that recognizes
the tyrosine 701 phosphorylation also in SUMOylated STAT1 (
11)
(
Figure 4A). It can be seen that interferon-β stimulation
induced Y701 phosphorylation of endogenous STAT1 and of the
STAT1-FRB fusion protein. However, pY701 could not be detected
in the gel region where SUMOylated STAT1-FRB migrates, neither
after 1 h nor 2 h or 4 h of interferon-β stimulation. A
subsequent western blot using a STAT1 specific antibody clearly
detects SUMOylated STAT1-FRB after 1, 2 and 4 h incubation with
AP21967. Hence, STAT1 phosphorylation at Y701 excludes K703
SUMOylation.
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Figure 4. Mutually exclusive in vivo phosphorylation of Y701 and SUMOylation of K703 in STAT1. (A–C) For USDDS, STAT1-FRB or STAT1-FKBP was coexpressed with Ubc9-FKBP or Ubc9-FRB and EGFP-SUMO1 in HEK293 cells. After 24 h, the transfectants were stimulated with interferon-β (1/2 h or 1 h) or left unstimulated (–) and were subsequently treated with AP21967 (1 µM). (B and C) Where indicated transfectants were treated (2 h) with AP21967 (1 µM) first and subsequently stimulated with interferon-β (1 h). The proteins of the transfectants were immunoblotted with a phospho (p)Y701 STAT1 antibody (-pY701 STAT1), stripped and re-probed with a STAT1 antibody (-STAT1) to detect also non-phosphorylated and SUMOylated STAT1. (D) Schematic representation of the role of mutually exclusive STAT1 modifications. The phosphorylation site Y701 of STAT1 and the SUMOylation site K703 are in close proximity. Receptor activation, e.g. by interferon-β induces phosphorylation at Y701. This is a prerequisite for the STAT1 dimerization, nuclear import and transcriptional activation and inhibits SUMOylation at K703. STAT1 is inactivated by a nuclear phosphatase (PPase). Dephosphorylated STAT1 is then a potential substrate for SUMOylation that inhibits nuclear re-phosphorylation of Y701 of STAT1 and is possibly involved in transcriptional reprogramming, nuclear export or regulation of further STAT1 modifications such as acetylation. SUMOylation of cytoplasmic STAT1 inhibits Y701 phosphorylation and could be involved in nuclear import and regulation of preceding STAT1 modifications like acetylation. E-S1-STAT1-FRB or -FKBP = STAT1-FRB or -FKBP fusion protein conjugated with coexpressed EGFP-SUMO1, P-STAT1-FRB or -FKBP = STAT1-FRB or -FKBP phosphorylated at Y701, E = EGFP-Tag. E-S1-STAT1-FRB or -FKBP, P-STAT1-FRB or -FKBP, endogenous P-STAT1, STAT1-FRB or -FKBP and endogenous STAT1 are indicated by black arrow heads. In the upper blot, the open arrow head indicates the positions of the E-S1-STAT1-FRB or -FKBP that are not decorated by the pY701-STAT1 antibody (-pY701-STAT1).
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Y701 phosphorylation and K703 SUMOylation are mutually exclusive
The results obtained by UFDS (
11) and USDDS (above) let us suppose
that Y701 phosphorylation and K703 SUMOylation are mutually
exclusive modifications. To further characterize the dynamic
interplay between Y701 phosphorylation and K703 SUMOylation
of STAT1 by USDDS
in vivo, we applied USDDS to analyse the influence
of K703 SUMOylation on Y701 phosphorylation of both STAT1-FRB
and STAT1-FKBP (
Figure 4B and C) and extended to analysis of
the influence of Y701 phosphorylation on K703 SUMOylation on
STAT1-FKBP (
Figure 4C). Therefore, we transfected HEK293 cells
with the respective expression plasmids and stimulated the transfectants
first with interferon-β and then with AP21967, or vice
versa. Western blot analysis of the transfectants for the STAT1-FRB
(
Figure 4B) or STAT1-FKBP (
Figure 4C) revealed that no double
modification of STAT1 by SUMOylation and Y701 phosphorylation
is detectable under any of the stimulation scenarios although
single Y701 phosphorylation or SUMOylation are clearly detectable.
Hence, STAT1 is phosphorylated at Y701 or SUMOylated at K703
but cannot carry both modifications at the same time.
A closer inspection of the blots also shows a weak decrease in the pY701 signal of STAT1-FRB and STAT1-FKBP when the cells were stimulated first with interferon-β and then with AP21967 (Figure 4A and B, P-STAT1-FRB; Figure 4C, P-STAT1-FKBP). This is possibly due to the kinetics of the interferon-β stimulation in HEK293 where the induced tyrosine 701 phosphorylation declines after about 2 h of stimulation. Furthermore, SUMOylation of STAT1-FRB (FKBP) could reduce the amount of protein that can be re-phosphorylated at Y701. Apart from this, we cannot completely rule out that there is also an effect of AP21967 on dephosphorylation or degradation of pY701-STAT1-FRB. However, USDDS clearly reveals that STAT1 SUMOylation at K703 and STAT1 phosphorylation at Y701 are mutually exclusive while interferon-β stimulation does not inhibit overall SUMOylation in the cell (Figure 4).
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DISCUSSION
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In the control of protein function, post-translational protein
modifications can act separately, together or even counteract
each other to integrate extracellular signals and to ensure
a specific function of a protein at the appropriate localization
within the cell (
1). Here, we have characterized the dynamic
interplay between Y701 phosphorylation and K703 SUMOylation
of STAT1 by USDDS. The results obtained together with the finding
that SUMOylation of STAT1 at K703 inhibits the phosphorylation
at Y701 (
11) support a model where phosphorylation of tyrosine
701 und SUMOylation of lysine 703 of STAT1 represent mutually
exclusive modifications which prevent signal integration at
these molecule and probably ensure the existence of differentially
modified subpopulations of STAT1, pY701-STAT1 and SUMO-K703-STAT1,
necessary for its regulated nuclear cytoplasmic activation/inactivation
cycle (
Figure 4D). pY701-STAT1 dimerizes, translocates to the
nucleus and becomes part of the transcriptional initiation complex
at STAT1-specific genes. Generation of SUMO-K703-STAT1 possibly
takes place in the nucleus after dephosphorylation of STAT1
and could be necessary to inhibit a fast re-phosphorylation
of STAT1 directly in the nucleus before it is exported to the
cytoplasm. Hence, a second round of STAT1 transcriptional activation
can only begin in the cytoplasm. We can also not exclude that
the two differently modified STAT1 populations, pY701-STAT1
and SUMO-K703-STAT1, are essential components of different transcriptional
complexes at different subsets of genes. Accordingly, generation
of SUMO-K703-STAT1 after dephosphorylation of pY701-STAT1 in
the nucleus could lead to transcriptional reprogramming where
SUMOylation could also act as prerequisite for further modifications
such as acetylation (
23). Apart from this, it is possible that
the STAT1 SUMOylation takes place in the cytoplasm, where it
could inhibit the Y701 phosphorylation or is involved in an
alternative nuclear import. USDDS will be probably helpful in
answering these open questions in further studies, where a detailed
characterization of the functional properties of the overexpressed
STAT1 fusion proteins will be also required.
Our results are partially confirmed by recent data that show a reduced SUMOylation of a STAT1 pY701-peptide in vitro (24) and are apparently in contrast to the described enhancement of STAT1 SUMOylation by interferon- stimulated phosphorylation of STAT1. However, very recently an interferon- induced STAT1 phosphorylation of S727 by the MKK6/p38 pathway has been described, which enhances SUMOylation at K703 (25). Hence, the intriguing possibility exists that, depending on the phosphorylated site, STAT1 K703 SUMOylation can be stimulated or inhibited offering the possibility for a complex regulation.
The analysis of the interplay of different modifications at one protein will be one of the difficult tasks to be solved in future description of signalling processes. We have developed the USDDS system that combines the effective and specific SUMOylation of UFDS with an inducible heterodimerization that makes it possible to reach controlled SUMOylation of a substrate protein at any time point within a sequential scenario of modification events. Although we can not exclude some forced artificial SUMOylation by the USDDS, we have demonstrated that this is a unique method for studying the kinetics and the dynamic interplay of protein SUMOylation with other post-translational modifications.
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FUNDING
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Institute for Physiological Chemistry/Biochemistry; the HiLF
program of the Hannover Medical School (to R.N.). Funding for
open access charge: Medizinische Hochschule Hannover, Institut
für Physiologische Chemie.
Conflict of interest statement. None declared.
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ACKNOWLEDGEMENTS
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We thank Alexey Kotlyarov for helpful discussions, Astrid Jacobs
for help in cell culture and ARIAD for providing the ARGENT
Regulated Heterodimerization system (
www.ariad.com/regulationkits).
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