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DMF (Sigma-Aldrich) was dissolved in 40% (v/v) DMSO (Merck) resulting in a
70 mM stock solution and diluted 10-fold in culture medium. All stock solutions
were freshly made 10 min before use. HEK293 cells (ATCC® CRL-1573) were either
left untreated or were pre-incubated with 140 μM of DMF for 1 h and stimulated12
for either 15 min (RSK2) or 30 min (MSK1) with 1 ng ml–1 of human recombinant
EGF (Pepro Tech, UK). Cells were stopped after one wash with ice-cold phosphate-
buffered saline and flash frozen in liquid nitrogen. Whole-cell extracts were
prepared by adding 400 μl of 1 × cell lysis buffer (Cell Signaling Technology) to
each 10 cm dish. The 1 × lysis buffer was supplemented with 22 μl protease
inhibitor cocktail (EDTA-free complete, Roche Diagnostics), and 10 µl PMSF per
ml buffer. The collected samples were added 1 µl Benzonase per 400 µl buffer
(MERCK, Denmark), sonicated and centrifuged for 10 min at 4 °C at 10,000 g and
the supernatants were saved for protein determination. Equal loads of protein (50
µg) were separated on pre-cast gels, SDS-Page 8–16% (Invitrogen). Proteins were
blotted onto Hybond nitrocellulose membrane (Amersham). Antibodies for
western blotting were all from Cell Signaling, Beverly, MA, USA: anti-phospho-
MSK1 (Ser376, #9591, 1:750), anti-phospho-RSK2 (Ser386, #9341, 1:750), and
mouse anti-GST (26H1, #2624, 1:2000); HRP anti-rabbit (#7074, 1:2000), anti-
mouse (#7076, 1:2000). GST-RSK2 and GST-MSK1 full-length protein of about
116 kDa were identified. Antibody binding was visualized13 by horseradish
peroxidase-conjugated second antibody in a standard ECL™ (RPN 2106) reagent on
Hyperfilm™ enhanced chemoluminescence (RPN 3103K, Amersham Bioscience),
and densitometric analysis of the band intensity was carried out using Kodak one-
dimensional imaging analysis software. Graphs were made with Sigma Plot v.11.
STATA v.14 was used to test for normal distribution and for performing Student’s
t-tests. A probability of P < 0.05 was regarded as statistically significant. Uncropped
western blots are shown in Supplementary Fig. 4.
Data collection, processing, and refinement. Crystals were mounted in nylon
loops from mother liquor supplemented with 20% (v/v) ethylene glycol and flash
cooled in liquid N2. A complete data set was collected at 100 K on the X06SA
beamline at the Swiss Light Source (Paul Scherrer Institute). The diffraction images
were indexed using XDS22 and the reflections were scaled in SCALA23. Molecular
replacement was performed with the program PHASER24 and a search model derived
from PDB ID 2QR819. Rigid body refinement, full model refinement, and calculation
of omit maps were performed in the PHENIX suite25. Model building and analysis
was performed with Coot26. Root mean square deviations were calculated, super-
positions were performed, and structural figures were prepared using PyMOL
residues in favourable region of the Ramachandran plot, and remaining 3% in the
additionally allowed region as indicated by Molprobity27
.
Bioinformatics. A multiple sequence alignment of kinases with at least 25%
sequence identity to RSK2 was prepared with PSI-BLAST with the CTKDRSK2 as
query against the human kinome and searched for kinases with a cysteine corre-
sponding to C599 (Supplementary Fig. 1). A homology models of the activated
state of RSK2 was prepared in Modeller28 based on PDB ID 3A62. The morph
between the inactive state of RSK2 (PDB ID 2QR8) and the homology model of the
.
In vitro assay. In vitro activity of murine RSK2CTKD was evaluated in the HTRF-
KinEASETM assay (Cisbio Bioassays). Evaluation of the effect of DMF and MMF
on activated RSK2CTKD was performed by activation of RSK2CTKD (2 μM) in
reaction buffer with ERK2 (0.1 μM) (Signalchem) with 200 μM ATP and 10 mM
MgCl2 in kinase buffer (50 mM HEPES pH 7.0, 2 mM TCEP, 3 mM NaN3, 0.01%
BSA, and 0.1 mM Na3VO4) for 1 h at 37 °C. In all, 50 ng activated RSK2CTKD was
mixed with DMSO or a concentration series of DMF in kinase buffer for 1 h at
room temperature in 384-well plates for fluorescence (Greiner). The reactions were
started by addition of ATP (100 μM), STK1 substrate (0.1 μM) resulting in 10 μl
reactions. The reactions were stopped after 20 min at room temperature with
Streptavidin-XL665 in EDTA (5 μl) and STK antibody cryptate (5 μl). Evaluation of
the effect of DMF on the inactive state of RSK2CTKD was evaluated by incubation
of RSK2CTKD in reaction buffer with DMSO or a concentration series of DMF in
kinase buffer followed by ERK2 activation and kinase reaction as described above.
IC50 values were calculated by non-linear regression using sigmoid concentration
response in Graph pad Prism 7. All experiments were repeated at least three times
(n = 3) and variance and two-tailed Student's t-test were applied in the statistical
analysis.
Mass spectrometry. Purified murine RSK2 was treated with 1 mM DMF or
DMSO (vehicle) and left for 2 weeks. Samples were digested with chymotrypsin.
From a single peptide mapping experiment (Alphalyse A/S, Odense, Denmark) it
was possible to identify C436, C560, C579, and C599 with DMF modifications,
whereas no peptides containing C439 were detected.
GST-RSK2 was expressed in HEK293 cells as for the mutational analyses (see
below) with the difference that HEK293 cells were transfected with 6 µg of human
GST-RSK2 plasmid DNA per dish. For affinity purification, an amount of 5.6 mg
total protein from not stimulated (empty vehicle) or from DMF-stimulated cells
was suspended in 8 ml of 1 × Lysis buffer and was added 1 ml of glutathione-
sepharose 4B beads (GE 17-0756-05, GE Healthcare Life Science) each and rotated
for 30 min on ice. Glutathione-sepharose 4B beads were previously equilibrated in
wash and binding buffer (100 mM Tris-Base pH 7.4, 0.15 M NaCl, 1 mM EDTA).
Unbound sample was removed by centrifugation 500 g for 2 min. Sepharose 4B
beads were washed with 10 ml of wash buffer two times and centrifuged at 500 g for
2 min. Proteins were eluted in two steps, step one: elution buffer of 50 mM Tris-
HCl and 20 mM reduced GSH (Sigma-Aldrich), pH 8.0, and step two: elution
buffer with 50 mM reduced GSH, 6 ml of each. The eluted protein samples were
immediately dialysed for wash buffer and overnight in Slide-A-Lyzer 10 K Dialysis
Casettes (No.66810 Thermo Fisher Scientific). The next day, proteins were freeze
dried in portions of 1 ml and re-dissolved in 40 µl of 50 mM TCEP in SDS loading
buffer + 60 µl of native buffer (Bio-Rad #161-0738) and analysed on PAGE-SDS
8–16% Tris-glycin gels. The proteins were stained by Page Blue Protein Staining
Solution (Thermo Fisher Scientific Inc). Purity was controlled by western blotting.
Proteins run on the same gel, treated by vehicle or DMF, were blotted to a
nitrocellulose membrane and tested with monoclonal antibody for RSK2 (Santa
Cruz Biotechnology sc-9986, 1:1000) and antibody for GST (Cell Signaling #2624S,
1:2000) to identify the full-length protein of about 116 kDa. Step two elution
contained a bulk amount of GST-RSK2 and gel pieces were cut out. After in-gel
digestion by trypsin or chymotrypsin and by nano-HPLC-ESI-MS/MS (Proteome
Factory, Berlin), DMF modifications of the five cysteine peptides were identified by
the chymotrypsin digests and comparing signals from the unmodified peptides in
the vehicle with DMF-treated samples in a single comparison experiment (n = 1).
The degree of modification of the peptide containing C436 and C439 could not be
determined due to the hydrophilic nature of this peptide.
Kinase panel screening. Kinase selectivity profiling for DMF was carried out as
carried out at room temperature (21 °C) and were linear with respect to time and
enzyme concentrations under the conditions used. Assays were performed for 40
min using a Biomek 2000 Laboratory Automation Workstation in a 96-well format
(Beckman Instruments, Palo Alto, CA, U.S.A.). The concentration of magnesium
acetate in the assays was 10 mM, while the concentration of [γ-33P]-ATP (800 cpm
pmol–1) used was selected to be close to the kinase’s Km for ATP. Assays were
initiated with Mg2+-ATP and stopped by the addition of 5 µl of 0.5 M ortho-
phosphoric acid. Aliquots were then spotted on to P30 filtermats, washed four
times in 75 mM phosphoric acid to remove ATP, once in methanol, then dried and
counted for radioactivity30
.
Analysis of mutants. Mutational analysis of MSK1 and RSK2 was performed in a
mammalian expression vector (pEBG2T) in which a glutathione S-transferase (GST)
domain and FLAG-tag (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) was fused to the N-
terminus of human MSK1 (GST-FLAG-MSK1). The positions corresponding to
C436, C579, and C599 in human RSK2 of human MSK1 (C440, C583, and C603)
were mutated to Val, Ser, and Thr using the QuikChange Lightning Kit (Agilent
Technologies). The Val mutants generally displayed the best ERK2 activation.
Human embryonic kidney cells (HEK293) were cultured in tissue culture flasks
(150 cm2) to 60% confluence in Dulbecco's modified Eagle's medium (DMEM,
Gibco) supplemented with 50 units ml–1 penicilin G (Gibco), 50 μg ml–1
streptomycin (Invitrogen), 5 μg ml–1 gentamycin (Gibco), 10% (v/v) foetal bovine
serum (FBS, Gibco), and 2.5% HEPES (Gibco). Cells were trypsinated and seeded
in 10 cm Petri dishes at a density of 6.5 × 106 cells per dish in 10 ml DMEM
supplemented with 50 μg ml–1 bovine pituitary growth hormone (BGH, Gibco),
antibiotics (penicilin G, streptomycin, gentamycin), 2% FBS, and 2.5% HEPES and
were incubated for 2 days. The culture medium was changed to DMEM
supplemented with 2.5% HEPES for 16 h. Transfection with plasmids was
performed as previously described31 with modifications. HEK293 cells were
transfected using 3.5 μg plasmid DNA/dish dissolved in 250 μl Optimem
(Invitrogen) and 30 μl Lipofectamine 2000 (Invitrogen) dissolved in 250 μl
Optimem added together for 20 min before transferring to cells. The Lipofectamine
and DNA complexes were incubated with cells for 6 h at 37 °C and 5% CO2. Cell
culture medium was then changed back to DMEM special growth medium with
BGH, antibiotics, 2% foetal calf serum, and 2.5% HEPES for 48 h.
Data availability
The data that support the findings of this study are available from the corresponding
author upon request. The refined coordinates and crystallographic data are available in
the Protein Data Bank (PDB) with access code 5O1S.
Received: 28 April 2017 Accepted: 23 September 2018
References
1. Mrowietz, U. et al. Efficacy and safety of LAS41008 (dimethyl fumarate) in
adults with moderate-to-severe chronic plaque psoriasis: a randomized,
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