(Received for publication, October 13, 1995)
From the
The NF-B transcription factor induces rapid transcription
of many genes in response to a variety of extracellular signals.
NF-
B is readily activated from normally inhibited cytoplasmic
stores by induced proteolytic degradation of I
B-
, a principal
inhibitor of this transcription factor. Following the inhibitor's
degradation, NF-
B is free to translocate to the nucleus and induce
gene transcription. The I
B-
inhibitor is targeted for
degradation by signal-induced phosphorylation of two closely spaced
serines in its NH
terminus (Ser
and
Ser
). Proteolytic degradation appears to be carried out by
proteasomes which can recognize ubiquitinated intermediates of the
I
B-
inhibitor. We provide evidence which supports a
ubiquitin-mediated mechanism. Amino acid substitutions of two adjacent
potential ubiquitination sites in the NH
terminus of
I
B-
(Lys
and Lys
) almost completely
block the rapid, signal-induced degradation of the mutant protein,
while they do not interfere with induced phosphorylation. The mutant
I
B-
also does not permit signal-induced activation of
NF-
B bound to it. The data suggest that ubiquitination at either
of the two adjacent lysines (21 and 22) is required for degradation
following induced phosphorylation at nearby serines 32 and 36. Such
dependence on ubiquitination of specific sites for protein degradation
is unusual. This mechanism of degradation may also apply to
I
B-
, an inhibitor related to and functionally overlapping
with I
B-
, as well as to cactus, an I
B homolog of Drosophila.
The transcription factor complexes known collectively as
NF-B function primarily as mediators of inducible transcription in
response to a variety of environmental signals (for recent reviews, see (1, 2, 3, 4, 5) ). Stress-
and pathogen-related signals in particular are known to activate
NF-
B, leading to induced expression of a large number of genes,
including many genes which encode functions relevant to immune
responses. In most cell types, p50-p65 heterodimers represent the vast
majority of the rapidly inducible NF-
B complexes, although several
other NF-
B dimers may coexist and may become activated also. All
NF-
B dimers are composed of members of the Rel/NF-
B family of
polypeptides, and in vertebrates this family is comprised of p50
(NF-
B1), p65 (RelA), c-Rel, p52 (NF-
B2), and RelB. The
various NF-
B dimers usually lie dormant in the cytoplasm of cells,
kept there by inhibitory ankyrin-containing members of the I
B
family of proteins, in particular I
B-
. I
B-
strongly
associates with p50/p65 heterodimers and appears to shield the nuclear
localization sequences contained in both subunits; this is presumed to
be the mechanism by which this protein retains the heterodimers in the
cytoplasm(6, 7, 8, 9) . Activation
of NF-
B proceeds via rapid, signal-induced proteolytic degradation
of the inhibitor, liberating the transcription factor which is now free
to translocate to the nucleus (10, 11, 12, 13, 14, 15, 16, 17) .
Degradation is carried out by proteasomes and is preceded by
signal-induced phosphorylation of I
B-
itself(18, 19, 20, 21, 22, 23, 24) .
Induced phosphorylation occurs on two closely spaced serines in the
NH
terminus of the protein (amino acids 32 and 36),
mediated by an as yet unknown
kinase(s)(25, 26, 27, 28) . It has
recently been shown that signal-induced phosphorylation can lead to
ubiquitination of I
B-
(29) . Since ubiquitin-tagged
proteins are generally subject to proteasome-mediated
proteolysis(30) , these observations suggest that degradation
of I
B-
is triggered by ubiquitination. However, a
ubiquitin-independent mechanism of degradation is not necessarily
excluded, since only a fraction of the total pool of I
B-
could be shown to be ubiquitinated (under conditions of proteolysis
inhibition). Furthermore, precedents exist for a ubiquitin-independent,
but proteasome-dependent degradation mechanism(31) . Therefore,
we sought to demonstrate ubiquitin-dependence by investigating the
requirement for lysines in I
B-
degradation, because lysines
are the sites at which ubiquitin is ligated(30) . Here we
provide evidence which strongly suggests that rapid, signal-regulated
degradation of I
B-
proceeds primarily via a
ubiquitin-dependent mechanism. An I
B-
mutant bearing
conservative substitutions at two potential ubiquitination sites is
remarkably resistant to signal-regulated degradation. The results imply
that two adjacent NH
-terminal lysines (Lys
and
Lys
) are the primary targets of signal-induced
ubiquitination. That specific lysines play such an important role in
ubiquitin-mediated protein degradation is uncommon(30) .
Since ubiquitin is ligated to proteins through lysine
residues, we substituted the lysines in IB-
by site-directed
mutagenesis and tested the resulting mutant proteins for defects in
signal-dependent degradation. Human I
B-
(Mad-3) contains
lysine at positions 21, 22, 38, 47, 67, 87, 98, 177, and 238 (32) . Lysines 22, 38, 87, and 238 are perfectly conserved in
pig, rat, and chicken (pp40) I
B-
; lysines 21, 47, 67, and 98
are absent in chicken; and lysine 177 is not conserved at
all(39) . We substituted each of the NH
-terminal
lysines (21, 22, 38, 47, 67, and 87) and the pair, 21 + 22, with
arginine residues to block ubiquitination of these sites which all lie
near the inducibly phosphorylated serines 32 and 36. Arginine was
chosen so as not to change the charge of the protein. As an initial
test of the mutant proteins, we transiently transfected expression
constructs for the various I
B-
mutants into Ntera-2 embryonal
carcinoma cells, together with an expression construct for
NF-
B/p65. Undifferentiated Ntera-2 cells do not express
significant levels of endogenous NF-
B or I
B proteins and are
thus ideal for evaluating the activities of the transfected proteins
without interference by endogenous
counterparts(33, 35, 36, 37, 38, 40) .
Transfected p65 potently transactivated a cotransfected
B-dependent CAT reporter, while coexpression of wild-type
I
B-
or any of the lysine-substituted mutants severely
inhibited p65-mediated transactivation (25) (data not shown).
Therefore, these mutations did not interfere with the inhibitory
activity of the protein, which was expected, since inhibition does not
require the NH
-terminal domain of
I
B-
(25, 41, 42, 43) . We
then tested whether the lysine mutations interfered with the
signal-induced degradation of the proteins bearing them, as measured by
the ability of a PMA stimulus to relieve inhibition and thus allow
p65-mediated transactivation of the CAT reporter (Fig. 1;
inducibility of mutants is shown as percent of inducibility of
wild-type I
B-
; inducibility is measured as the ratio of CAT
activity of PMA-stimulated cells/unstimulated cells). None of the
individual lysine mutations significantly interfered with
signal-induced transactivation, suggesting that no single lysine is
critical for the signal-induced degradation of I
B-
(Fig. 1; column 1, wild-type (wt); columns
2, 3, 5-8, mutants K21R, K22R, K38R, K47R,
K67R, and K87R (R
, R
, R
, R
, R
, and R
, respectively). However, an
I
B-
mutant bearing substitutions at both lysines 21 and 22
(K21R/K22R (R
R
mutant) had
a dramatic effect; this mutant did not allow significant activation of
NF-
B in PMA-stimulated, transfected Ntera-2 cells (Fig. 1, column 4). The data suggest that at least one of the two
lysines at positions 21 and 22 has to be present for rapid
signal-induced degradation of I
B-
to occur, since elimination
of both lysines effectively blocked NF-
B activation, while
substitution of either lysine alone was of little consequence.
Figure 1:
Inducibility of a B-dependent CAT
reporter in the presence of wild-type (wt) or mutant
I
B-
s in transiently transfected NTera-2 cells. NTera-2 cells
were transfected with PMT2T-NF-
B/p65, wild-type or mutant
PMT2T-I
B-
expression vectors and the
B-dependent CAT
reporter plasmid (see ``Materials and Methods''). (p65,
transfected alone, potently stimulated CAT activity, and cotransfection
of the I
B-
vectors inhibited this transactivation to
near-background levels(25) .). The p65/I
B-
cotransfected cells were stimulated with PMA, and inducibility was
calculated as the ratio of PMA-stimulated CAT activity to unstimulated
activity. The inducibilities are shown as a percent of that seen with
matched, wild-type I
B-
, which represents an at least 10-fold
stimulation. In several independent experiments, only the K21R/K22R (R
R
)
mutant blocked PMA induction of CAT
activity.
To
confirm these interpretations and to rule out a potential defect in
phosphorylation of the K21R/K22R mutant, we directly evaluated mutants
for phosphorylation and degradation. Murine EL-4 T cells were
permanently transfected with the various IB-
mutants, and
then the cells were stimulated with PMA and ionomycin. We showed
previously that exogenously derived human I
B-
is subject to
the same signal-induced phosphorylation and degradation as the
endogenous murine I
B-
(25) . Endogenous murine
I
B-
serves as an internal positive control in these
experiments. Since it migrates slightly faster than the transfected
human protein, both proteins could be simultaneously
visualized(25) . Among the mutants tested, only the K21R/K22R
mutant was resistant to signal-induced degradation in the EL-4 cells (Fig. 2), while all other transfected human (h) mutant
proteins bearing individual lysine substitutions appeared to be
degraded as efficiently as the endogenous murine (m)
I
B-
(Fig. 2, K21R, K22R, K38R, K47R, K67R, and K87R (R
, R
, R
, R
, R
, and R
).
All mutant proteins, including the double mutant K21R/K22R, were
rapidly phosphorylated in response to signals, as indicated by the
shift in mobility of I
B-
in the presence of calpain inhibitor
I, which inhibits proteasomes (20, 21, 22, 23, 24) (Fig. 2,
data not shown for K38R, K47R, K67R, and K87R). This is as expected for
the rapidly degraded I
B-
proteins. In the case of the double
mutant (K21R/K22R), the proteasome inhibitors were not needed to see
the phosphorylation, since this mutant was not efficiently degraded.
The presence of the proteasome inhibitor did, however, increase the
amount of the K21R/K22R I
B-
mutant observed, suggesting that
these mutations may not completely block induced degradation.
Nonetheless, the K21R/K22R mutation afforded this I
B-
significant protection from degradation, and the results are consistent
with the Ntera-2 experiments shown in Fig. 1.
Figure 2:
Signal-induced degradation and
phosphorylation of wild-type and mutant IB-
expressed in
stably transfected EL-4 cells. EL-4 murine T cells were permanently
transfected with wild-type (wt) or mutated (mt),
human (h) I
B-
expression vectors (see
``Materials and Methods''), as indicated. Cells were
stimulated with PMA and ionomycin (Iono) for 15 min in the
presence (+) or absence of calpain inhibitor I (Calp.
Inhib.), which inhibits proteasome activity. Both transfected
human and endogenous murine (m) I
B-
, as well as the
inducibly phosphorylated form of human I
B-
(I
B-
) were visualized by Western analysis (see
``Materials and Methods''). (The inducibly phosphorylated
mutated I
B-
migrates to almost the same position as the
uninduced form and is not readily distinguished here(25) .)
Only the K21R/K22R (R
R
)
mutation in human I
B-
blocked rapid degradation (while the
murine wild-type protein was degraded in the same cells), which
resulted in an accumulation of the inducibly phosphorylated form, even
in the absence of proteasome inhibitors.
We have demonstrated a critical requirement for the presence
of either of two lysines at positions 21 and 22 in signal-induced
degradation of IB-
and, as a consequence, in signal-induced
transactivation by NF-
B/p65. The substitution of both lysines 21
and 22 with arginines in I
B-
(K21R/K22R) caused a severe
block to rapid signal-induced degradation of that I
B-
in
stably transfected EL-4 cells. The mutant protein was, however, still
phosphorylated at nearby serine sites (S
and
S
), indicating that the defect lies downstream of the
phosphorylation step; it also suggests that the protein was not grossly
altered by these conservative substitutions, since the kinase(s)
acitivity on this substrate appears unaffected. The K21R/K22R mutant
prevented the signal-induced, p65-mediated transactivation of a
B-dependent reporter in transient transfection experiments using
Ntera-2 cells. In contrast to the K21R/K22R mutant, mutants bearing
substitutions of individual lysines, including those at residue 21 or
22, had no measurable effect and behaved like wild type. Taken together
these data demonstrate that either of the two lysines at positions 21
and 22 is necessary for rapid degradation (but not for phosphorylation)
and they provide a compelling argument for obligatory ubiquitination
prior to degradation of I
B-
: at least one of the two
potential ubiquitination sites must be present for rapid signal-induced
degradation to proceed. Although degradation is dramatically inhibited,
it appears not to be absolutely blocked (see Fig. 2); this may
indicate that ubiquitination can also occur at other sites, albeit less
efficiently, or that another mechanism allows for a slower degradation.
Finally, the data do not tell us if Lys
or Lys
are sufficient for ubiquitin-mediated degradation. It is
possible, for example, that some other, not necessarily specific,
lysine is necessary also. To formally test this less likely possibility
would require a mutant I
B-
bearing substitutions of all
lysines other than 21 and 22.
Individual ubiquitination sites do not
usually play a dominant role in protein degradation, where often
multiple functional ubiquitination sites exist and targeted mutations
have little effect(30) , although the degradation of Mos may be
another exception to this rule(44) . It is possible that
ubiquitination of IB-
may be specifically directed to lysines
21 and 22, or, alternatively, that these lysines are the only ones
accessible for ligation (no other lysines exist NH
-terminal
to the phosphorylation sites). This latter possibility may be supported
by the observation that ubiquitination occurs with I
B-
still
bound to NF-
B(29) , which should partially shield the
inhibitor. The central part of I
B-
consists of 6 ankyrin
repeats whose primary function is to interact with NF-
B; this part
may be largely buried in the cleft between the two NF-
B subunits,
as suggested by x-ray crystallographic data of p50
homodimers(45, 46) . The fairly short COOH-terminal
region of I
B-
is required for inhibition of DNA binding by
NF-
B, implying that it too may interact with NF-
B
proteins(25, 43) . By contrast, the
NH
-terminal part of the protein is not required for these
functions, rather, it must be accessible to a kinase(s) to allow
inducible phosphorylation. An as yet undetermined protein may then
recognize the phosphorylated protein, presumably by recognizing the
phosphorylated serines or local changes induced in the protein as a
consequence of phosphorylation (no major conformational changes are
expected, since the phosphorylated species remains tightly bound to
NF-
B and continues to
inhibit)(18, 19, 20, 21, 22, 23, 24) .
Thus, the two lysines NH
-terminal to the two serines may
present the only obvious targets. It remains to be shown whether highly
ubiquitinated I
B-
is removed from the complex just prior to
degradation, or if degradation is initiated while ubiquitinated
I
B-
is still in the complex. In contrast to bound
I
B-
, the free unbound form may present additional sites for
ubiquitination.
Chicken IB-
(pp40) contains only one of
the two lysine residues important for degradation (the Lys equivalent
to that at position 22 in the human protein), suggesting that a single
substitution of that lysine may be sufficient to block rapidly
inducible degradation of pp40. Recently I
B-
was cloned and
shown to be inducibly degraded in response to certain signals, such as
interleukin-1 and lipopolysaccharide(47) . While I
B-
and I
B-
share high overall similarity, their
NH
-terminal regions are surprisingly different, save for a
few conserved amino acids; however, these few amino acids appear to be
highly significant in that they suggest shared regulatory features of
these proteins (Fig. 3). Both inducibly phosphorylated serines
and a few surrounding residues are conserved as is the lysine
equivalent to that at position 22 in I
B-
. (This is the only
lysine in the entire NH
-terminal part of the I
B-
protein, which may suggest that it is absolutely required for signaling
in that protein). This limited but significant conservation of
functional sites can also be found in cactus, the Drosophila homolog of I
B proteins (48, 49) (see Fig. 3), suggesting that all three proteins may be regulated in
a similar fashion. Cactus, which contains a much larger
NH
-terminal domain than either I
B-
or
I
B-
, may offer additional sites for regulation.
Figure 3:
Sequence comparisons of IB-
,
I
B-
, and cactus. Functionally important residues of
I
B-
are conserved in I
B-
and cactus. This includes
both inducibly phosphorylated serines, 32 and 36, a few surrounding
residues, and at least one of the two NH
-terminal lysines
important for degradation.