Phosphorylation and ubiquitination of the IKK complex by two distinct signaling
Prashant B. Shambharkar, Marzenna Blonska, Bhanu P. Pappu, Hongxiu Li, Yun You,
Hiroaki Sakurai, Bryant G. Darnay, and Xin Lin
1. Supplemental Methods
2. Supplemental Figures
1. Supplemental Methods
Immunoprecipitation and immunoblotting
To prepare whole cell lysates, cells were lysed in a lysis buffer containing 50 mM
HEPES (pH7.4), 250 mM NaCl, 1% Nonidet P-40, 1 mM EDTA, 1 mM Na3VO4, 1 mM
NaF, 1 mM PMSF, 1 mM dithiothreitol, and a protease inhibitor cocktail (Roche
Diagnostics, Mannheim, Germany). Detection of polyubiquitination of NEMO was
previously described (Wu et al., 2006), Briefly, the lysis buffer was supplemented with 5
mM N-ethlymaleimide. The cell lysates were pre-cleared with Protein A Sepharose beads
for 4 hours, followed by overnight incubation with anti-NEMO antibody-conjugated
Protein A Sepharose beads. The immunoprecipitates were washed with lysis buffer 4
times and eluted with 2 X SDS loading buffer. The samples were fractionated on 10%
SDS-PAGE and transferred to nitrocellulose membranes for Western analysis using
In vitro kinase assay
TAK1 or IKKα/β was immunoprecipitated from variously stimulated cells.
Immunoprecipitation was performed at 4oC for 3 hrs. Resulting immunoprecipitates were
washed 3 times with lysis buffer and once with kinase buffer containing 10 mM HEPES
(pH 7.4), 1 mM MnCl2, 5 mM MgCl2, 12.5 mM glycerol-2-phosphate, 0.1 mM
Na3VO4, 4 mM NaF, and 1 mM dithiothreitol. FLAG-tag IKKβ(K44A) was expressed in
HEK293T cells, then purified by FLAG antibody-conjugated beads, and eluted using
FLAG peptides. This purified FLAG-tagged IKKβ(K44A) or bacterially expressed and
purified His-MKK6 was used as the substrate for TAK1, whereas purified GST-
IκB(1-62) was used as the substrate for IKK. For auto-phosphorylation of IKK complex
the immunoprecipitates were incubated with32P-γ-ATP alone. The immunoprecipitated
TAK1 or IKK were incubated with their substrates in the kinase buffer with 20 µM of
cold or 32P-γ-ATP at 300C for 30 min. The reactions were stopped by adding 2 X SDS
loading buffer and the samples were boiled for 5 min. The samples were fractionated on
SDS-PAGE and transferred to nitrocellulose membranes, followed by immunoblotting or
Nuclear extracts were prepared from Jurkat cells after various stimulations. Cells (1 x
107) were resuspended in 400 μl of lysis buffer (10 mM HEPES (pH 7.9), 10 mM KCl,
0.1 mM EDTA, 1mM dithiothreitol, 0.5 mM PMSF, 0.4% Nonidet P-40, and 1% protease
inhibitor mixture). Nuclear pellets were washed in lysis buffer and then resuspended in
80 μl of extraction buffer (20 mM HEPES (pH 7.9), 0.4 M NaCl, 1 mM EDTA, 1 mM
dithiothreitol, 0.5 mM PMSF, and 1% protease inhibitor mixture). After vortexing for 15
min at 4 °C, the samples were centrifuged, and the nuclear proteins in the supernatant
were collected. Protein concentrations were determined by the Bio-Rad protein assay
(Bio-Rad) using BSA standards. EMSA analysis was performed with 10 µg nuclear
extract and 32P-labeled NF-κB or Oct-1 probe.
Detection of immunological synapse formation was performed as described previously
(Wang et al., 2004). JPM50.6 and JPM50.6WT cells were transfected with 20 µg of
pEGFP-TAK1 with a Gene Pulser at 250V (950mF). 24 hr after transfection, the
transfected cells were labeled with ALEXA594-conjugated cholera toxin B (CTxB) (6µg/
ml) (Molecular Probes, Eugene, OR) at 4ºC for 20 min. Cells were then washed twice
and resuspended in serum-free RPMI1640 at 106 cells per ml. Raji cells were incubated in
serum-free RPMI1640 (Hyclone, Logan, UT) with or without 8 mg of Staphylococcus
enterotoxin E (SEE) (Toxin Technology, Inc., Sarasota, FL) per ml at 37ºC for 1.5 hr.
Raji cells were then washed and resuspended in serum-free RPMI1640 at 106 cells per
ml..A total of 0.5 ml of Raji cells were mixed with an equal volume of JPM50.6 cells,
and the cells were mixed thoroughly by pipetting and then pelleted at 500 rpm for 5 min
at room temperature. A total of 0.5 ml of supernatant was removed, and pellets were
incubated for 45 min at 37ºC. Cells were fixed in 4% paraformaldehyde at room
temperature. The cells were transferred to poly-L-lysine-coated microscope slides using
cytospin. For lipid raft aggregation experiments, primary T cells (2 X 106 /ml) were
incubated with 6µg of Cholera toxin B subunit conjugated to FITC (Sigma, St. Louis,
MO). CTx–FITC-stained T cells were washed twice and divided into two equal fractions.
The experimental cells were incubated with 5µg/ml goat anti-CTx (Calbiochem, La Jolla,
CA) and 50 ng/ml PMA plus100 ng/ml calcium ionophore for 10 min at 37 oC. The
control cells were left untreated. Cells were fixed with 4% paraformaldehyde, transferred
onto slides using cytospin, and permeabilized with 0.1% Triton X-100 in PBS. 3% BSA
in PBS was used as blocking agent. Cells were then stained with mouse anti-TAK1 (C-2)
or rabbit anti NEMO(FL-419) antibodies. Goat anti–mouse/rabbit IgG secondary
antibodies conjugated to Alexa Fluor 594 (Molecular Probes, Eugene, OR) was used to
visualize the primary antibody. Confocal images were obtained and analyzed by
OLYMPUS FLUOVIEW FV300 confocal laser scanning microscope. All images were
obtained by using sequential laser scanning mode. Figures are representative of about 100
cells from at least 3 independent experiments.
2. Supplemental Figures
Figure S1. IKK phosphorylation in Jurkat and JPM50.6 cells. Jurkat or JPM50.6
cells (1 x 107) were stimulated with or without anti-CD3-28 or PMA/ionomycin for 15
min. Total cell lysates were subjected to immunoprecipitation with a combination of
IKKα/β and IKKα antibodies for 4 hrs. The immunoprecipitate were resolved by SDS-
PAGE and analysed by immunoblotting with indicated antibodies. Lane ‘L’- Anti-CD3-
CD28 stimulated Jurkat cell lyaste. Lane ‘M’- Molecular weight.
Figure S2. The kinetics of IκBα and IκBβ phosphorylation and degradation in
Jurkat and JPM50.6 cells. Jurkat or JPM50.6 cells (1 x 107) were stimulated with or
without Anti-CD3-CD28 or TNFα for indicated time period. Total cell lysates were
subjected to SDS-PAGE and analyzed by immunoblotting by indicated antibodies.
Figure S3. IKK activity is dependent on CARMA1. Jurkat or JPM50.6 cells
(8x106/sample) were stimulated with anti-CD3-CD28, PMA/Ionomycin, or TNFα for
various time points. The IKK complex was immunoprecipitated using anti-IKKα/β, and
the immunoprecipitate was incubated with 32P-γ-ATP in the kinase reaction buffer for 30
min at 30oC. The reaction mixtures were subjected to SDS-PAGE and analyzed by
Figure S4. Signal-induced phosphorylation of IKKα/β is independent of CARMA1
or BCL10. Splenic B cells from wild type, CARMA1-/- or Bcl10-/- mice were stimulated
with PMA and Ionomycin (20ng/ml and 200 ng/ml). Whole cell lysates were subjected to
SDS-PAGE and analyzed by immunoblotting using indicated antibodies.
Figure S5. Anti-IgM/CD40-induced phosphorylation of IKKα/β is independent of
CARMA1 or BCL10. Splenic B cells from wild type, CARMA1-/- or Bcl10-/- mice were
stimulated with anti-IgM (Sigma) and CD40 (Biolegend) (15µg/ml and 7µg/ml
respectively). Whole cell lysates were subjected to SDS-PAGE and analyzed by
immunoblotting using indicated antibodies.
Figure S6. Inducible ubiquitination of NEMO in response to CD3-CD28
costimulation or PMA/ionomycin stimulation. JPM50.6WT (3 X 107) cells were
stimulated with or without CD3/28 or PMA/ionomycin for 20 min. The cell lysates were
subjected to the immunoprecipitation using anti-NEMO, antibodies and then analyzed by
immunoblotting using indicated antibodies. Lane ‘L’: lysate alone. To avoid detection of
IgG heavy chain mouse anti-NEMO antibodies was used for immunobloting, whereas,
rabbit anti-NEMO antibodies were used for immunoprecipitation.
Figure S7. CARMA1 induces K63-linked, but not K48-linked, NEMO
ubiquitnation. NEMO was co-expressed with or without CARMA1 along with plasmids
encoding wild type, K63 only, or K48 only variant of HA-tagged ubiquitin in HEK293
cells. Twenty-four hours after transfection, NEMO was immunoprecipitated and analyzed
by immunoblotting with mentioned antibodies. To avoid the detection of IgG heavy
chain, Myc-NEMO proteins were immunoprecipitated with Rabbit anti-Myc antibodies
and immunoblotted with mouse anti-NEMO antibodies.
Figure S8. CARMA1-L808P mutant is defective in its sub-cellular localization.
JPM50.6, JPM50.6WT or JPM50.6L808P (2 X 107) cells were stimulated with anti-CD3-
CD28 (10 and 5 µg/ml, respectively) for 6 min and lysed in 1% Triton-X buffer. The
lysates were subjected to sucrose density centrifugation to obtain lipid rafts and soluble
fractions. These fractions were pooled and subjected to SDS-PAGE and analyzed by
immunoblotting using the mentioned antibodies. Total cell lysates were also analyzed by
SDS-PAGE and immunoblotting using indicated antibodies.
Figure S9. Statistical analysis of TAK1-GFP recruitment to lipid rafts in
JPM50.6WT and JPM50.6 cells. The localization of TAK1-GFP in c-SMAC vs p-
SMAC of the SEE-treated Jurkat-Raji conjugates in Figures 7C and D were counted. The
aggregated lipid raft area was considered as c-SMAC and outside of the aggregated lipid
raft area was considered as p-SMAC.
Figure S10. Signal-induced subcellular localization of TAK1 is independent of
CARMA1 in primary mice T cells. Peripheral lymph nodes T cells from wild type (A)
and CARMA1-/- (B) mice were labeled with FITC-CTxB. Lipid raft aggregation was
induced by stimulating the cells with PMA and ionomycin (50ng/ml and 100ng/ml,
respectively) and crosslinked by anti-CTxB antibodies. Control cells were left untreated.
The labeled cells were stained with mouse anti-TAK1 antibodies, and followed with
ALEXA-conjugated goat anti-mouse IgG antibodies (Red). The localization of lipid rafts
(Green) and TAK1 (Red) was examined by confocal microscopy. Yellow in merged
images indicates the colocalization of TAK1 and lipid rafts. All results are representative
of three independent experiments.