From the
Department of Cell Biology and Genetics,
College of Life Sciences, Peking University, Beijing 100871, China and the
Department of Immunology and Cancer Center,
National Jewish Medical and Research Center, University of Colorado Health
Sciences Center, Denver, Colorado 60206
Received for publication, September 24, 2002 , and in revised form, May 2, 2003.
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ABSTRACT |
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INTRODUCTION |
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There are five members of the NF-B family in mammals, including Rel
(c-Rel), RelA (p65), RelB, NF-
B1 (p50 and its precursor p105), and
NF-
B2 (p49 and its precursor p100)
(4). The major cellular form of
NF-
B is a heterodimer consisting of the DNA-binding subunit p50 and the
transactivator p65. Normally, NF-
B is retained in the cytoplasm through
association with its inhibitor, I
B. Upon stimulation by various
NF-
B activating signals, I
B is phosphorylated and degraded
through an ubiquitin-dependent process. This process frees NF-
B, which
is then translocated into the nucleus to activate transcription of downstream
genes.
Regulation of NF-B activation does not rely entirely on I
B
phosphorylation and degradation. Various studies have shown that
phosphorylation of p65 by serine/threonine protein kinases, particularly the
catalytic subunit of protein kinase A
(PKAc),1 is critical
for its interaction with the transcriptional co-activator p300/CBP
(CREB-binding protein) and the transcriptional competence of nuclear
NF-
B (4,
913).
Gene knock-out studies of the serine/threonine protein kinases GSK-3
,
NAK/T2K/TBK, and NIK provide additional evidence of a role for nuclear
NF-
B modification in its transcriptional competence
(1417).
These studies indicate that a deficiency of these kinases does not affect
I
B degradation, NF-
B translocation into the nucleus, or binding
to DNA, but it severely impairs NF-
B-mediated transcription induced by
various stimuli.
TNF is one of the major proinflammatory cytokines
(18). This effect of TNF is
mediated through activation of NF-B
(18). The signaling pathway
that is responsible for TNF-induced NF-
B activation has been resolved
for the most part. Upon TNF stimulation, TNF receptor 1 (TNF-R1) trimerizes
and recruits the downstream death domain-containing protein, TRADD
(TNF-receptor-associated death domain), to the receptor
(19). TRADD then functions as
an adaptor to recruit several downstream proteins, including FADD
(Fas-associated death domain), TRAF2, and RIP to the TNF-R1 signaling complex
(2022).
Although FADD is critically involved in TNF-R1-mediated apoptosis, TRAF2 and
RIP recruit IKK, the I
B kinase, to the TNF-R1 complex
(23,
24). IKK contains three
subunits, the catalytic subunits IKK
and IKK
and the regulatory
subunit IKK
(4). Once
IKK is recruited to the TNF-R1 complex, it is activated and subsequently leads
to I
B phosphorylation and NF-
B activation
(2,
4).
In addition to TNF-R1, most members of the TNF receptor family can also
activate NF-B (18).
Recently, we and others
(2529)
identified a new member of the TNF family of ligands, TALL-1, also called
BAFF, Blys, THANK, and zTNF4, which is expressed by macrophages and monocytes
and is critically involved in peripheral B cell survival. TALL-1 signals
through three receptors, including BCMA, TACI, and BAFF-R
(29). All the three TALL-1
receptors belong to the TNF receptor family and are capable of activating the
transcription factor NF-
B
(3036).
In a yeast two-hybrid screening for BCMA-associated molecules, we
identified SINK, an NF-B-inducible protein sharing sequence homology to
serine/threonine protein kinases. Surprisingly, our results suggest that SINK
interacts with p65 and inhibits NF-
B-dependent transcription through
blocking p65 phosphorylation by the protein kinase PKAc. Our findings reveal a
novel negative feedback control pathway of NF-
B-dependent gene
expression.
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EXPERIMENTAL PROCEDURES |
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Yeast Two-hybrid ScreeningTo construct a BCMA bait vector, a cDNA fragment encoding amino acids 119184 of BCMA was inserted in-frame into the Gal4 DNA-binding domain vector pGBT (Clontech, Palo Alto, CA). The human B cell cDNA library (ATCC, Manassas, VA) was screened as described (1921, 38, 39).
Northern Blot HybridizationHuman multiple tissue mRNA blots were purchased from Clontech. The blots were hybridized with labeled SINK cDNA (spanning the coding region) in Rapid Hybridization Buffer (Clontech) under high stringency conditions.
Vectors and TransfectionMammalian expression vectors for HA- or FLAG-tagged SINK and PKAc were constructed by PCR amplification of the corresponding cDNA fragments and subsequently cloned into CMV promoter-based vectors containing an HA or FLAG tag.
Expression plasmids for HA-BCMA and HA-BAFF-R were previously described
(48). Expression plasmids for
TNF-R1, RIP, TRAF2, TRAF5, TRAF6, IKK, p65, p50, I
B
, and
I
B
(SS/AA) (provided by David Goeddel), MEKK1 (provided by Gary
Johnson), and NF-
B and AP1 luciferase reporter plasmids (provided by
Gary Johnson) were provided by the indicated investigators. Transfection of
293 cells was performed using the standard calcium phosphate precipitation
method (40).
Reporter Gene Assays293 cells (2 x
105) were seeded on 6-well (35-mm) dishes and were transfected the
following day by standard calcium phosphate precipitation. Within the same
experiment, each transfection was performed in triplicate, and where
necessary, a sufficient amount of empty control plasmid was added to ensure
that each transfection continued to receive the same amount of total DNA. To
normalize for transfection efficiency, 0.3 µg of RSV-
-galactosidase
plasmid was added to each transfection. Luciferase reporter assays were
performed using a luciferase assay kit (BD PharMingen) and following the
manufacturer's protocols.
-Galactosidase activity was measured using the
Galacto-Light chemiluminescent kit (Tropix, Bedford, MA). Luciferase
activities were normalized on the basis of
-galactosidase expression
levels.
Co-immunoprecipitation and Western Blot AnalysisTransfected 293 cells from each 100-mm dish were lysed in 1 ml of lysis buffer (20 mM Tris (pH 7.5), 150 mM NaCl, 1% Triton, 1 mM EDTA, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride). For each immunoprecipitation, 0.4-ml aliquots of lysates were incubated with 0.5 µg of the indicated antibody or control IgG and 25 µl of a 1:1 slurry of GammaBind G Plus-Sepharose (Amersham Biosciences) for at least 1 h. The Sepharose beads were washed three times with 1 ml of lysis buffer containing 500 mM NaCl. The precipitates were fractionated on SDS-PAGE, and subsequent Western blot analyses were performed as described (1921, 39, 40). Endogenous co-immunoprecipitation was performed as described previously (2022).
RNAi ExperimentsWe have used the RNAi system purchased from
Oligoengine Inc. A cDNA fragment containing the SINK targeting sequence
(AAGCTGTGTCGCTTTGTCTTC) was cloned into the pSuper.Retro retroviral vector,
which contains a puromycin selection maker. The resulting vector or control
empty vector was transfected into 293-10A1 packaging cells. The
retrovirus-containing supernatant was then used to infect 293 cells. The
infected 293 cells were selected by puromycin (2 µg/ml) for 3 days. The
puromycin-resistant cells were amplified. NF-B luciferase reporter gene
assays were performed as described above.
Apoptosis Assay-Galactosidase co-transfection assays
for determination of cell death were performed as described previously
(1921,
41). Briefly, 293 cells
(
2 x 105) were seeded on 6-well (35-mm) dishes and were
transfected the following day by calcium phosphate precipitation with 0.1
µg of pCMV-
-galactosidase plasmid and the indicated testing plasmids.
Within the same experiment, each transfection was performed in triplicate, and
where necessary, a sufficient amount of empty control plasmid was added to
keep each transfection receiving the same amount of total DNA. Approximately
14 h after transfection, the cells were treated with TNF (20 ng/ml) or TRAIL
(200 ng/ml) or left untreated for 12 h. Cells were then stained with X-gal as
described previously
(1921,41).
The numbers of survived blue cells from five representative viewing fields
were determined microscopically. Data shown are the averages and standard
deviations of one representative experiments in which each transfection was
performed in triplicate.
Electrophoretic Mobility Shift AssayThe electrophoretic
mobility shift assay was performed as described previously
(37). Briefly, 293 cells
stably transfected with SINK or empty control plasmid were treated with TNF
for 30 min. Cells were then harvested and incubated in 500 µl Buffer A (10
mM Hepes, 10 mM NaCl, 5 mM EDTA, 1.5
mM MgCl2,10 µg/ml aprotinin and leupeptin, 2
mM dithiothreitol, and 1 mM phenylmethylsulfonyl
fluoride, pH 7.8) at 4 °C for 10 min. 500 µl of Buffer B (Buffer A plus
1.2% Nonidet P-40) was added, and the sample was vortexed vigorously for 10 s.
The pellet was spun down, washed with Buffer A, and resuspended in 4.5 µl
of Buffer C (Buffer A plus 10% glycerol). After the addition of 5 µl of 4.1
M NaCl and incubation at 4 °C for 30 min, the supernatant
sample was centrifuged and the nuclear extract was quantified with Bio-Rad
protein assay kit. The NF-B targeting oligonucleotide, GGGGACTTTCCC
(Santa Cruz Biotechnology), was labeled with [
-32P]ATP by T4
DNA kinase and incubated with 20 µg of nuclear extract and 0.8 µg of
poly(dI-dC) in binding buffer (20 mM Tris, 50 mM KCl, 1
mM EDTA, 1 mM dithiothreitol, 0.1% Nonidet P-40, 5%
glycerol, pH 7.5) at room temperature for 15 min. The mixture was fractionated
in 5% acrylamide gel prepared in 0.5x Tris borate-EDTA. The gel was
dried and subjected to autoradiography.
In Vitro Kinase AssayCell transfection and
immunoprecipitation were carried out as described above. The
immunoprecipitates were washed twice with kinase buffer (25 mM
Tris, 5 mM -glycerophosphate, 2 mM dithiothreitol,
0.1 mM Na3VO4, 10 mM
MgCl2, pH 7.5) and then incubated in 30 µl of kinase buffer plus
100 µM ATP and 10 µci of [
-32P]ATP for 30
min at 30 °C. The beads were spun down and washed once with kinase buffer.
The proteins were fractionated by SDS-PAGE. The gel was then dried and
subjected to autoradiography.
Immunofluorescent Staining293T cells cultured on glass coverslips were plunged sequentially into methanol and acetone at -20 °C, each for 10 min. The cells were rehydrated in PBS, blocked with 1% bovine serum albumin in PBS for 15 min, and stained with a monoclonal anti-FLAG antibody (2 µg/ml) in blocking buffer for 1 h at room temperature. The cells were rinsed with PBS and stained with Texas Red-conjugated Affinipure goat anti-mouse IgG (1:200 dilution) for 45 min at room temperature. The cells were then rinsed with PBS containing Hercus and mounted in Prolong Antifade (Molecular Probes). The cells were observed with a Leica DMR/XA immunofluorescent microscope using a 100x plan objective.
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RESULTS |
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BLAST searches of the GenBankTM data bases suggested that the sequence of full-length SINK is identical to that of an uncharacterized or hypothetical protein called SKIP3 (GenBankTM accession numbers AF250310 [GenBank] , AK026945 [GenBank] , NM021156, BC027484 [GenBank] , AL034548 [GenBank] , and BC019363 [GenBank] ). It seems that SKIP3 is the human ortholog of the rat protein NIPK, which was identified as a protein up-regulated during neuronal apoptosis induced by NGF withdrawal (42), an uncharacterized mouse protein TRB3 (GenBankTM accession number AF358868 [GenBank] ), and the Drosophila protein Tribbles, which is critically involved in inhibition of cell cycle progression (4346) (data not shown). Data base searches also identified two uncharacterized or hypothetical human proteins, GS3955 (GenBankTM accession number NP_067675 [GenBank] ) and SKIP1 (GenBankTM accession number AAK58174 [GenBank] ), which are highly homologous to human SINK (Fig. 1A). Taken together, these data suggest that SINK is a member of an evolutionally conserved protein family.
Sequence analysis suggests that SINK contains a kinase-like domain and shares significant homology to serine/threonine protein kinases such as PKAc and PKA-C (Fig. 1B). However, SINK is probably not a functional kinase because it contains only 5 of the 12 subdomains found in most serine/threonine protein kinases and lacks a conserved ATP-binding site (47).
Northern blot analysis suggests that human SINK mRNA is weakly expressed in spleen, thymus, prostate, liver, and pancreas (Fig. 1C) and is undetectable in other examined tissues, including testis, ovary, small intestine, colon, leukocyte, heart, brain, placenta, lung, skeletal muscle, and kidney.
Expression of SINK Is InducibleTo detect endogenous human
SINK protein, we raised a peptide-directed rabbit polyclonal anti-SINK
antibody. Human SINK protein was barely detectable in several tested cell
lines by Western blot analysis (Fig.
2 and data not shown). Because rat NIPK is induced during neuronal
apoptosis (42), we reasoned
that human SINK is probably also an inducible protein. As shown in
Fig. 2, SINK protein expression
could be induced potently by stimulation of 293 cells with the heavy metal ion
Cd2+, TNF, and phorbol 12-myristate 13-acetate plus ionomycin.
Also, overexpression of BCMA in 293 cells could induce SINK expression, and
this was inhibited by blocking NF-B activity with an undegradable
I
B
mutant (Fig.
2). Because TNF and phorbol 12-myristate 13-acetate plus ionomycin
can also activate NF-
B in 293 cells (data not shown), collectively
these data suggest that SINK can be induced through an NF-
B-dependent
process.
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SINK Does Not Interact with BCMA in Mammalian CellsTo determine whether SINK interacts with BCMA in mammalian cells, we transfected expression plasmids for C-terminal HA-tagged BCMA and N-terminal FLAG-tagged SINK into 293 cells. We then performed co-immunoprecipitation with anti-FLAG antibody and Western blot analysis with anti-HA antibody. This experiment indicated that SINK could interact with BCMA (data not shown). However, reverse co-immunoprecipitation experiments failed to detect an interaction between BCMA and SINK (data not shown). In combination with data shown below, we conclude that SINK does not interact specifically with BCMA.
SINK Inhibits NF-B-dependent Transcription but Not
NF-
B Translocation into the Nucleus or Binding with
DNA Because SINK is homologous to PKAc, a serine/threonine kinase
involved in p65 phosphorylation and NF-
B activation, we examined the
effect of SINK on NF-
B activation by NF-
B luciferase reporter
gene assays. The results indicated that SINK inhibits NF-
B-induced
transcription triggered by overexpression of BCMA, TNF-R1, and its downstream
signaling proteins RIP, TRAF2, IKK
, and p65. These experiments indicate
that SINK inhibits NF-
B activation induced by overexpression of all the
tested proteins (Fig.
3A). SINK also inhibited NF-
B activity induced by
TRAF5 and TRAF6 (Fig.
3A), two signaling proteins involved in NF-
B
activated by multiple TNF receptor family members
(18). However, SINK did not
significantly inhibit MEKK1-induced AP1 activation
(Fig. 3B), suggesting
that SINK specifically inhibits NF-
B-dependent transcription.
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We also tested whether SINK could inhibit NF-B activation triggered
by TNF and IL-1 stimulation. The data suggest that SINK can inhibit TNF- and
IL-1-triggered NF-
B activation in a dose-dependent manner
(Fig. 3C).
To determine whether SINK has a physiological role in NF-B-induced
transcription, we obtained 293 cell lines stably expressing SINK RNAi. Western
blot analysis indicated that SINK RNAi decreased SINK protein expression
induced by Cd2+ (Fig.
3D). We then determined the effect of SINK RNAi on
TNF-induced NF-
B activation. The results indicated that SINK RNAi
expression could potentiate basal and TNF-induced NF-
B activation
(Fig. 3E). These data
suggest that SINK is a physiological inhibitor of NF-
B activation.
Because SINK can inhibit NF-B activation induced by downstream
signaling proteins, including the NF-
B transactivator p65, we reasoned
that SINK functions in the nucleus to inhibit nuclear NF-
B
transcriptional competence. To test this hypothesis, we made 293 cell lines
that stably express SINK and then examined the effect of overexpression of
SINK on TNF-induced NF-
B nuclear translocation and binding to DNA.
These experiments indicated that SINK does not affect TNF-induced NF-
B
translocation into the nucleus or binding to DNA
(Fig. 3F). We also
transfected SINK into 293 cells and performed immunofluorescent staining
experiments. The results indicated that SINK was localized primarily in the
nucleus (Fig. 3G).
Taken together, our results suggest that SINK inhibits NF-
B-dependent
transcription but not NF-
B translocation and DNA binding.
SINK Interacts with p65Because SINK inhibited the
transcriptional competence but not translocation and DNA binding of
NF-B, we reasoned that SINK might directly bind to NF-
B. To test
this possibility, we co-transfected SINK and p50, p65, or I
B
into 293 cells and performed immunoprecipitation experiments. The results
suggest that SINK interacts with p65 but not with p50 or I
B
(Fig. 4, A and
B).
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To determine whether SINK interacts with p65 under physiological condition, we induced SINK expression by Cd2+ and then performed endogenous co-immunoprecipitation. The results indicated that the induced SINK could interact constitutively with endogenous p65 (Fig. 4C).
SINK Inhibits p65 Phosphorylation by PKAcThe next question
we asked is why SINK binding to p65 affects NF-B transcriptional
competence. We first determined whether SINK could disrupt p50-p65
interaction. The results indicated that overexpression of SINK did not affect
p50-p65 interaction (data not shown). Previously, it has been shown that p65
phosphorylation by PKAc is important for the transcriptional competence of
nuclear NF-
B (4,
913).
Because SINK shares homology with PKAc
(Fig. 1B) and
interacts with p65, we tested whether SINK affects the phosphorylation of p65
by PKAc in in vitro kinase assays. As shown in
Fig. 5, PKAc could potently
phosphorylate both p65 (lanes 4 and 6) and SINK (lanes
7 and 911) in these assays. Moreover, p65 phosphorylation
by PKAc was significantly inhibited by SINK (compare lane 3 with
lane 4), and this inhibition was dose-dependent (lanes
811). These data are consistent with the conclusion that SINK
inhibits NF-
B transcription competence through blocking p65
phosphorylation by PKAc.
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SINK Sensitizes Cells to TNF- and TRAIL-induced
ApoptosisNF-B promotes cell survival through
transcriptional induction of anti-apoptotic genes
(38).
Because SINK inhibits NF-
B-dependent transcription, we determined
whether SINK affects NF-
B-dependent cell survival. Therefore, we
transfected 293 cells with SINK and examined its effect on TNF- and
TRAIL-induced apoptosis. The results suggest that SINK can significantly
sensitize 293 cells to TNF- and TRAIL-induced apoptosis
(Fig. 6).
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DISCUSSION |
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SINK shares significant sequence homology with serine/threonine protein kinases. However, SINK may not be a functional kinase because it contains only 5 of 12 conserved kinase subdomains and lacks a conserved ATP-binding site found in all serine/threonine kinases. We transfected SINK into 293 cells and found that it was weakly phosphorylated in in vitro kinase assays (Fig. 5). It is possible that SINK has intrinsic kinase activity, or alternatively, SINK is phosphorylated by an unidentified kinase. In this context, we found that SINK could be phosphorylated by PKAc (Fig. 5). We also mutated several conserved amino acids critical for kinase activity of many well studied serine/threonine kinases, including K97A, K118A, D182A, K184A, and found that mutation of the sites did not affect its phosphorylation in in vitro kinase assays (data not shown). Based on these data, we believe that SINK is not an authentic kinase.
Rat NIPK was identified in a study of genes up-regulated during nerve
growth factor withdrawal-induced neuronal apoptosis
(42). It was also found that
rat NIPK was up-regulated by other apoptotic stimuli, such as serum depletion
or DNA damage in PC6-3 and NIH3T3 cells
(42). These studies suggest
that rat NIPK is involved in either the promotion or inhibition of apoptosis.
In human cells, SINK is weakly expressed or undetectable. Interestingly, we
found that various stimuli, including Cd2+, TNF, phorbol
12-myristate 13-acetate plus ionomycin, and overexpression of BCMA, could
induce expression of SINK protein. Because IB
could inhibit
BCMA-induced SINK expression, we conclude that SINK can be induced in a
NF-
B-dependent manner.
Our results suggest that overexpression of SINK inhibits
NF-B-dependent transcription but not NF-
B translocation and DNA
binding, whereas SINK RNAi potentiates basal and TNF-induced NF-
B
activation. Co-immunoprecipitation experiments suggest that SINK interacts
directly with p65 and inhibits p65 phosphorylation by PKAc. Previously, it has
been well documented that PKAc-mediated p65 phosphorylation is critically
involved in NF-
B transcriptional competence. Thus, we reason that SINK
inhibits NF-
B-dependent transcription through blocking p65
phosphorylation by PKAc or its related kinases. Consistent with its role in
inhibiting NF-
B-dependent transcription, SINK also sensitizes cells to
TNF and TRAIL-induced apoptosis, probably through inhibition of expression of
NF-
B activated anti-apoptotic genes.
In conclusion, we have identified an NF-B-inducible protein that can
inhibit NF-
B-dependent transcription. Our findings reveal a novel
negative feedback control pathway for NF-
B-dependent transcription and
cell survival.
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FOOTNOTES |
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¶ To whom correspondence should be addressed: Dept. of Immunology, National Jewish Medical and Research Center, 1400 Jackson St., k516c, Denver, CO 80206. Tel.: 303-398-1329; Fax: 303-398-1396; E-mail: shuh{at}njc.org.
1 The abbreviations used are: PKAc, catalytic subunit of protein kinase A;
CREB, cAMP-responsive element-binding protein; TNF, tumor necrosis factor;
TNF-R1, TNF receptor 1; TRAF, TNF receptor-associated factor; RIP,
receptor-interacting protein; IKK, the IB kinase; RNAi, RNA
interference; HA, hemagglutinin; IL-1, interleukin 1; TRAIL, TNF-related
apoptosis-inducing ligand; BCMA, B cell maturation protein; CMV,
cytomegalovirus; BAFF, B cell-activating factor; MEKK1, mitogen-activated
protein kinase/extracellular signal-regulated kinase kinase kinase 1; RSV,
Rous sarcoma virus; X-gal,
5-bromo-4-chloro-3-indolyl-
-D-galactopyranoside; PBS,
phosphate-buffered saline; SINK, p65-interacting
inhibitor of NF-
B; NIPK, nerve growth
factor-induced protein kinase.
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ACKNOWLEDGMENTS |
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REFERENCES |
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