(Received for publication, October 17, 1995; and in revised form, December 21, 1995)
From the
The p105 Rel protein has dual functions; it is the precursor of
the p50 subunit of NF-B, and it acts as an I
B-like inhibitor
to retain other Rel subunits in the cytoplasm. We have investigated the
posttranslational regulation of p105 following activation of Jurkat T
cells and find that a rapid and sustained phosphorylation of p105 is
induced. The inducible phosphorylation occurs on multiple serines in
the C-terminal-most 150 amino acids of the molecule, a region rich in
Pro, Glu, Ser, and Thr residues. Phosphorylation of p105 in Jurkat
cells treated with phorbol 12-myristate 13-acetate/ionomycin or with
okadaic acid, another activator of NF-
B, is correlated with an
increase in proteolytic processing to p50. Intact PEST sequences are
required for the phorbol 12-myristate 13-acetate/ionomycin-induced p105
processing, as a 68-amino acid C-terminal deletion abolishes the
response to stimulation. When compounds that block I
B
phosphorylation and degradation were tested, the serine protease
inhibitors L-1-tosylamido-2-phenylethyl chloromethyl ketone
and 1-chloro-3-tosylamido-7-amino-2-heptanone blocked inducible p105
phosphorylation, but the antioxidants pyrrolidine dithiocarbamate and
butylated hydroxyanisol did not. Thus, while regulation of the p105
I
B resembles that of I
B
, involving inducible serine
phosphorylation and proteolysis of the inhibitory ankyrin repeat
domain, it depends on a different, redox-insensitive, signaling
pathway.
The Rel proteins are a ubiquitous family of transcription
factors that regulate the expression of many genes, particularly those
induced during an immune response (see (1) and (2) for recent reviews). The members of this family bind to
specific DNA sequences, B sites, as homo- or heterodimers to
regulate the transcription of target cellular genes and several
viruses, including human immunodeficiency virus. In mammalian cells,
five proteins containing the Rel homology domain characteristic of
these factors have been cloned. Although in some cases Rel proteins are
constitutively nuclear, notably the p50 homodimer, most
B-binding
activity is in an inactive form in the cytoplasm bound to specific
inhibitory proteins, known as I
Bs. Upon receipt of a specific
activating stimulus, Rel dimers are rapidly freed from these
cytoplasmic complexes and translocated to the nucleus, mostly in the
form of NF-
B, a heterodimer of p50 and p65 (also called RelA). A
large variety of stimuli induce nuclear
B binding activity,
including mitogens such as PHA (
)and lipopolysaccharide,
cytokines like tumor necrosis factor and interleukin-1
, and stress
stimuli such as double-stranded RNA or ultraviolet
light(1, 2, 3) . The two signals required for
T cell activation, mimicked by phorbol esters and agents that increase
intracellular Ca
, synergistically activate prolonged
nuclear
B-binding activity (reviewed in (2) ).
In a
given cell, several IBs are expressed. All I
Bs contain
multiple copies of a motif called the ankyrin repeat, which is also
found in many functionally unrelated proteins and may facilitate
protein-protein interactions (see (4) and (5) for
review). There are two types of I
B. Those initially described are
separate protein subunits that are believed to bind as monomers to a
Rel dimer. In mammalian cells these include I
B
, -
, and
-
(derived from the p105/p50 gene and present in some murine
cells)(6, 7, 8, 9) . p105 and p100,
the precursors of the Rel subunits p50 and p52, respectively, form the
other class of I
Bs. The N-terminal half of each is a Rel
DNA-binding subunit, which is liberated by specific proteolysis of the
C-terminal portion of the molecule(10) , which contains the
ankyrin repeats. These precursors most likely retain a single
associated Rel subunit in the cytoplasm via interactions involving the
dimerization domains of both Rel homology domains and the ankyrin
repeats of the precursor(11, 12) .
The mechanisms
involved in the release of Rel proteins from cytoplasmic Rel-IB
complexes in response to an activating stimulus is best understood for
I
B
. Activators of NF-
B typically induce rapid
phosphorylation and degradation of I
B
, although the kinetics
of the response depends on the activating stimulus. Two N-terminal
serines were recently shown to be the targets of the inducible
phosphorylation of I
B
, and the presence of constitutively
phosphorylated C-terminal PEST sequences is required as well for the
inducible
degradation(13, 14, 15, 16, 17) .
Phosphorylation of I
B
does not cause it to dissociate from
NF-
B, as was initially hypothesized; rather I
B
must be
degraded from the complex to liberate the associated Rel dimer (18, 19, 20, 21, 22, 23) .
The regulation of cytoplasmic complexes containing either precursor protein is less well understood but clearly involves proteolysis of the ankyrin repeat domain in the C-terminal half of the molecules. Increased rates of processing of p105 have been reported in response to lipopolysaccharide(24) , PMA and ionomycin(25) , and double-stranded RNA(26) , and phosphorylation has been implicated in up-regulation of p105 processing following stimulation with tumor necrosis factor(26, 27) . However, neither the target residues nor the kinase responsible have been identified. Previous studies have reported increased serine and threonine phosphorylation of p105 in activated Jurkat T cells. One group also detected phosphotyrosine(28) , while another did not (29) .
We report here that activation of Jurkat T cells by
PMA/ionomycin treatment rapidly induces phosphorylation of multiple
serines in the C-terminal-most 150 amino acids of p105, a PEST-rich
domain following the ankyrin repeats. We show that phosphorylation is
correlated with increased processing of p105 in Jurkat cells treated
with PMA/ionomycin or with okadaic acid, a serine/threonine phosphatase
inhibitor and activator of NF-B(30) . p105 phosphorylation
is critical for the effect of PMA/ionomycin on processing, as the
latter is abolished by deletion of 68 amino acids in the inducibly
phosphorylated PEST domain. Finally, we show that treatment of cells
with antioxidants, which blocks phosphorylation and degradation of
I
B
(21, 31, 32) , does not affect
PMA/ionomycin-induced p105 phosphorylation. The differential
sensitivity of p105 and I
B
phosphorylation to cellular redox
status indicates that, although both inhibitors undergo inducible
serine phosphorylation and proteolysis following T cell activation,
they are regulated by different signaling pathways.
Whole-cell
extracts were made from 3-5 10
cells lysed in
a modified radioimmune precipitation buffer (50 mM Tris-HCl,
pH 7.4, 400 mM NaCl, 1% SDS, 1% Triton X-100, 1% deoxycholate,
5 mM EDTA) and then briefly sonicated to shear DNA. All
solutions for extract preparation were supplemented with phosphatase
inhibitors (15 mM
-glycerol phosphate, 2 mM sodium pyrophosphate, 1 mM Na
VO
)
and protease inhibitors (4-(2-aminoethyl)-benzene sulfonyl fluoride
hydrochloride, aprotinin, leupeptin, and phenylmethylsulfonyl fluoride)
just prior to use.
Immunoprecipitation was carried out in 500-1000
µl of radioimmune precipitation buffer (50 mM Tris-HCl, pH
7.5, 150 mM NaCl, 0.1% SDS, 1% Triton X-100, 0.5%
deoxycholate) with 10 µl of polyclonal rabbit antiserum for every 2
10
extracted cells 1-14 h at 4 °C, to
which an excess of Protein A-Sepharose beads (Sigma) was added for
1-2 h at 4 °C.
Our earlier investigation of the IB-like function of
p105 showed that p50/c-Rel and p50/p65 dimers derived from complexes
with p105 could be detected in the nuclear and cytoplasmic fractions of
Jurkat T cells several hours after stimulation with PMA and
PHA(11) . Similar p105-derived dimers were difficult to detect
during the same period in unstimulated cells, suggesting that the
processing of p105 is up-regulated by activation. On the basis of
structural and functional similarities between p105 and I
B
,
we hypothesized that phosphorylation might be involved in increasing
p105 processing in activated T cells.
To begin to test this
hypothesis, we labeled Jurkat cells with P-orthophosphate
and treated or mock-treated them with PMA and PHA for 3 h. Cytoplasmic
and nuclear extracts were prepared from the labeled cells and boiled in
1% SDS before immunoprecipitation with an antiserum directed against an
N-terminal p105/p50 peptide. As shown in Fig. 1, the very low
basal level of
P phospholabeling of p105 in the cytoplasm (lane 1) was greatly enhanced by PMA/PHA treatment (lane
2). (The 98-kDa phosphoprotein also seen in the immunoprecipitate
may represent an alternatively spliced p105 form similar to one
described in mouse, which shares N- but not C-terminal sequences with
the full-length p105(35, 36) .) Precipitation of
P-labeled Jurkat extracts with a preimmune serum did not
reveal a labeled band in the 110-kDa range, and two rounds of
immunoprecipitation with different p105/p50 antibodies further
confirmed the identity of the 110-kDa phosphorylated band as p105 (data
not shown).
Figure 1:
p105 is hyperphosphorylated and p50
levels are increased in Jurkat T cells 3 h postactivation.
Phosphorylation of p105 and p50 in activated and unactivated Jurkat T
cells is shown. Equivalent numbers of Jurkat T cells were treated or
not treated with 20 ng/ml PMA and 1 mg/ml PHA for 1 h in phosphate-free
medium and then labeled with [P]orthophosphate
for 2 h with or without PMA and PHA. Cytoplasmic and nuclear extracts
were prepared as described under ``Materials and Methods.''
The
P-labeled cytoplasmic extracts were boiled to
dissociate complexed proteins and immunoprecipitated with an antiserum
directed against the N terminus of p105/p50. The washed
immunoprecipitates were analyzed by SDS-PAGE and subsequent
autoradiography (left panel). A fraction of each extract was
separated by SDS-PAGE and analyzed by immunoblotting with an
anti-p105/p50 antiserum (right
panel).
The increase in p105 phosphorylation could not be
accounted for by an increase in protein amount, as determined by
immunoblotting of fractions of the labeled extracts (Fig. 1, lanes 3 and 4). Immunoblotting also revealed
increased p50 in both the cytoplasmic and nuclear fractions of
stimulated cells (lanes 4 and 6) relative to the
unstimulated condition (lanes 3 and 5). We did not
detect phospholabeled p50, in contrast to other
reports(29, 37) . The increased p50 seen in the
PMA/PHA-treated cells must be derived from processing of the precursor,
and the apparently unchanged amount of p105 observed in these cells is
likely accounted for by neosynthesis following activation of the p105
promoter by NF-B(39, 42) .
The correlation
between increased phospholabeling of p105 and increased p50 several
hours after PMA/PHA treatment led us to examine the effects of T cell
activation on p105 at earlier times. In these and subsequent
experiments we used ionomycin instead of PHA to provide the
Ca signal. As shown in Fig. 2A,
P-phospholabeling of p105 in Jurkat T cells was increased
severalfold by a 20-min PMA/ionomycin treatment (lane 2). In
this experiment p105 was immunoprecipitated with an antiserum to the
C-terminal epitope; thus, the only specific band is full-length p105.
Cells were similarly labeled with
[
P]orthophosphate in the presence of the peptide N-acetyl-Leu-Leu-norleucinal (aLLnL), which inhibits the
proteolytic activities of the 26 S proteasome and calpain and has been
shown to inhibit both proteolysis of I
B
and processing of
p105 (41) . The PMA/ionomycin-induced phospholabeling of p105
was increased in cells treated with aLLnL (Fig. 2A, lane 4), and a slight increase in basal phosphorylation of
p105 was also observed in the presence of aLLnL (lane 3).
Figure 2:
p105 is phosphorylated, and its migration
in SDS-PAGE is retarded at early times following PMA/ionomycin
treatment. A, [P]phosphate-labeled
p105, immunoprecipitated with an anti-C-terminal antiserum from
extracts of
P-labeled Jurkat T cells treated with aLLnL
for 1 h and/or PMA/ionomycin for 20 min, as indicated (ns,
nonspecific band). B, immunoblot of p105 from cytoplasmic
extracts of Jurkat T cells pretreated or not pretreated with the
protease inhibitor aLLnL for 1 h prior to stimulation with PMA and
ionomycin for 15 min, as indicated (lanes 1-4). Lanes 5 and 6 show the effect of treatment with calf
intestine alkaline phosphatase of the extract shown in lane 4,
in the absence (lane 5) or presence (lane 6) of
phosphatase inhibitors (40 mM NaF, 15 mM
-glycerol phosphate, 2 mM sodium pyrophosphate, and
1 mM sodium orthovanadate).
In immunoblots of p105 in cytoplasmic extracts from Jurkat cells treated with PMA/ionomycin for 15 min, with or without aLLnL, the p105 band is upshifted (Fig. 2B, lanes 2 and 4) relative to unstimulated controls (lanes 1 and 3). This change in the migration of p105 in SDS-PAGE is due to phosphorylation, as determined by treatment with calf intestine alkaline phosphatase. When the extract in lane 4 was treated with calf intestine alkaline phosphatase, the p105 band migrated more rapidly (lane 5), and this effect was blocked by the addition of phosphatase inhibitors to the reaction (lane 6).
Using
the slower migration of p105 in SDS-PAGE as a marker for the addition
of phosphate to the protein, we examined the kinetics of p105
phosphorylation in PMA/ionomycin-stimulated Jurkat cells. The slower
migrating form was observed in anti-p105 immunoblots within a few
minutes of activation, as reported by Li et al.(29) ,
as well as at the latest times examined, 3-4 h poststimulation
(data not shown). Immunoblots of these extracts with an antiserum
against the human IB
, MAD3, showed a short lived upshifted
form within minutes of stimulation and complete degradation of the
protein within 10-15 min (data not shown). In contrast, p100
migration was unaffected at early times following PMA/ionomycin
treatment, although its migration on SDS-PAGE gels was slowed at later
times (3 h), coincident with the appearance of nuclear p52 (data not
shown).
We next sought to determine the region of p105 phosphorylated in PMA/ionomycin-stimulated Jurkat cells. Our finding that processed p50 is not phosphorylated suggested the C-terminal half of p105 as the likely target of inducible phosphorylation in the full-length molecule. To begin to identify the phosphorylated domain, we took advantage of our finding that thrombin treatment of p105 generates a 30-kDa C-terminal fragment, probably by cleaving at a site (RGS) in the sixth ankyrin repeat. Our approach was to perform the cleavage reaction on a Sepharose bead-bound immunoprecipitate and then load the supernatant and the immunoglobulin-bound fragment separately on SDS gels.
In Fig. 3A, an in vitro translated S-labeled p105 C-terminal construct, containing amino
acids 367-969 of p105 (p105C), was immunoprecipitated with an
anti-C-terminal antiserum and bound to Protein A-Sepharose beads. The
precipitate was washed and then treated with thrombin, leaving a 30-kDa
C-terminal fragment bound to the beads (Fig. 3A, lane 2) and absent in the supernatant of the cleavage reaction (lane 3). A similar protocol was applied to
P-orthophosphate-labeled p105 immunoprecipitated with an
anti-C-terminal antiserum from extracts of Jurkat cells stimulated for
15 min with PMA/ionomycin in the presence of aLLnL. Following thrombin
cleavage of the bead-bound immunoprecipitate, the supernatant and
bead-bound C-terminal fragment were resolved separately on an SDS-PAGE
gel, and the
P-phosphate content was assessed by
autoradiography. As shown in Fig. 3B, the bound
C-terminal fragment (lane 7) contained all of the detectable
P-phosphate incorporated in p105 in vivo following PMA/ionomycin treatment (lane 5).
Figure 3:
A C-terminal 30-kDa thrombin cleavage
fragment of p105 contains all PMA/ionomycin-induced phosphorylation
sites. A, a S-labeled in vitro translated p105 C-terminal protein (amino acids 367-969) (lane 1) was immunoprecipitated with an anti-C-terminal p105
antibody and treated with thrombin on the Protein A-Sepharose beads, as
described under ``Materials and Methods.'' Antibody-bound
C-terminal fragment (lane 2) and half of the resulting
supernatant (lane 3) were run separately on an SDS-PAGE gel
and exposed for autoradiography. B, p105 was
immunoprecipitated with an anti-C-terminal antiserum from
P-labeled Jurkat T cells pretreated with aLLnL (lanes
4-7) and stimulated or not stimulated with PMA and ionomycin
for 15 min, as indicated. Lane 7 shows the antibody-bound
C-terminal fragment produced by thrombin treatment of an
immunoprecipitate identical to that shown in lane 5. Lane
6 contains half the supernatant of the thrombin-treated
immunoprecipitate.
To
identify the type of amino acid phosphorylated in p105 in these cells,
both constitutively and following PMA/ionomycin treatment, the P-labeled p105 and the thrombin-generated C-terminal
fragment were subjected to hydrolysis and electrophoresis in
two-dimensions on thin-layer cellulose plates. p105 from unstimulated
cells is phosphorylated on serine and to a far lesser extent on
threonine, and the phosphorylation induced by PMA/ionomycin treatment
is likewise almost entirely on serine (data not shown). In contrast
with the report of Neumann et al.(28) , we did not
detect phosphotyrosine in p105, either by phosphoamino acid analysis of
P-labeled p105 or by immunoblotting of p105
immunoprecipitates with an anti-phosphotyrosine antiserum (data not
shown).
Immunoblotting of extracts from Jurkat cells stimulated with PMA/ionomycin in both one- and two-dimensional gels revealed at least four differently migrating p105 isoforms (data not shown), as previously reported(28) . These results and those of preliminary tryptic peptide mapping experiments suggest PMA/ionomycin treatment induces phosphorylation of multiple C-terminal serines (data not shown; see results below).
As the C terminus of p105 is extremely rich in serine residues, with 20 in the presumed thrombin fragment, we chose not to attempt a mutational analysis but rather to further delimit the region of PMA/ionomycin-induced p105 phosphorylation using two C-terminally truncated constructs, ending at amino acids 901 and 819 of p105. For these experiments we transiently transfected Tag Jurkat cells, which stably express SV40 large T antigen(33) , with plasmids containing the above constructs under control of the CMV promoter. Immunoblots of cytoplasmic and nuclear extracts of Tag Jurkat cells expressing either C-terminally truncated protein showed that, like the full-length (969-amino acid) protein, these molecules are almost entirely cytoplasmic.
Cells
transiently transfected with plasmids encoding either the 901- or
819-amino acid construct were labeled with P-orthophosphate in the presence of aLLnL and then treated
with PMA and ionomycin for 20 min. Cell extracts were prepared as
before, boiled in SDS, and immunoprecipitated with an anti-N-terminal
antiserum, which recognizes both the endogenous p105 and the C-terminal
truncations. As shown in Fig. 4, in the Tag Jurkat cell line, as
in our normal Jurkat line, a short PMA/ionomycin treatment induced an
upshift in p105 and a large increase in phospholabeling of the
molecule. In contrast, the 901 construct, which lacks the last 68 amino
acids of the protein, including 10 serines, shows only a slight
increase in
P phospholabeling in response to the
PMA/ionomycin treatment (left panel) and no change in
migration in SDS-PAGE (right panel). The 819 construct, which
deletes another 82 amino acids, of which six are serines, abolishes
both low level constitutive and PMA/ionomycin-induced phosphorylation.
Immunoblotting of a fraction of the labeled immunoprecipitate (Fig. 4, right panel) shows that the differences in
phospholabeling cannot be accounted for by differences in the amount of
protein but rather reflect the deletion of target phosphorylation sites
in the C-terminal truncations.
Figure 4:
p105 constructs lacking C-terminal PEST
sequences are not phosphorylated in response to PMA/ionomycin. P-Phosphate incorporation into p105 and transfected
C-terminally truncated p105 constructs immunoprecipitated with an
anti-N-terminal antiserum from cytoplasmic extracts of
P-labeled Tag Jurkat T cells treated with aLLnL (50
µM, 1 h) and with or without PMA/ionomycin for 20 min, as
indicated (left panel), is shown. The migration of p105 (square) and truncated constructs ending at amino acid 901 (open circle) and 819 (carat) is indicated, and the
number of serine residues deleted in each truncated construct is shown below the corresponding lanes. The right panel is an anti-p105/p50 immunoblot of a fraction of the labeled
immunoprecipitate shown in the left
panel.
These results localize the PMA/ionomycin-inducible and low level constitutive phosphorylation of p105 in Jurkat cells to more than one of the 16 serine residues in the C-terminal-most 150 amino acids of the protein, a PEST-rich domain, with most of the target serines in the final 68 amino acids. The phosphorylated region does not overlap the ankyrin repeats, which end at amino acid 807. This PEST domain contains multiple consensus serine phosphorylation sites, including Pro-Ser sequences and sites for protein kinase A and casein kinase II. The possible role of these kinases in p105 phosphorylation is discussed below.
We next
investigated the effect of p105 phosphorylation on processing to p50 at
early times following PMA/ionomycin stimulation of Jurkat T cells. To
avoid any transcriptional effect due to up-regulation of p105 and
IB
via
B sites in their
promoters(38, 39, 40, 42, 43, 44) ,
we determined the levels of p105 and p50 in cells treated with CHX. We
compared the ratio of p105 to p50 in stimulated and unstimulated cells
by densitometric measurement of the relevant bands in immunoblots of
SDS-PAGE separations of whole-cell extracts. We used this approach
rather than pulse-chase labeling methods, as it reveals changes in the
total pool of p105 rather than that of a newly synthesized subset of
molecules. As the precursor-product relation of p105 and p50 is well
established, any increase in p50 can be assumed to derive from
processing of p105. Further, comparison of the ratios of p105 and p50
within each extract corrects for any variability in total extract
amount.
Quantification of multiple experiments shows that PMA/ionomycin treatment increases p105 processing at early times (20 min to 1 h) following stimulation, extending our initial observations of increased cytoplasmic and nuclear p50 3 h poststimulation (Fig. 1). As shown in Fig. 5A, the ratio of p105 to p50 in whole-cell extracts from Jurkat T cells drops from 1.2 to 0.7 within 40 min of PMA/ionomycin treatment, reversing the initial excess of inhibitor over activator. Cells treated with CHX alone showed no significant change in the ratio of p105/p50 in this interval. Although the absolute ratios of p105/p50 measured using this approach varied, the decrease of p105 relative to p50 induced by PMA/ionomycin treatment was consistently observed.
Figure 5: Stimulus-induced p105 phosphorylation is correlated with increased processing to p50. A, immunoblot of endogenous p105 and p50 in Jurkat T cells pretreated 1 h with CHX (lane 1) and then stimulated with PMA and ionomycin for 20 min (lane 2) and 40 min (lane 3). The ratio of p105 to p50 is shown below the corresponding lane. B, immunoblot of p105 and p50 in extracts from Tag Jurkat cells overexpressing p105. The extract in lane 1 was treated with CHX for 1 h. In lanes 2 and 3 the cells were treated for an additional hour with CHX, in the absence (lane 2) or presence (lane 3) of PMA and ionomycin. C, immunoblot of p105 and p50 in extracts from Jurkat cells treated for 3 h with CHX alone (lane 1) or CHX and okadaic acid (0.6 µM; lanes 2 and 3). The cells in lane 3 were additionally treated with aLLnL (100 µM) 3 h.
Similarly, p105 transiently overexpressed in Tag Jurkat cells underwent increased processing to p50 within the first hour following PMA/ionomycin stimulation (Fig. 5B, lane 1, ratio 1.7, versus lane 3, ratio 1.1), whereas the p105/p50 ratio in a control treated with CHX alone reflected only low level constitutive processing (compare lane 1, ratio 1.7, and lane 2, ratio 1.6).
We also observed a slower migrating phosphorylated form of p105 in
Jurkat T cells treated with okadaic acid, an inhibitor of
serine/threonine phosphatases of the 1 and 2A classes and an activator
of NF-B. As shown in Fig. 5C (lane 2),
okadaic acid treatment also induced processing of p105, as evidenced by
a decrease in the p105/p50 ratio relative to a control treated with CHX
alone (0.3 in lane 2, versus 0.9 in the control, lane 1). The okadaic acid-induced processing of p105 was
blocked by aLLnL (ratio 0.8 in lane 3), suggesting that it
depends on the same proteolytic machinery as other p105 processing. The
kinetics of the change in relative amount of p105 induced by okadaic
acid was identical to that of the accumulation of the upshifted form (3
h in Fig. 5C), although the exact timing of the upshift
varied, perhaps due to variable drug potency.
If
PMA/ionomycin-induced phosphorylation of the C-terminal PEST sequences
of p105 up-regulates processing to p50, the removal of all or part of
this domain would be predicted to abolish the response. To investigate
this hypothesis, we used a convenient restriction enzyme site to
introduce a small (36-amino acid) N-terminal deletion in full-length
p105 and in the 901 and 819 C-terminal truncations described above;
these N-terminally truncated forms are designated Np105,
N901, and
N819. The N-terminal deletion allows us to separate
the processed form of the transfected plasmid (
Np50) from the
endogenous p50 in SDS-PAGE, and thus the influence of the C-terminal
PEST domain on processing can be analyzed.
The Tag Jurkat cell line
was transiently transfected with the plasmid encoding Np105 (Fig. 6A),
N901 (Fig. 6B), or
N819
(not shown), and 24 h later the cells were pretreated with CHX for 1 h
and then stimulated or not stimulated with PMA/ionomycin for an
additional hour. As shown in Fig. 6A, the N-terminal
deletion in
Np105 did not interfere with the response to
PMA/ionomycin, as
Np50 increased in response to stimulation.
Np50 in the extract prepared from stimulated cells (lane
2) is 165% that in the unstimulated control (lane 1) by
densitometry. Unfortunately, the ratio of
Np105 to
Np50 could
not be determined due to comigration of
Np105 with endogenous p105
in stimulated cells.
Figure 6:
PMA/ionomycin-induced p105 processing
requires intact PEST sequences. A, immunoblot with antiserum
against a p50 Rel homology domain peptide (peptide 1157) of extracts
from Tag Jurkat cells transiently transfected with N-terminally
truncated p105 (Np105). Twenty-four hours posttransfection, cells
were pretreated with CHX for 1 h and then treated with PMA and
ionomycin 1 h (lane 2) or left in CHX alone (lane 1). B, immunoblot of extracts from Tag Jurkat cells transiently
transfected with N-terminally truncated p901 (
N901). Twenty-four
hours posttransfection, cells were pretreated with CHX for 1 h. The
extract in lane 2 and 4 was treated with PMA and
ionomycin for an additional hour, and that in lanes 1 and 3 was left in CHX alone. A shorter exposure of the endogenous
p50 in the same blot is shown as an internal control for the effect on
processing of the wild-type molecule in these cells (lanes 3 and 4). The ratio of
N901/
Np50 is shown in lanes 1 and 2, and the endogenous p105/p50 ratio is
listed in lanes 3 and 4.
In contrast to Np105, processing of
N901 was not induced by PMA/ionomycin treatment, as levels of
N901 and
Np50 in stimulated cells were similar to those of
unactivated controls (Fig. 6B, lanes 1 and 2). The effectiveness of the PMA/ionomycin treatment is
reflected by the upshifting of the endogenous p105 in these extracts
and the increase in p50 in stimulated cells visible in a shorter
exposure of the blot (lane 4 versus lane 3). The densitometric
analysis of this and other blots confirms that while the ratio of
endogenous p105/p50 drops in response to PMA/ionomycin treatment, that
of
N901/
Np50 is unaffected. In this experiment
N901/
Np50 remains unchanged at 0.5 (Fig. 6B, lanes 1 and 2), while in the same extracts the
p105/p50 ratio drops from 0.8 to 0.4 in stimulated cells (lanes 3 and 4). Interestingly, immunoblots of extracts from cells
transfected with the
N819 plasmid revealed
Np50, but
unprocessed
N819 was barely detectable, suggesting that the
absence of the PEST domain renders the protein highly susceptible to
constitutive processing.
Our finding that PMA/ionomycin treatment
induces phosphorylation of p105 as well as IB
suggests that
the two inhibitors might be the targets of identical signaling
pathways. We used pharmacological agents known to block phosphorylation
of I
B
to investigate whether the signal transduction elements
leading to phosphorylation of p105 are similarly affected. These agents
are of two types, antioxidants and certain serine protease inhibitors,
both of which have been shown to act upstream of the serine
phosphorylation that induces I
B
degradation (see (1) and (2) and references therein).
We examined
the effects of antioxidants on the PMA/ionomycin-induced
phosphorylation of p105 in Jurkat T cells as evidenced by altered
migration on SDS-PAGE. As shown in Fig. 7B, pretreatment of
Jurkat cells with the antioxidants PDTC (lane 3) or BHA (lane
4) prior to stimulation with PMA/ionomycin inhibited the
phosphorylation and degradation of IB
such stimulation
usually induces (lane 2). In contrast, neither antioxidant
blocked the PMA/ionomycin-induced phosphorylation of p105 (Fig. 7A, lanes 3 and 4), nor did
treatment with these agents alone affect the migration of p105 (not
shown).
Figure 7: PMA/ionomycin-induced p105 phosphorylation is blocked by TPCK but not antioxidants. A, immunoblot of p105 and p50 in whole-cell extracts from Jurkat T cells pretreated with CHX 1 h (all lanes) alone or with the antioxidants BHA and PDTC or the serine protease inhibitor TPCK. In lanes 2-5 cells were stimulated with PMA and ionomycin for 20 min. Pretreatments were as follows: CHX only (lanes 1 and 2); 250 µM BHA (lane 3); 100 µM PDTC (lane 4); 50 µM TPCK (lane 5). B, immunoblot of MAD3 in whole-cell extracts of Jurkat T cells pretreated with CHX 1 h (all lanes). Cells in lanes 2-4 were stimulated with PMA/ionomycin for 20 min. Pretreatments were as follows:, CHX only (lanes 1 and 2); 100 µM PDTC (lane 3); 250 µM BHA (lane 4).
Pretreatment of Jurkat T cells with the serine protease
inhibitor TPCK (Fig. 7A, lane 5) or TLCK (not
shown) blocked PMA/ionomycin-induced p105 phosphorylation, much as they
block that of IB
(20) . TPCK and TLCK have previously
been shown to block the degradation of I
B
(26, 45, 46) and proteolytic processing of
p105 (26) . However, this probably depends not on their
anti-serine protease activity but rather on their alkylating
properties(20) .
These results show that the signaling
pathway responsible for inducibly phosphorylating the p105 C terminus
following PMA/ionomycin stimulation is not identical to that regulating
IB
, on the basis of their different sensitivities to the
redox state of the cell. Their common sensitivity to TPCK and TLCK may
indicate a shared signaling element upstream of the redox-sensitive
step in the I
B
pathway, or it may simply reflect a
nonspecific inhibition due to alkylation.
p105 phosphorylation
induced by okadaic acid was also blocked by the serine protease
inhibitor TPCK (not shown). Interestingly, it was recently reported (47, 48) that NF-B activation induced by okadaic
acid or calyculin A is insensitive to antioxidants. The similar
sensitivity of okadaic acid- and PMA/ionomycin-induced p105
phosphorylation to pharmacological agents suggests they may utilize
common signaling elements.
The above results show that p105 processing is not simply a
constitutive process but that it can be up-regulated by PMA/ionomycin
or okadaic acid, confirming similar reports with other stimuli (24, 25, 26) . The up-regulation requires the
presence of the 68 amino acids in the C-terminal PEST domain that
contain multiple inducibly phosphorylated serines. These findings
underline fundamental similarities in the regulation of p105- and
IB
-containing complexes (see Fig. 8). In both cases
release of Rel dimers from the inactive complex with an I
B depends
on serine phosphorylation and ensuing proteolysis of the inhibitor
domain. The target serine residues are located in unique domains of
each I
B outside the conserved ankyrin repeats critical for Rel
protein binding. Both inhibitors also contain PEST sequences, which, in
other proteins, have been correlated with rapid protein
turnover(59) . These regions are essential for stimulus-induced
proteolysis, which probably occurs in the 26 S proteasome in the
context of Rel-I
B
complexes(9, 18, 19, 21, 22, 23, 41, 49) .
The inhibitory effect of aLLnL is not restricted to the proteasome, as
this compound has been originally described as an inhibitor of calpain.
However experiments carried out with more specific inhibitors suggest
that the proteasome is indeed involved in p105 processing and
I
B
degradation(41) .
Figure 8:
Schematic of regulation of p105-Rel and
IB
-Rel complexes. RelX refers to any Rel DNA-binding
subunit (p65, c-Rel, p50, or p52); stripes indicate ankyrin
repeats; asterisk represents phosphoserine or
phosphothreonine.
Although the basic scheme of
inducible serine phosphorylation and proteolysis is conserved between
the p105 precursor and IB
, evidence presented here shows that
the phosphorylation of each is regulated by a different signaling
pathway. The finding that antioxidants do not block phosphorylation of
p105 induced by PMA/ionomycin, as they do that of I
B
, proves
the two inhibitors cannot share all upstream signaling elements,
despite the similar kinetics of their induced phosphorylation. The p105
processing reportedly induced by H
O
(27) likely acts via a different mechanism, as suggested
by the cell specificity of this response, in contrast with the
generalized effects of antioxidants (50) . The insensitivity of
p105 to redox status in Jurkat cells suggests that even if oxygen
radicals act as second messengers in the activation of NF-
B, as
has been proposed(31) , they do not regulate all Rel/I
B
complexes in the cell.
This difference in the sensitivity of p105
and IB
to antioxidants suggests that the contribution of
p105-containing complexes to nuclear Rel activity induced by
PMA/ionomycin stimulation could be determined in their presence. In
Jurkat cells treated with 200 µM PDTC and activated with
PMA/ionomycin, a reduced but easily detectable increase in nuclear
B binding activity, mainly in the form of p65/p50, was
reproducibly observed, as was a corresponding increase in nuclear p50
in immunoblots (see, e.g.(51) ). (
)In
contrast, aLLnL blocked all inducible
B binding activity and p50
translocation. However, the interpretation of these experiments is
problematic for two reasons. First, antioxidants do not completely
block I
B
degradation, even at high concentrations, although
no upshifted phosphorylated form is observed (see Fig. 7B and (1) and (2) ). Second, when cells are
activated after preincubation with antioxidants, the vast majority of
I
B
is still present and could associate with dimers released
by processing of p105. Under normal physiological conditions,
I
B
is totally absent during the first hour following T cell
activation, which would presumably allow direct translocation to the
nucleus of dimers derived from processing of p105-containing complexes.
Although the effects of PMA/ionomycin treatment on p105 processing
are less dramatic than the total degradation of IB
, usually
on the order of a 2-fold change in the p105/p50 ratio, this shift in
the balance of inhibitor and DNA-binding forms of the protein would be
expected to have a significant effect on Rel-dependent transcription.
p105 processing likely contributes to
B binding activity both
directly, when I
B
is absent, and indirectly, by generating
dimers that associate with resynthesized I
B
. This
interpretation is consistent with the results of earlier pulse-chase
experiments showing dimers derived from
S-labeled p105
complexes in both the nuclear and cytoplasmic fraction of Jurkat cells
several hours after activation(11) . In contrast to the release
of NF-
B from I
B
, which is total and transient, the
hyperphosphorylation, and presumably the up-regulated processing, of
p105 continues for several hours. This sustained effect on p105 may
explain the continued increase in nuclear
B binding activity
observed after I
B
has been resynthesized(53) .
In contrast to the immediate effects of T cell activation on p105
and I
B
, p100 appears relatively stable (12, 25) and may be phosphorylated and processed a few
hours after activation.
These results suggest that each
I
B plays a specific role in coordinating Rel-dependent
transcription.
The PEST sequences of p105 that are the target of
PMA/ionomycin-induced phosphorylation contain consensus phosphorylation
sites for multiple serine/threonine kinases, including one for protein
kinase A, two for casein kinase II, and three for proline-directed
kinases such as Cdc2, ceramide-activated and the mitogen-activated
protein kinases. In in vitro tests using purified
serine/threonine kinases, protein kinase A and casein kinase II, but
neither mitogen-activated protein kinase from Xenopus, protein
kinase C, Cdc2 kinase, nor calmodulin-dependent protein kinase II
phosphorylated recombinant p105. ()
p105 phosphorylation in vitro by protein kinase A has previously been reported to
prevent association of the p105 C terminus or p105 with other Rel
proteins(27, 54) . However, these in vitro effects probably do not reflect events in vivo, as proved
to be the case for similar results with in vitro phosphorylation of IB
(55) . Protein kinase A is
unlikely to be responsible for the PMA/ionomycin-induced
phosphorylation of p105, as protein kinase A is not activated by this
stimulus and actually inhibits activation of NF-
B in T
cells(56) .
Casein kinase II has been implicated in the
phosphorylation of multiple transcription factors, although there is
some controversy as to whether its activity responds to
signals(57) . Interestingly, the inducible phosphorylation of
IB
occurs on two consensus casein kinase II sites in the N
terminus, although biochemical and immunological evidence indicates
casein kinase II constitutively phosphorylates only C-terminal sites in
I
B
in vivo(58) . Casein kinase II could
conceivably be responsible for the inducible p105 phosphorylation
described here if the increased phosphorylation of p105 is due to
inactivation of a constitutive phosphatase, as discussed below.
We
have evidence that a serine kinase activity that acts on the PEST
domain of p105 is specifically associated with that protein, ()and in vitro phosphorylation of p105 with Jurkat
extracts has been reported(29) . The association of a kinase
and a transcription factor substrate has precedent in the
mitogen-activated protein kinase family, notably the Jun kinase 1/c-Jun
association(60, 61) . However, unlike Jun kinases, the in vitro activity of the p105-associated kinase we identified
is not altered by prior PMA/ionomycin treatment or other Rel-activating
stimuli. If the associated kinase activity is responsible for the
inducible p105 phosphorylation described here, there are at least two
possibilities. Either the associated kinase is activated by
PMA/ionomycin in vivo but deregulated in the in vitro kinase assays, or it phosphorylates p105 constitutively and
stimulation inactivates constitutive p105 phosphatase activity. Current
experimental evidence is consistent with either model, and other
kinases not stably associated with p105 could phosphorylate the
molecule as well.
If there is constitutive p105 kinase and
phosphatase activity in cells, the balance of the two may also regulate
the basal rate of processing of p105. The processing to Np50 of
nearly all
N819, the p105 construct lacking the phosphorylated
C-terminal PEST domain, in the absence of stimulation suggests that
this domain may also be involved in regulating constitutive processing.
In this model, the PEST sequences in their unphosphorylated state
protect full-length p105 from extensive processing, and phosphorylation
of this domain alters the conformation of the protein, making it more
accessible to the proteolytic machinery (see Fig. 8).
The
results reported here not only correlate increased phosphorylation and
processing of p105 in a model of T cell activation but also for the
first time identify the C-terminal PEST sequences as the target of
PMA/ionomycin-induced phosphorylation and show that they are required
for up-regulation of processing. These findings indicate the general
regulatory scheme of inducible serine phosphorylation and proteolysis
is conserved between p105 and IB
inhibitor complexes.
However, the insensitivity of p105 phosphorylation to antioxidants
demonstrates that each inhibitor is independently regulated, and the
response induced is graded to reflect the specific role of each in
determining the Rel response to changes in cell status.