(Received for publication, January 3, 1997, and in revised form, May 20, 1997)
From the University of Colorado Health Sciences
Center, Department of Pharmacology, School of Medicine, Denver,
Colorado 80262 and the ¶ Gladstone Institute of Virology and
Immunology and
Departments of Medicine and Microbiology and
Immunology, University of California, San Francisco, San Francisco
General Hospital, San Francisco, California 94141-9100
Nuclear factor B (NF-
B) is a eukaryotic
member of the Rel family of transcription factors whose biological
activity is post-translationally regulated by its assembly with various
ankyrin-rich cytoplasmic inhibitors, including I
B
. Expression of
NF-
B in the nucleus occurs after signal-induced phosphorylation,
ubiquitination, and proteasome-mediated degradation of I
B
. The
induced proteolysis of I
B
unmasks the nuclear localization signal
within NF-
B, allowing its rapid migration into the nucleus, where it
activates the transcription of many target genes. At present, the
identity of the I
B
kinase(s) that triggers the first step in
I
B
degradation remains unknown. We have investigated the
potential function of the 90-kDa ribosomal S6 kinase, or
pp90rsk, as a signal-inducible I
B
kinase.
pp90rsk lies downstream of mitogen-activated protein (MAP)
kinase in the well characterized Ras-Raf-MEK-MAP kinase pathway that is induced by various growth factors and phorbol ester. We now show that
pp90rsk, but not pp70S6K or MAP kinase,
phosphorylates the regulatory N terminus of I
B
principally on
serine 32 and triggers effective I
B
degradation in
vitro. When co-expressed in vivo in COS cells,
I
B
and pp90rsk readily assemble into a complex that
is immunoprecipitated with antibodies specific for either partner.
While phorbol 12-myristate 13-acetate produced rapid activation of
pp90rsk, in vivo, other potent NF-
B
inducers, including tumor necrosis factor
and the Tax
transactivator of human T-cell lymphotrophic virus, type I, failed to
activate pp90rsk. These data suggest that more than a
single I
B
kinase exists within the cell and that these I
B
kinases are differentially activated by different NF-
B inducers.
Nuclear factor B
(NF-
B)1 is a transcription
factor whose function is regulated by a family of cytoplasmic
inhibitors termed the I
Bs (reviewed in Refs. 1 and 2). At present,
nine I
B family members have been identified (I
B
, I
B
,
I
B
, I
B
, I
B
, p105, p100, Bcl-3, and Cactus), each
distinguished by the presence of multiple ankyrin repeats. The
prototypic and best studied of the I
Bs is I
B
(3), which binds
to the heterodimeric NF-
B complex (p50/Rel A) (4), masks the nuclear
localization signal present in Rel A (5, 6), and sequesters NF-
B in
the cytoplasm (4-6). When appropriate inductive signals are delivered
to the cell, phosphorylation of I
B
ensues (7-10), followed by
the conjugation of multiple ubiquitin molecules and the degradation of
the ubiquitinated I
B
phosphoprotein by the 26 S proteasome
complex (11-13). Of note, I
B
degradation proceeds while the
inhibitor is still physically associated with the NF-
B heterodimer
(10, 14-17). However, the NF-
B complex is ultimately liberated,
allowing its rapid translocation into the nucleus, where it engages
cognate enhancer elements and alters the transcriptional activity of
various target genes.
Although phosphorylation of IB
is required for its proteolysis
and the subsequent activation of NF-
B, the nature of the cellular
protein kinase(s) mediating this reaction remains unknown. Signal-induced phosphorylation involves two serine residues located at
positions 32 and 36 near the N terminus of I
B
. Substitution of
these serines with alanine residues generates a constitutively acting
I
B
repressor that readily binds to NF-
B but fails to undergo
signal-induced phosphorylation and degradation (7, 8, 10, 18).
Studies in Drosophila have also yielded valuable insights
into the biology of the Rel proteins and their control by the IBs. In the dorsal-ventral signal transduction pathway of
Drosophila, dorsalizing signals mediated through the
receptor Toll (an IL-1 receptor homologue) target Cactus (a member of
the I
B family) for degradation and result in activation of Dorsal (a
Rel family member) (19). In this pathway, Pelle, a serine/threonine
protein kinase, regulates the degradation of Cactus through
phosphorylation, although it is unknown whether Pelle acts directly or
indirectly on Cactus (20). Recently, a human IL-1 receptor-associated
kinase has been cloned that is homologous to Pelle (21). This kinase appears to participate in the IL-1-induced signaling pathway leading to
NF-
B induction, but no evidence yet exists for its direct phosphorylation of I
B
.
Casein kinase II (CKII) also phosphorylates IB
in vivo
(22-24). However, phosphopeptide mapping of phosphorylated I
B
has shown that residues within the C-terminal PEST region, rather than
the N-terminal serines, are targeted by CKII. Phosphorylation of the C
terminus of I
B
by CKII or other kinases may play a role in the
constitutive degradation of uncomplexed I
B
. Additionally, CKII-mediated phosphorylation appears important for the accelerated turnover of I
B
and the persistent induction of NF-
B observed following HIV infection of macrophages (24). Immunodepletion of CKII
from these cell extracts results in an inhibition of I
B
degradation in vitro (24).
Recently, a novel ubiquitination-stimulated protein kinase has been
identified that phosphorylates IB
in a serine
32/36-dependent manner (25). This kinase resides in a large
700-kDa multiprotein complex, and ubiquitination of some component of
the complex results in increased I
B
phosphorylating activity.
Whether ubiquitination directly activates the kinase or, alternatively,
acts indirectly to alter another component of the complex remains
unresolved.
We have investigated the potential role of a known intracellular
protein kinase in the Ras-Raf-MEK-MAP kinase signaling pathway as an
IB kinase. This enzyme, the 90-kDa ribosomal S6 kinase, or
pp90rsk, lies immediately downstream of MAP kinase in the
phorbol ester and growth factor signaling pathway (26, 27). We now show that pp90rsk phosphorylates the N terminus of I
B
principally on serine 32 and functionally induces I
B
degradation
in vitro. We further show that pp90rsk and
I
B
can physically associate in vivo. Finally, we show
that only a subset of the known NF-
B-inducing signals leads to the activation of pp90rsk. These findings suggest that rather
than a single I
B kinase, a family of I
B kinases may exist within
the cell that are differentially activated by different inducers of
NF-
B.
The expression
vector containing a hemagglutinin-epitope-tagged pp90rsk
cDNA (pMT2-HA-RSK1) was provided by Dr. Joseph Avruch (Harvard University and Massachusetts General Hospital, Boston, MA). Wild type
IB
cDNA provided by Dr. Al Baldwin (University of North Carolina, Chapel Hill, NC) was cloned into the HindIII and
XbaI sites of the pCMV4 eukaryotic expression vector
provided by Dr. Mark Stinski (University of Iowa, Iowa City, IA). For
in vitro translation, wild type I
B
or mutant I
B
containing alanine for serine substitutions at position 32 and/or 36 (4) cloned into the HindIII and XbaI or
XmaI sites of the pBluescript SK(+) vector (Stratagene).
Biosynthetically radiolabeled I
B
or its mutant analogues were
synthesized by transcription-coupled in vitro translation in
wheat germ extracts (Promega).
A bacterial expression plasmid encoding hexahistidine (His)-tagged
IB
was constructed by cloning the I
B
cDNA into the pTrcHisC vector (Invitrogen). The N-terminal
1-36 I
B
deletion mutant was cloned into the pRSETC vector (identical to the pTrcHis vector except in the promoter region). Wild type I
B
and the S32/36A I
B
mutant containing alanine for serine substitutions at
both residues 32 and 36 were isolated from Escherichia coli lysates by purification on a nickel chelate column (Ni-NTA, Qiagen). Following an initial wash in high salt buffer (50 mM
Tris-HCl, pH 7.5, 300 mM NaCl), successive washes were
performed with elution buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl) containing increasing concentrations of imidazole
(0, 10, 50, 100 mM). The His-tagged proteins were eluted in
buffer containing 200 mM imidazole. The fractions
containing the desired proteins were dialyzed overnight in 50 mM Tris-HCl, pH 7.5, and 2 mM DTT.
Jurkat cells and
Jurkat cells stably expressing either HTLV-I Tax or Tax antisense
cDNA constructs and a neomycin resistance gene were maintained in
RPMI 1640 supplemented with 10% fetal calf serum and
penicillin/streptomycin at 37 °C in 5% CO2; 800 µg/ml
G418 was added to the Jurkat-Tax and anti-Tax cell culture media. Cells
were treated with phorbol 12-myristate 13-acetate (PMA) (50 ng/ml) or
TNF- (50 ng/ml) for various periods of time. Vehicle controls
corresponding to the amounts of added Me2SO for PMA and
water for TNF-
were performed in parallel. The cells were washed
once with ice-cold phosphate-buffered saline and lysed in ELB buffer
(50 mM HEPES, pH 7.4, 250 mM NaCl, 0.2%
Nonidet P-40, 5 mM EDTA, 0.5 mM DTT, 1.0 mM phenylmethylsulfonyl fluoride, and protease inhibitor
mixture containing 0.75 µg/ml bestatin; 0.5 µg/ml each of
aprotinin, antipain, leupeptin, and trypsin inhibitor; 0.4 µg/ml
phosphoramidon; and 0.05 µg/ml pepstatin). The cell lysates were
clarified by centrifugation at 4 °C for 15 min at 15,000 × g, and the supernatant was used for immunoprecipitation as
described below.
Phosphorylation
of bacterially expressed IB
(0.5 µg) was performed in 50-µl
reaction mixtures incubated for 15 min at room temperature. For
Xenopus laevis egg-derived MAP kinase, X. laevis egg-derived pp90rsk, and mouse pp70S6K, the
reaction buffer contained 20 mM HEPES, pH 7.0, 10 mM MgCl2, 2 mM DTT, 100 µM EGTA, 0.1 mg/ml bovine serum albumin, 100 µM ATP, 25 µCi of [
32P]ATP (specific
activity 3000 Ci/µmol). Myelin basic protein (0.1 mg/ml, Sigma) or 40 S ribosomal subunits (0.05 mg/ml) generously provided by Dr. James
Maller (Howard Hughes Medical Institute and University of Colorado
Health Sciences Center, Denver, CO) were used as positive controls for
these kinase reactions. Aliquots of the reactions were mixed in
SDS-PAGE sample buffer, heated to 98 °C, and microcentrifuged at
15,000 × g for 2 min, followed by analysis of the
supernatants by SDS-PAGE. Alternatively, I
B
was
immunoprecipitated from the reactions with peptide-specific polyclonal
rabbit antisera (2.5 µl) recognizing the C-terminus of I
B
.
Immune complexes were reacted with formalin-fixed Staphylococcus aureus cells (Pansorbin A, 50 µl, Calbiochem) and collected by centrifugation. The Protein A-bound immune complexes were washed three
times and similarly analyzed by SDS-PAGE and autoradiography. In the
case of pp90rsk, three independently purified preparations
were tested. The specific activities of these pp90rsk
preparations were 4.6, 8.9, and 11.2 nmol of ATP incorporated/min/mg of
protein assayed using Kemptide as substrate.
Bacterially produced His-tagged IB
was
phosphorylated with purified pp90rsk as described above and
subjected to mild V8 proteolysis at room temperature for 10 min. The
protease inhibitor
N
-p-tosyl-L-lysine
chloromethyl ketone was added to the reaction mixture together with 100 µg/ml (final concentration) of bovine serum albumin to terminate
proteolytic cleavage. I
B
peptides containing the N-terminal
His-epitope were immunoprecipitated from the digest using
his-tag-specific antibodies. The immunoprecipitates were analyzed with
Tris-Tricine gels by the method of Schägger and von Jagow
(28).
In vitro
synthesized [35S]-radiolabeled IB
was
phosphorylated with pp90rsk in the presence of unlabeled
ATP as described. The phosphorylated or control I
B
proteins (25 µl) were incubated in 100 µl of reticulocyte lysates containing 5 mM DTT, 2.5 mM ATP, 1 mM creatine
phosphate, and 200 µg/ml creatine phosphokinase. Samples were removed
at various times and quickly frozen in liquid nitrogen. The samples were then immunoprecipitated with C-terminalspecific anti-I
B
antibodies and analyzed by SDS-PAGE followed by autoradiography.
Monkey kidney COS7 cells,
maintained in complete Dulbecco's modified Eagle's medium, were
transfected with pMT2-HA-RSK1 (encoding pp90rsk-1) or
pCMV4-HA-PP2A (encoding the A regulatory subunit of protein phosphatase
2A) and CMV4-IB
using LipofectAMINE (Life Technologies, Inc.).
After 48 h, the cells were starved for 1 h in
methionine/cysteine-free Dulbecco's modified Eagle's medium and then
metabolically radiolabeled with [35S]methionine and
[35S]cysteine for 2 h. Whole-cell extracts were
prepared by lysis in ELB buffer, followed by immunoprecipitation
analyses as described above using either anti-HA-epitope antibody
(BAbCo, Berkeley, CA) or anti-I
B
antisera specific for the C
terminus of this inhibitor. Nonradioactive COS cell lysates were also
prepared and immunoprecipitated with the anti-HA or I
B
antibodies
followed by immunoblotting with anti-pp90rsk-specific
antibodies. These immunoblots were developed with a horseradish
peroxidase-conjugated secondary antibody and enhanced chemoluminescense
(ECL) as described by the manufacturer (Amersham).
Cell lysates
derived from about 5 × 106 Jurkat cells were
precleared for 1 h and incubated with 10 µl of
anti-pp90rsk antibody (Santa Cruz Biotechnology) at 4 °C
for 1 h. 30 µl of Protein A-agarose (Boehringer Mannheim) was
then added to the mixture, and the incubation was continued for an
additional 1 h. The mixture was centrifuged at 4 °C, followed
by washing of the Protein A-agarose resin three times in ELB buffer.
Immune complexes were washed three times with kinase buffer (25 mM glycerol-2-phosphate, 20 mM HEPES, pH 7.4, 10 mM MgCl2, 4 mM NaF, 2 mM MnCl2, 1 mM dithiothreitol, 0.1 mM Na3VO4, and either 20 µM ATP (specific activity 3000 Ci/µmol for
[32P]labeling of recombinant IB
) or 100 µM ATP (for phosphorylation of
[35S]-labeled I
B
)). 30 µl of the kinase buffer
containing 0.3 µg of bacterially expressed I
B
or 2 µl of
wheat germ-translated reactions of wild type or mutant I
B
was
added to the immune complexes, and the mixtures were incubated at
30 °C for 30 min for [32P]radiolabeling of I
B
or
2 h for phosphorylation of [35S]-labeled I
B
.
The reaction products were then analyzed by SDS-PAGE and
autoradiography.
Following
the phosphorylation reactions, IB
was immunoprecipitated with
anti-I
B
antiserum specific for the C-terminus and Protein
A-agarose. The bound immune complexes were washed three times in
dephosphorylation buffer (50 mM Tris-HCl, pH 8.0, 1 mM EDTA) followed by the addition of 10 units of calf
intestinal alkaline phosphatase (Boehringer Mannheim). The samples were
then incubated at 37 °C for 30 min and analyzed by SDS-PAGE and
autoradiography.
Phorbol ester is both a potent inducer of
NF-B and a known activator of the MAP kinase pathway. To determine
whether kinases positioned along this or related pathways are capable
of phosphorylating I
B
, the ability of MAP kinase,
pp70S6K, and pp90rsk to phosphorylate I
B
in vitro was examined. These kinases phosphorylate residues
that lie within specific consensus sequences. For example, MAP kinase
phosphorylates serines and threonines that precede proline residues
(proline-directed kinase consensus sequence), while both
pp90rsk and pp70S6K phosphorylate serines or
threonines within the consensus sequence Arg-X-X-Ser/Thr (29).
Recombinant His-tagged IB
was used as the substrate in these
in vitro kinase assays. Following the kinase reaction,
I
B
was immunoprecipitated from the reaction using polyclonal
anti-I
B
antibodies (30). Immunoprecipitation was performed to
ensure that the phosphorylated protein was indeed recombinant I
B
.
As shown in Fig. 1, pp90rsk
(lanes 7 and 8), but not pp70S6K
(lanes 3 and 4) or p42MAPK
(lanes 11 and 12), phosphorylated recombinant
His-tagged I
B
in vitro. The failure of the
pp70S6K and p42MAPK preparations to
phosphorylate I
B
was not due to inactivation of these enzymes
during their purification, as each capably phosphorylated known protein
substrates, including the 40S ribosomal subunit for pp70S6K
(lanes 1 and 2) and myelin basic protein for
p42MAPK (lanes 9 and 10). Of note,
while both pp90rsk and pp70S6K recognize the
same consensus phosphoacceptor site and phosphorylate the S6 protein of
the 40S ribosome, only pp90rsk phosphorylated I
B
.
Another kinase, Ca2+-calmodulin-dependent
protein kinase II (CaMKII) also shares the same consensus
phosphoacceptor site as pp90rsk and pp70S6K
(31). However, like pp70S6K, CaMKII failed to phosphorylate
I
B
in vitro, although it did phosphorylate one of its
physiological substrates, synapsin II (data not shown). These data
highlight the ability of pp90rsk to utilize I
B
as a
substrate for phosphorylation in vitro.
Immunoprecipitation of a Phosphorylated N-terminal Fragment of I
To ascertain whether pp90rsk phosphorylates
IB
at either of the two critical N-terminal serine residues
located at positions 32 and 36, a 6xhis-I
B
and a similarly
epitope-tagged S32/36A I
B
mutant in which both of these serines
were substituted with alanine, was subjected to in vitro
phosphorylation as described above. The protein was then subjected to
mild V8 (endoprotease Glu C) proteolysis with a sequencing-grade
protease. The cleaved proteins were immunoprecipitated with antiserum
specific for the N-terminal his-epitope. This antiserum recognizes both
wild type I
B
and the S32/36A His-tagged mutants. Phosphorylation
of I
B
at either serine 32 or 36 should result in a fragment of
84/87 amino acids (including the epitope tag) when exposed to V8
protease, which cleaves at residue 48 or 51. Fig.
2A depicts the recombinant I
B
protein showing the signal-dependent N-terminal
phosphorylation sites in relation to the V8 cleavage sites. The
smallest band in the immunoprecipitate shown in the leftmost two
lanes of Fig. 2B, indicated by the arrow,
migrates between the 8.16- and 10.6-kDa myoglobin fragment, consistent
with the molecular size of the 84/87 N-terminal fragment of I
B
.
In contrast, the S32/36A I
B
protein failed to yield a similarly
sized band (Fig. 2, fourth and fifth lanes). The
even smaller band detected with these samples was also detected in the
absence of added V8 protease. These results thus demonstrate that
pp90rsk is capable of phosphorylating the regulatory N
terminus of I
B
involving serine 32 and/or 36.
pp90rsk-mediated Phosphorylation Promotes I
Although a variety of kinases can
phosphorylate IB
in vitro, the critical functional
issue is whether these kinases promote I
B
degradation. Using an
in vitro degradation system, we studied whether
pp90rsk-mediated phosphorylation of I
B
triggers its
destruction. The reticulocyte lysate degradation system employed in
these experiments contains all of the component proteins and
macromolecules necessary for ubiquitin-dependent and
-independent 26S proteasome-mediated degradation (31). A variety of
proteins have been shown to be degraded in this reticulocyte lysate
system, including ornithine decarboxylase, antizyme, and the
transcription factors Fos, Jun, and Myc (32-38).
[35S]-Radiolabeled wild type IB
(Fig.
3, upper panel) and the
S32/36A I
B
mutant (lower panel) were synthesized
in vitro and preincubated with Xenopus
egg-derived pp90rsk (lanes 1-4), mammalian
pp70S6K (lanes 5-8), or control kinase buffer
lacking an added kinase (lanes 9 and 10). The
I
B
proteins were then incubated in a degradation-competent reticulocyte lysate that had specifically not been pretreated with
RNase or hemin. Hemin is known to inhibit the 26S proteasome but is
often added to reticulocyte lysates to improve translation since it
prevents premature peptide chain termination (39). Degradation of
[35S]-I
B
was monitored by immunoprecipitation with
specific anti-I
B
antisera over the course of a 120-min incubation
conducted in the presence of an ATP-regenerating system. As shown in
lanes 1-4 of the upper panel,
pp90rsk-treated wild type I
B
was significantly
degraded during the 2-h incubation. However, I
B
treated with
pp70S6K (lanes 5-8) or control buffer
(lanes 9 and 10) was not degraded. In contrast to
wild type I
B
, the S32/36A I
B
mutant was not degraded when
incubated with pp90rsk (lower panel, lanes
1-4), indicating that I
B
degradation in this in
vitro system is dependent on the presence of these N-terminal regulatory serines, as it is in vivo. Together, these
results suggest that pp90rsk-mediated phosphorylation of
I
B
is biologically relevant since it leads to serine 32- and/or
serine 36-dependent degradation of wild type I
B
protein in vitro.
pp90rsk Phosphorylates I
Previous in vivo studies have
shown that both S32 and S36 at the N terminus of IB
are required
for signal-induced phosphorylation and degradation of this inhibitor
(7-10, 18). Additionally, both of these serine residues are directly
phosphorylated in vivo by an unknown kinase(s) following
cellular stimulation with PMA or TNF-
(45). Phosphorylation at these
sites in vivo results in a slower electrophoretic mobility
for the I
B
protein that is readily detectable on SDS-PAGE gels.
In contrast, no mobility shift is observed when cells containing the
S32/36A I
B
mutant is similarly induced. To assess whether
pp90rsk mediates phosphorylation on S32, S36, or both
sites, wild type or mutant I
B
proteins altered at one or both of
these N-terminal serines were used as substrates in the in
vitro kinase reactions. Activated pp90rsk was obtained
by immunoprecipitating this kinase from PMA-stimulated HeLa cells. As
shown in Fig. 4A, wild type
I
B
displayed a mobility shift when incubated with activated
pp90rsk (lane 2), while the S32/36A I
B
mutant containing alanine substitutions at both positions 32 and 36 did
not (lane 4). The S36A single-substitution mutant of
I
B
also exhibited a significant change in mobility in the
presence of pp90rsk (lane 8); however, the S32A
I
B
mutant displayed only a minimal change in migration
(lane 6). These data suggest that the principal site of
pp90rsk-mediated phosphorylation in vitro
corresponds to serine 32. Assuming that in vitro and
in vivo degradation requirements are the same for I
B
,
this finding suggests either that phosphorylation at this single site
is sufficient for in vitro degradation or, alternatively, that a second kinase present in the reticulocyte lysate may act in
concert with pp90rsk to modify serine 36 and thus complete
the phosphorylation requirements for degradation. Alternatively,
pp90rsk phosphorylation of serine 32 may facilitate
subsequent modification of serine 36 by pp90rsk.
Phosphatase Treatment of Phosphorylated I
To confirm that the observed mobility
shifts reflect phosphorylation of IB
, pp90rsk-treated
wild type and mutant S32/36A I
B
proteins were incubated with calf
intestinal alkaline phosphatase (Fig. 4B). The retarded mobility of pp90rsk-treated wild type I
B
(lane
3) was lost following treatment with phosphatase (lane
4). In contrast, phosphatase treatment of the S32/36A I
B
mutant, which did not display a mobility shift in the presence of
pp90rsk, did not alter its electrophoretic mobility
(compare lanes 7 and 8). These results confirm
that pp90rsk-mediated phosphorylation of I
B
is
responsible for the altered migration of the wild type I
B
proteins.
Under basal conditions, IB
is normally complexed with
NF-
B in the cytosol. The Rel A subunit of NF-
B is directly
associated with I
B
in this complex. Studies by Chen et
al. (11) suggest that I
B
is not only phosphorylated, but
also ubiquitinated and degraded while still complexed to NF-
B. To
investigate whether the presence of Rel A affects the pattern of
phosphorylation of I
B
, the pp90rsk-mediated in
vitro kinase assays were performed with I
B
in both the
presence and absence of in vitro cotranslated Rel A. These proteins assembled in vitro as indicated by the ability of
anti-Rel A antibodies to coimmunoprecipitate I
B
from the
translation mix (data not shown). As shown in Fig. 4C,
I
B
displayed the same mobility shift when incubated with
pp90rsk in the presence or absence of Rel A (compare
lanes 2 and 6). No changes in mobility were
obtained when the S32/36A I
B
mutant was incubated with
pp90rsk in the absence of Rel A, suggesting that Rel A did
not occlude additional pp90rsk phosphorylation sites whose
modification would affect I
B
mobility (data not shown).
Proteins that participate in a linear pathway of signaling
may sometimes physically associate with each other. Some protein kinases associate with their substrates under circumstances where the
kinase is inactive or where ATP is limiting. If the association between
a protein kinase and its substrate is sufficiently avid, their
interaction may be detected by coimmunoprecipitation of the two
proteins. To assess whether IB
can physically associate with
pp90rsk in vivo, COS cells were cotransfected
with expression vectors encoding HA-tagged pp90rsk or
control HA-tagged protein phosphatase-2A A regulatory subunit (HA-PP2A)
and I
B
. Following transfection, proteins were metabolically radiolabeled with [35S]methionine and cysteine, and the
resultant cell lysates were immunoprecipitated with nonspecific
preimmune, anti-HA, or anti-I
B
antibodies. Immunoprecipitation of
HA-PP2A and I
B
-transfected cells with anti-HA antibodies revealed
a major band corresponding to HA-PP2A but no coimmunoprecipitation of
I
B
(Fig. 5A, compare lanes 1 and 2). Similarly, addition of
anti-I
B
antibody immunoprecipitated I
B
but not PP2A
(compare lane 3 to lanes 1 and 2). In
contrast, cotransfection of cells with HA-pp90rsk and
I
B
led to coimmunoprecipitation of both of these molecules using
either the HA- or I
B
-specific antibodies (lanes 5 and 6). The in vivo association of
pp90rsk and I
B
was confirmed in experiments involving
initial immunoprecipitation with anti-HA or anti-I
B
followed by
immunoblotting with an anti-pp90rsk antibody (Fig.
5B). As shown in lanes 2 and 3,
anti-I
B
coimmunoprecipitated significant amounts of
pp90rsk in these cotransfected cells, while nonspecific
preimmune sera yielded no detectable pp90rsk (lane
1). Together, these results indicate that I
B
can physically associate with pp90rsk in vivo and thus provide
further support for the possibility that pp90rsk functions
as a physiologically relevant I
B
kinase.
Induction of pp90rsk Kinase Activity by Phorbol Ester but Not by TNF-
Studies were next performed to
assess whether pp90rsk is activated by various well known
inducers of NF-B in vivo (Fig.
6). Activation of pp90rsk was
monitored either by a very small but perceptible change in its
electrophoretic mobility, reflecting autophosphorylation (Fig. 6A), or by its ability to phosphorylate recombinant I
B
when the latter was added as an exogenous substrate to an in
vitro kinase assay performed with anti-pp90rsk
immunoprecipitates (Fig. 6B). PMA stimulation of Jurkat
cells for 5 or 15 min resulted in the rapid activation of
pp90rsk (panel A, lanes 3 and
4) and phosphorylation of I
B
(panel B, lanes 3 and 4). In contrast, two other recognized
inducers of NF-
B, TNF-
and HTLV-I Tax, did not significantly
activate pp90rsk autophosphorylation (panel A,
lanes 5-8) or induce phosphorylation of I
B
in the
in vitro kinase assay (panel B, lanes
5-8). However, this particular preparation of TNF-
(see
panel C, lanes 5-7) and HTLV-I Tax (data not
shown) stimulated phosphorylation and degradation of I
B
. These
findings indicate that only a subset of the known inducers NF-
B
leads to the activation of pp90rsk, suggesting that other
kinases are likely activated by different NF-
B inducers, such as
TNF-
and HTLV-I Tax. This result argues against the notion of a
single cellular I
B
kinase.
Phosphorylation and dephosphorylation represents a general
strategy frequently employed for the dynamic regulation of eukaryotic transcription factor function. The enhancer-binding protein is often
the specific target of such post-translational modifications that lead
to an alteration in its DNA binding or functional activity. However, in
the NF-B system, primary regulation is exerted through phosphorylation of I
B
, an ankyrin-rich inhibitor that sequesters the NF-
B complex in the cytoplasm. Phosphorylation targets I
B
for ubiquitination and subsequent degradation by the 26 S proteasome. Although a necessary step in the activation of transcription, phosphorylation alone does not result in the release of I
B
from the NF-
B complex and thus is insufficient for activation of
NF-
B-mediated transcription. Thus far, the identity of the kinase(s)
responsible for coupling cellular activation to phosphorylation of
I
B
or other members of the I
B family has remained elusive.
In the current paper, we have explored the possible function of
pp90rsk as a stimulus-coupled IB
kinase. In quiescent
cells, inactive pp90rsk resides in the cytoplasm and is
partially complexed with its upstream regulator, p42/44MAPK
(41). Cellular activation mediated by various growth factors operating
through the Ras-Raf-MEK-MAPK pathway or phorbol ester leads to the
activation of MAP kinase, the phosphorylation and activation of
pp90rsk, and the import of these kinases into the nucleus.
Activated forms of pp90rsk have already been implicated in
the regulation of various nuclear transcription factors, including
c-Fos (40) and Nur77 (42, 43). Recently, pp90rsk has been
reported to produce both positive and negative effects on another
family of inducible transcription factors, the cyclic AMP response
element-binding proteins (CREB). Specifically, pp90rsk2,
one of three closely related rsk genes (rsk1,
rsk2, rsk3), has been shown to function as a
stimulus-coupled CREB kinase modulating CREB activity by
phosphorylating this factor on a key regulatory serine located at
position 133 (44). Conversely, pp90rsk appears to oppose
CREB action by inducibly but stably associating with the CREB-binding
protein and blocking the interaction of this co-activator with CREB
(45). Of note, the enzymatic function of pp90rsk is not
required for these latter inhibitory effects. Together, these various
results provide an experimental precedent for the participation of
pp90rsk as a regulatory interface between the signals
induced by the ligation of various growth factor receptors on the
membrane and specific transcription factors that modulate the activity
of target genes within the nucleus.
Using a purified activated enzyme preparation in initial in
vitro kinase assays, we found that pp90rsk mediates
phosphorylation of IB
. Furthermore, based on V8 protease analysis, this phosphorylation involves the N-terminus of I
B
, where two critical residues for signal-induced phosphorylation, Ser-32
and Ser-36, reside. pp90rsk-mediated phosphorylation of
I
B
proved biologically relevant since this post-translational
modification stimulated proteasome-dependent degradation of
I
B
. In contrast, the S32/36A double-substitution mutant of
I
B
was not degraded in the presence of activated
pp90rsk. The stoichiometry of I
B
phosphorylation by
pp90rsk appeared quite high, as assessed by the ability of
pp90rsk to elicit a gel mobility shift for I
B
. By
this criterion, one-third to one-half of the I
B
molecules
displayed an altered mobility in the presence of pp90rsk.
Interestingly, the closely related S6 kinase, pp70S6K,
which recognizes the same consensus phosphoacceptor site,
Arg-X-X-Ser/Thr, as pp90rsk is
incapable of phosphorylating I
B
. Likewise, CaMKII, a
calmodulin-dependent kinase that also phosphorylates within
the same consensus phosphoacceptor site as pp90rsk and
pp70S6K, fails to phosphorylate I
B
. These findings
indicate that the recognition of I
B
by these protein kinases
involves determinants beyond the consensus phosphoacceptor site. It is
likely that the overall three-dimensional structures of I
B
and
the kinase play a pivotal role in the effectiveness of this
protein-protein interaction.
Since a "purified" pp90rsk preparation was used in
these in vitro studies, we considered the possibility that
the IB
phosphorylation might be due to a contaminating kinase.
However this possibility is unlikely because: 1) each of three
independently purified pp90rsk preparations displayed the
same I
B
kinase activity in at least three assays; 2) overloading
of an SDS-PAGE gel with the pp90rsk preparation followed by
silver staining revealed only pp90rsk and no other bands;
and 3) autophosphorylation reactions with the pp90rsk
preparation revealed no other bands. The observed I
B
kinase activity in the pp90rsk preparations thus appears to
reflect the activity of pp90rsk rather than a
contaminant.
Since serines 32 and 36 located near the N terminus of IB
are key
regulatory sites that must be directly phosphorylated to trigger
subsequent ubiquitination and degradation of this inhibitor (7-10, 18,
46), potential phosphorylation of these sites by pp90rsk
was studied. Serine 32 is embedded within a sequence that conforms to
the consensus phosphoacceptor site for phosphorylation by
pp90rsk; however, serine 36 is not. With wild type I
B
or the S32A and S36A single-substitution mutants of this inhibitor as
substrates, pp90rsk was shown to readily phosphorylate
I
B
proteins containing serine 32. In contrast, as judged by
mobility shift, serine 36 functioned as a very poor substrate for
pp90rsk. Since both serine 32 and serine 36 must be
directly phosphorylated for subsequent degradation (46), these findings
suggest that the action of pp90rsk alone may not be
sufficient to trigger the subsequent ubiquitination and degradation of
I
B
. It is possible that a second, yet unidentified, kinase
present in the reticulocyte lysate mediates phosphorylation at serine
36, thus promoting I
B
degradation. Although unproven, it is
intriguing to consider the possibility that phosphorylation in
vivo at the first serine site by one kinase may facilitate sequential phosphorylation at the second serine site by a different kinase. Alternatively, pp90rsk phosphorylation at serine 32 may enhance its activity at serine 36, producing a polarity to the
sequence of modifications. Precedence for regulation by such sequential
phosphorylation is found in the case of both pp90rsk and
pp70S6K phosphorylation of S6 peptide (47). Finally,
degradation of I
B
in vitro may proceed with
phosphorylation at serine 32 only.
To test the potential in vivo relevance of
pp90rsk as an IB
kinase, we investigated whether
these proteins can physically associate within a cell. Using COS cells
for cotransfection with HA epitope-tagged pp90rsk and
I
B
expression vectors, we demonstrated that these two proteins, but not similarly epitope-tagged control proteins, are
coimmunoprecipitated using either anti-HA or anti-I
B
-specific
antibodies. Using mutants of I
B
to study this interaction
further, our preliminary results indicate that the N terminus of
I
B
spanning the regulatory serines at positions 32 and 36 is not
required for I
B
binding to pp90rsk. In contrast,
deletion of ankyrin repeats 1-5 of I
B
severely impairs the
interaction of I
B
with pp90rsk.
Our final series of studies explored what NF-B-inducing signals
operate through pp90rsk-mediated phosphorylation of
I
B
. These studies revealed that PMA induced activation of
pp90rsk, phosphorylation of I
B
, and induction of
NF-
B. In sharp contrast, neither TNF-
nor the Tax trans-activator
protein of HTLV-I activated pp90rsk. However, both of the
latter inducers potently stimulated phosphorylation and degradation of
I
B
and activated nuclear NF-
B expression. This result clearly
indicates that not all NF-
B inducers operate through
pp90rsk activation. These results predict the likely
existence of multiple I
B kinases that are differentially coupled to
various signaling pathways. Thus, the critical kinase(s) ultimately
responsible for phosphorylating serines 32 and 36 may vary, depending
on the particular NF-
B-inducing signal. We propose that
pp90rsk represents one such kinase in a larger set of
enzymes that regulate I
B
phosphorylation.
We thank Dr. Joseph Avruch, Dr. Al Baldwin, Dr. Dean Ballard (Vanderbilt University, Nashville, TN), Dr. James Maller and Dr. Michael Browning (University of Colorado Health Sciences Center, Denver, CO), and Dr. Mark Stinski for reagents, Dr. Dean Ballard and Dr. James Maller for helpful scientific discussions, and Mark Dettle for preparation of the manuscript. We also thank Richard Ruhlen and David Brand for technical assistance.