SUPPLEMENTAL INFORMATION
SUPPLEMENTAL MATERIALS AND METHODS
Plasmids.
Human TRIM45 cDNAs were amplified from a human liver cDNA library by PCR with
BlendTaq
(Takara)
using
the
5’-AGTATGTCAGAAAACAGAAAACCG-3’
5’-CCATCAGAGAGCCACAGTCCTAAG-3′
following
primers:
(TRIM45-sense)
(TRIM45-antisense).
The
and
amplified
fragments were subcloned into pBluescript II SK+ (Stratagene). Deletion mutants of
TRIM45
were
amplified
using
the
following
forward
primers:
5’-CCCTTCTCAGTAGTGGACATC-3’
(TRIM45
RING),
5’-AGCAATGTCATCCACAAGCAT-3’
(TRIM45
B-box),
5’-CAATATAGCACCCGTCCTGGA-3’ (TRIM45 FLMN). TRIM45 (BBC) cDNA
was amplified using the following primers: 5’-TACATTTCCTACACCCCCAAG-3’
and 5’-GCCGCTGGTCAGCAAGTG-3’. cDNAs of Flag-tagged or HA-tagged
TRIM45 and its deletion mutants were then subcloned into pCR (Invitrogen) and pCGN
vectors. cDNAs of human RACK1 and its deletion mutants were amplified using
combinations of the following primers: 5’-GCCATGACTGAGCAGATGACC-3’
(RACK1-sense),
5’-CTTCTAGCGTGTGCCAATGGT-3’
(RACK1-antisense),
5’-GAGAGCCACTCAGAGTGGGTG-3’
(RACK1
WD1-3),
5’-CAGGTTCCATACCTTGACCAG-3’
(RACK1
WD5-7),
1
5’-GAGATCCCATAACATGGCCTG-3’
WD6-7),
(RACK1
and
5’-TAAATCCCAGATCTTGATGCT-3’ (RACK1 WD7). Human RACK1 ΔWD4 was
amplified using the following primers and full-length construct of human RACK1 that
was
subcloned
into
pBluescript
II
5’-ATTGGCCACACAGGCTATCTG-3’
5’-GGTATTCCATAGCTTGATGGT-3’
SK+
vector
(RACK1
(RACK1
as
a
template:
WD4-sense)
WD4-antisense).
cDNAs
and
of
Flag-tagged or HA-tagged RACK1 and its deletion mutants were subcloned into pCR
and pCGN vectors. A plasmid encoding for human PKCβII was amplified using the
following primers: 5’-AAGATGGCTGACCCGGCTGCG-3’ (PKCβII-sense) and
5’-TACTTAGCTCTTGACTTCGGGTTT-3’ (PKCβII-antisense). HA-tagged PKCβII
cDNAs were subcloned into pCGN vectors. HA-tagged PKCβII NPS1 was from
Addgene (Cambridge, MA, USA). His6-tagged ubiquitin was described previously2. All
of these constructs was confirmed by DNA sequencing.
Transfection, immunoprecipitation, and immunoblot analysis.
Cells were transfected by the calcium phosphate method and lysed in a solution
containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Nonidet P-40, leupeptin (10
μg ml-1), 1 mM phenylmethylsulfonyl fluoride, 400 μM Na3VO4, 400 μM EDTA, 10
mM NaF, and 10 mM sodium pyrophosphate. The cell lysates were centrifuged at
16,000 × g for 15 min at 4°C, and the resulting supernatant was incubated with
antibodies for 2 h at 4°C. Protein A-sepharose (GE Healthcare) that had been
equilibrated with the same solution was added to the mixture, which was then tumbled
2
for 1 h at 4°C. The resin was separated by centrifugation, washed five times with
ice-cold lysis buffer, and then boiled in SDS sample buffer. Immune complexes were
detected with primary antibodies, horseradish peroxidase–conjugated antibodies to
mouse or rabbit IgG (1:10,000 dilutions, Promega) and an enhanced chemiluminescence
system (GE Healthcare). For small-scale transfection, Fugene HD reagent (Roche) was
used according to the manufacturer’s protocol.
Ni-NTA pull-down assay.
Cell
lysates
containing
8
M
urea
were
used
for
purification
of
His6-ubiquitin–conjugated proteins by chromatography on ProBond resin (Invitrogen),
and proteins were then eluted from the resin with a solution containing 50 mM sodium
phosphate buffer (pH 8.0), 100 mM KCl, 20% glycerol, 0.2% NP-40, and 200 mM
imidazole2.
PKC kinase assay
HeLa cell lines stably expressing Flag-tagged TRIM45 or mock cells were transfected
with plasmids encoding HA-tagged PKC or an empty vector by lipofection, and cellular
proteins were extracted by cell lysis in PKC extraction buffer (50 mM HEPES, pH 7.5,
150 mM NaCl, 0.1% Tween-20, 1 mM EDTA, 2.5 mM EGTA, 10% glycerol) that
contained protease inhibitors and phosphatase inhibitors. HA-tagged PKC proteins were
immunoprecipitated with antibodies for 2 h at 4°C. Protein A-sepharose (GE
Healthcare) that had been equilibrated with the same solution was added to the mixture,
3
which was then tumbled for 1 h at 4°C. The resin was separated by centrifugation, and
washed five times with ice-cold lysis buffer and then twice with kinase buffer (50 mM
HEPES (pH 7.5), 10 mM MgCl2, 1 mM DTT). The kinase assay was initiated by adding
kinase buffer containing 1 mM CaCl2, 1 mM ATP, and PMA (10 ng ml-1). Reactions
were performed at 30°C for 30 min. The reactions were terminated by adding SDS
sample buffer and boiling for 5 min. The reaction products were analyzed by
SDS-PAGE and Western blotting.
In vitro ubiquitylation assay.
In vitro ubiquitylation assays were performed as previously described3. In brief,
portions of column fractions were mixed with 50 ng of recombinant E1 (Boston
Biomedica), 100 ng of Ubc2B, 3 μg of ubiquitin (Sigma), and 50 ng of recombinant
TRIM45 in a final volume of 10 μl containing 40 mM Hepes-NaOH (pH 7.9), 60 mM
potassium acetate, 2 mM dithiothreitol, 5 mM MgCl2, 0.5 mM EDTA, 10% glycerol
and 1.5 mM ATP. Reaction mixtures were incubated for 30 min at 26 °C. The reaction
was terminated by the addition of SDS sample buffer containing 4% β-mercaptoethanol
and heating at 95°C for 5 min. Samples were subjected to immunoblotting with
anti-Flag-TRIM45 antibodies.
Real-time quantitative PCR.
The
sequences
of
primers
used
5’-ATAAACAGTGCCCTCCAGAAGCGA-3’
4
are
as
follows:
TRIM45,
and
5’-AATGGCCTTAATGTAGCCCTCCGA
-3’;
5’-AGATGAACTCTTTCTGGCCTGCCT-3’
c-Jun,
and
5’-ACACTGGGCAGGATACCCAAACAA-3’;
JunB,
5’-AATGGAACAGCCCTTCTACCACGA-3’
and
5’-GGCTCGGTTTCAGGAGTTTGTAGT-3’;
Fos,
5’-AGATTGCCAACCTGCTGAAGGAGA-3’
and
5’-TCAGATCAAGGGAAGCCACAGACA-3’;
EGR1,
5’-AGCCAAGCAAACCAATGGTGATCC-3’
5’-ACGGAACAACACTCTGACACATGC-3’;
and
Cyclin
5’-ATGAGCATGTCACCGTTCCTCCTT-3’
5’-TCAGCTGGCTTCTTCTGAGCTTCT-3’;
and
Cyclin
5’-TCATGGCTGAAGTCACCTCTTGGT-3’
5’-TCCACTGGATGGTTTGTCACTGGA-3’;
D1,
and
Cyclin
5’-AGGAACCACACCACATCTAAGCCT-3’
5’-TGACTAGCCACCGAAATGCAGACA-3’;
A2,
D3,
and
Cyclin
5’-TGTCCTGGATGTTGACTGCCTTGA-3’
E,
and
5’-TGTCGCACCACTGATACCCTGAAA-3’;
ACTB,
5’-AATGTGGCCGAGGACTTTGATTGC-3’
and
5’-AGGATGGCAAGGGACTTCCTGTAA-3’;
ACAGCTACGGAACTCTTGTGCGTA-3’
CAGCCAAGGTTGTGAGGTTGCATT-3’;
Myc,
and
5’5’BDNF,
5’-TAACGGCGGCAGACAAAAAGA-3’ and 5’-GAAGTATTGCTTCAGTTGGCCT;
5
A20,
5’-ATTCAGCCCAAGGTTCCTC-3’
and
5’-AGCCAAGACGATGAAGCAGT-3’.
Protein stability analysis with cycloheximide.
Cells were cultured with cycloheximide (Sigma) at a concentration of 50 μg mL-1 and
then incubated for various times. Cell lysates were then subjected to SDS-PAGE and
immunoblot analysis with antibodies to RACK1, c-Myc, FLAG and β-actin.
Yeast two-hybrid screening.
Yeast strain L40 (MATa LYS2::lexA-HIS3 URA3::lexA-lacZ trp1 leu2 his3) (Invitrogen)
was transformed both with plasmid pBGK1 encoding Flag-tagged TRIM45 and with a
human B cell cDNA library in the pACT2 vector (Clontech). The cells were then
streaked on plates of a medium lacking histidine to detect interaction-dependent
activation of HIS3 according to the protocol of the manufacturer (Clontech).
ChIP assay.
We performed ChIP as previously described4. Briefly, cells from one 10 cm dish (∼1 ×
107) of HeLa cells grown to 70% – 80% confluence were used for each
immunoprecipitation. Cells were cross-linked with 1% formaldehyde in PBS for 20 min
at room temperature. The cells were resuspended and lysed in lysis buffer (0.5% SDS,
10 mM EDTA, 150 mM NaCl, 50 mM Tris-HCl pH 8.0) and were sonicated with a
Bioruptor Sonicator (Diagenode) for 30 × 30 s at the maximum power setting to
6
generate DNA fragments of ∼150-400 bps. Sonicated chromatin was incubated at 4°C
overnight with 10 µg of normal IgG or anti-c-Fos antibody (0.2 mg ml-1, K-25, Santa
Cruz). Then protein A agarose was added and incubated for 2 h at 4°C. Beads were
washed twice with IP buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM EDTA,
1% Triton X-100), 2 times with high-salt buffer (20 mM Tris-HCl pH 8.0, 500 mM
NaCl, 2 mM EDTA, 1% Triton X-100), once with LiCl buffer (250 mM LiCl, 20 mM
Tris-HCl pH8.0, 1 mM EDTA, 1% Triton X-100, 0.1% NP40 and 0.5% NaDOC), and
twice with TE buffer. Bound complexes were eluted from the beads with 100 mM
NaHCO3 and 1% SDS by incubating at 50°C for 30 min with occasional vortexing.
Crosslinking was reversed by overnight incubation at 65°C. Immunoprecipitated DNA
and input DNA were treated with RNase A and Proteinase K by incubation at 45°C.
DNA was purified using a QIAquick PCR purification kit (28106, QIAGEN) or
MinElute PCR purification kit (28006, QIAGEN). Immunoprecipitated and input
material was analyzed by quantitative PCR. ChIP signal was normalized to total input.
The primer sequences of the promoter region of the TRIM45 gene used for ChIP-qPCR
are
5’-AGGAATCAAGTGAAACAGTCGCCC-3’
and
5’-TCCAGTTGCGTCAGCTTGTCTACT-3’.
SUPPLEMENTAL REFERENCES
1.
Soh JW, Weinstein IB. Roles of specific isoforms of protein kinase C in the
transcriptional control of cyclin D1 and related genes. J Biol Chem 2003; 278:
34709-16.
2.
Okumura F, Hatakeyama S, Matsumoto M, Kamura T, Nakayama KI.
7
Functional regulation of FEZ1 by the U-box-type ubiquitin ligase E4B contributes to
neuritogenesis. J Biol Chem 2004; 279: 53533-43.
3.
Kamura T, Hara T, Matsumoto M, Ishida N, Okumura F, Hatakeyama S, et al.
Cytoplasmic ubiquitin ligase KPC regulates proteolysis of p27(Kip1) at G1 phase. Nat
Cell Biol 2004; 6: 1229-35.
4.
Takahashi H, Parmely TJ, Sato S, Tomomori-Sato C, Banks CA, Kong SE, et
al. Human mediator subunit MED26 functions as a docking site for transcription
elongation factors. Cell 2011; 146: 92-104.
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SUPPLEMENTAL FIGURE LEGENDS
Figure S1 RACK1 binds to TRIM45 in a yeast expression system, related to Figure 1.
Yeast two-hybrid screening for TRIM45 interacting proteins. RACK1 was identified as
a TRIM45-interacting protein using a human B cell cDNA library. pBGK1 and pACT2
vectors were used as negative controls. TRIM6 and Myc cDNAs were used as positive
controls . BD, binding domain; AD, activating domain.
Figure S2 In vivo binding assay between HA-tagged full-length TRIM45 (WT) and
Flag-tagged deletion mutants of TRIM45. Immunoprecipitated HA-TRIM45 (WT) was
probed for co-precipitating Flag-TRIM45.
Figure S3 Ubiquitin ligase activity of TRIM45. (a) E2 preference of TRIM45.
His6-Flag-TRIM45 was purified and incubated with ubiquitin, ATP, recombinant E1
enzyme and various types of recombinant E2 enzymes as described in Methods.
Ubiquitylation was detected by immunoblotting using an antibody against
Flag-TRIM45. (b) In vivo ubiquitylation assay of HA-RACK1 by Flag-TRIM45.
Expression vectors encoding Flag-tagged TRIM45, HA-tagged RACK1, and
His6-tagged ubiquitin were transfected into HEK293T cells. Cell lysates were used for
pull-down assays with nickel-affinity resin and immunoblotted with antibodies against
HA-tag, His6-tag or Flag-tag. (c) An in vitro ubiquitylation assay was performed with
the indicated combinations. (d) Protein stability analysis of endogenous RACK1 in
HeLa S3 cells stably expressing Flag-TRIM45. Twenty-four h after seeding on dishes,
the cells were cultured in the presence of cycloheximide (50 g ml-1) for the indicated
times.
Figure S4 TRIM45 negatively regulates NF-κB-mediated transcription.
(a) Silencing
of TRIM45 upregulates TNF-indued NF-B-mediated A20 gene transcription in HeLa
cells. Thirty-six hours after transfection, cells were treated with TNFα (20 ng ml-1) and
cultured for an additional 6 h. The relative A20 gene mRNA expression level in control
cells that had been treated without TNFα was defined as 1. Data are means ± s.d. of
values from three independent experiments. P values for indicated comparisons were
determined by Student’s t test. (b) PKC does not bind TRIM45. HA-tagged PKC and
9
Flag-tagged TRIM45 were transiently transfected into HEK293T cells. (c) Exogenous
Flag-TRIM45 does not affect endogenous levels of RACK1 and PKC in a
dose-dependent fashion in HeLa cells. HeLa cells were transfected with plasmids
encoding Flag-TRIM45 (5, 10 g) or an empty vector. The cell lysates were checked by
immunoblot analysis using anti-PKC antibody, anti-RACK1 antibody and anti-Flag
antibody. Anti--actin antibody was used as an internal control. (d) Exogenous
expression of TRIM45 represses phosphorylation of the PKC substrate in a kinase assay.
An expression vector encoding HA-PKC or the corresponding empty vector was
transfected into HeLa S3 cells stably expressing Flag-tagged TRIM45 protein or mock
cells. The anti-phospho-PKC substrate antibody that was used in this assay detects
endogenous levels of cellular phospho-PKC substrates in HeLa S3 cells. (e) TRIM45
inhibited phosphorylation of PKC substrates under the condition of PMA stimulation in
HeLa cells.
Figure S5 TRIM45 does not affect mRNA levels of c-Myc and BDNF genes, which are
also considered to be regulated by RACK1 gene activity, after stimulation with sera .
Figure S6 Silencing of TRIM45 does not affect mRNA levels of BDNF genes after
stimulation with sera.
10