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(Received for publication, October 13,
1995) From the
The NF-
The transcription factor complexes known collectively as
NF-
Since ubiquitin is ligated to proteins through lysine
residues, we substituted the lysines in I
Figure 1:
Inducibility of a
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 I
Figure 2:
Signal-induced degradation and
phosphorylation of wild-type and mutant I
We have demonstrated a critical requirement for the presence
of either of two lysines at positions 21 and 22 in signal-induced
degradation of I 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 I Chicken I
Figure 3:
Sequence comparisons of I
Volume 271,
Number 1,
Issue of January 5, 1996 pp. 376-379
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
B-
(*)
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
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 IB-
, 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 IB- 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
IB- (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
IB-
, an inhibitor related to and functionally overlapping
with IB-, as well as to cactus, an IB homolog of Drosophila.
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 IB
family of proteins, in particular IB-. IB- 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 IB-
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 IB-(29) . Since ubiquitin-tagged
proteins are generally subject to proteasome-mediated
proteolysis(30) , these observations suggest that degradation
of IB- is triggered by ubiquitination. However, a
ubiquitin-independent mechanism of degradation is not necessarily
excluded, since only a fraction of the total pool of IB-
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 IB- 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 IB- proceeds primarily via a
ubiquitin-dependent mechanism. An IB- 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) .
Site-directed Mutagenesis
Mutations in
full-length, human IB- cDNA (32) were generated
essentially as described previously(25, 33) . In each
case, lysine codons were substituted with arginine codons (for
positions 21, 22, 47 and 67: AAG was changed to AGG; for 38: AAA was
changed to CGA; for 87: AAG was changed to CGG). The mutated
IB- cDNAs were excised from a Bluescript vector (Stratagene,
La Jolla, CA) with EcoRI (1550 base pairs) and subcloned into
the PMT2T mammalian expression vector(34) . Mutations were
confirmed by subsequent DNA sequence analysis.Transient Transfections
Ntera-2, human embryonal
carcinoma cells were transfected via calcium phosphate-mediated
transfer as described elsewhere (35) with the following
vectors: a CAT (
)reporter plasmid containing the tandemly
repeated B sites of human immunodeficiency virus (5
µg)(35) ; the p65-PMT2T expression vector (0.2
µg)(35) ; PMT2T vectors carrying the wild-type or mutant
IB-s (see above) (depending on the experiment, between 0.15
and 0.6 µg were found to be required for near-maximal inhibition,
which was the point used in the experiments). Cells were stimulated
with PMA (10 ng/ml) for 6 h prior to harvesting, stimulation starting
at about 36 h post-transfection, and CAT activity was measured as
previously
described(25, 35, 36, 37, 38) . Permanent Transfections
EL-4 murine T lymphoma
cells were maintained in RPMI medium in the presence of 10% FCS (Life
Technologies, Inc.) and were stably transfected with the various
PMT2T-IB- vectors, together with a plasmid conferring
neomycin resistance, as described elsewhere(25) . Briefly, 20
µg of PMT2T-IB- DNA and 2 µg of neomycin-resistant
plasmid DNA were electroporated into 10
EL-4 cells using
the Bio-Rad Gene Pulser, set at 250 V, 960 microfarads. Stable
neomycin-resistant transfectants were selected after 3-4 weeks of
incubation with geneticin (G418, Life Technologies, Inc.), used at 400
µg/ml during the first week and at 200 µg/ml thereafter. The
resulting cell lines were screened for expression of human
IB- using a rabbit polyclonal antibody directed against
full-length IB- (10, 25) and ECL technology
(Amersham Corp.). Cells were stimulated with PMA (40 ng/ml) and
ionomycin (2 µM), and calpain inhibitor I was used at 100
µM, starting 30 min prior to stimulation.
B- by site-directed
mutagenesis and tested the resulting mutant proteins for defects in
signal-dependent degradation. Human IB- (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) IB-; 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 IB- 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 IB 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
IB- 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
IB-(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 IB-; 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 IB- (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 (RR
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 IB- to occur, since elimination
of both lysines effectively blocked NF-B activation, while
substitution of either lysine alone was of little consequence.
B-dependent CAT
reporter in the presence of wild-type (wt) or mutant
IB-s in transiently transfected NTera-2 cells. NTera-2 cells
were transfected with PMT2T-NF-B/p65, wild-type or mutant
PMT2T-IB- expression vectors and the B-dependent CAT
reporter plasmid (see ``Materials and Methods''). (p65,
transfected alone, potently stimulated CAT activity, and cotransfection
of the IB- vectors inhibited this transactivation to
near-background levels(25) .). The p65/IB-
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 IB-, which represents an at least 10-fold
stimulation. In several independent experiments, only the K21R/K22R (RR
)
mutant blocked PMA induction of CAT
activity.
B- mutants, and
then the cells were stimulated with PMA and ionomycin. We showed
previously that exogenously derived human IB- is subject to
the same signal-induced phosphorylation and degradation as the
endogenous murine IB-(25) . Endogenous murine
IB- 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)
IB- (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 IB- 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 IB- mutant observed, suggesting that
these mutations may not completely block induced degradation.
Nonetheless, the K21R/K22R mutation afforded this IB-
significant protection from degradation, and the results are consistent
with the Ntera-2 experiments shown in Fig. 1.
B- expressed in
stably transfected EL-4 cells. EL-4 murine T cells were permanently
transfected with wild-type (wt) or mutated (mt),
human (h) IB- 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) IB-, as well as the
inducibly phosphorylated form of human IB-
(IB-
) were visualized by Western analysis (see
``Materials and Methods''). (The inducibly phosphorylated
mutated IB- migrates to almost the same position as the
uninduced form and is not readily distinguished here(25) .)
Only the K21R/K22R (RR
)
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.
B- and, as a consequence, in signal-induced
transactivation by NF-B/p65. The substitution of both lysines 21
and 22 with arginines in IB- (K21R/K22R) caused a severe
block to rapid signal-induced degradation of that IB- 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 IB-: 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.B- 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 IB- still
bound to NF-B(29) , which should partially shield the
inhibitor. The central part of IB- 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 IB- 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 IB- is removed from the complex just prior to
degradation, or if degradation is initiated while ubiquitinated
IB- is still in the complex. In contrast to bound
IB-, the free unbound form may present additional sites for
ubiquitination.B- (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 IB- was cloned and
shown to be inducibly degraded in response to certain signals, such as
interleukin-1 and lipopolysaccharide(47) . While IB-
and IB- 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 IB-. (This is the only
lysine in the entire NH-terminal part of the IB-
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 IB 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 IB- or
IB-, may offer additional sites for regulation.
B-,
IB-, and cactus. Functionally important residues of
IB- are conserved in IB- 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.
)
We are grateful to A. S. Fauci for support and review
of the manuscript.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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A. Leonardi, H. Ellinger-Ziegelbauer, G. Franzoso, K. Brown, and U. Siebenlist Physical and Functional Interaction of Filamin (Actin-binding Protein-280) and Tumor Necrosis Factor Receptor-associated Factor 2 J. Biol. Chem., January 7, 2000; 275(1): 271 - 278. [Abstract] [Full Text] [PDF]
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J. R. Burke, M. K. Wood, R.-P. Ryseck, S. Walther, and C. A. Meyers Peptides Corresponding to the N and C Termini of Ikappa B-alpha , -beta , and -epsilon as Probes of the Two Catalytic Subunits of Ikappa B Kinase, IKK-1 and IKK-2 J. Biol. Chem., December 17, 1999; 274(51): 36146 - 36152. [Abstract] [Full Text] [PDF]
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 D. M. Rothwarf and M. Karin The NF-{kappa}B Activation Pathway: A Paradigm in Information Transfer from Membrane to Nucleus Sci. Signal., October 26, 1999; 1999(5): re1 - re1. [Abstract] [Full Text] [PDF]
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T. Uehara, J. Matsuno, M. Kaneko, T. Nishiya, M. Fujimuro, H. Yokosawa, and Y. Nomura Transient Nuclear Factor kappa B (NF-kappa B) Activation Stimulated by Interleukin-1beta May Be Partly Dependent on Proteasome Activity, but Not Phosphorylation and Ubiquitination of the Ikappa Balpha Molecule, in C6 Glioma Cells. REGULATION OF NF-kappa B LINKED TO CHEMOKINE PRODUCTION J. Biol. Chem., May 28, 1999; 274(22): 15875 - 15882. [Abstract] [Full Text] [PDF]
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M. Algarté, H. Nguyen, C. Heylbroeck, R. Lin, and J. Hiscott Ikappa B-Mediated Inhibition of Virus-Induced Beta Interferon Transcription J. Virol., April 1, 1999; 73(4): 2694 - 2702. [Abstract] [Full Text]
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S. Montaner, R. Perona, L. Saniger, and J. C. Lacal Activation of Serum Response Factor by RhoA Is Mediated by the Nuclear Factor-kappa B and C/EBP Transcription Factors J. Biol. Chem., March 26, 1999; 274(13): 8506 - 8515. [Abstract] [Full Text] [PDF]
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M. S. Rodriguez, J. Thompson, R. T. Hay, and C. Dargemont Nuclear Retention of Ikappa Balpha Protects It from Signal-induced Degradation and Inhibits Nuclear Factor kappa B Transcriptional Activation J. Biol. Chem., March 26, 1999; 274(13): 9108 - 9115. [Abstract] [Full Text] [PDF]
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M. Kroll, F. Margottin, A. Kohl, P. Renard, H. Durand, J.-P. Concordet, F. Bachelerie, F. Arenzana-Seisdedos, and R. Benarous Inducible Degradation of Ikappa Balpha by the Proteasome Requires Interaction with the F-box Protein h-beta TrCP J. Biol. Chem., March 19, 1999; 274(12): 7941 - 7945. [Abstract] [Full Text] [PDF]
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N. Li and M. Karin Ionizing radiation and short wavelength UV activate NF-kappa B through two distinct mechanisms PNAS, October 27, 1998; 95(22): 13012 - 13017. [Abstract] [Full Text] [PDF]
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J. R. Burke, K. R. Miller, M. K. Wood, and C. A. Meyers The Multisubunit Ikappa B Kinase Complex Shows Random Sequential Kinetics and Is Activated by the C-terminal Domain of Ikappa Balpha J. Biol. Chem., May 15, 1998; 273(20): 12041 - 12046. [Abstract] [Full Text] [PDF]
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F. Hirano, M. Chung, H. Tanaka, N. Maruyama, I. Makino, D. D. Moore, and C. Scheidereit Alternative Splicing Variants of Ikappa Bbeta Establish Differential NF-kappa B Signal Responsiveness in Human Cells Mol. Cell. Biol., May 1, 1998; 18(5): 2596 - 2607. [Abstract] [Full Text]
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S. L. Schauer, R. E. Bellas, and G. E. Sonenshein Dominant Signals Leading to Inhibitor {kappa}B Protein Degradation Mediate CD40 Ligand Rescue of WEHI 231 Immature B Cells from Receptor-Mediated Apoptosis J. Immunol., May 1, 1998; 160(9): 4398 - 4405. [Abstract] [Full Text] [PDF]
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J. Ochoa-Garay, J. Kaye, and J. E. Coligan Nuclear Factor {kappa}B Is Required for Peptide Antigen-Induced Differentiation of a CD4+CD8+ Thymocyte Line J. Immunol., April 15, 1998; 160(8): 3835 - 3843. [Abstract] [Full Text] [PDF]
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R.-M. Dai, E. Chen, D. L. Longo, C. M. Gorbea, and C.-C. H. Li Involvement of Valosin-containing Protein, an ATPase Co-purified with Ikappa Balpha and 26 S Proteasome, in Ubiquitin-Proteasome-mediated Degradation of Ikappa Balpha J. Biol. Chem., February 6, 1998; 273(6): 3562 - 3573. [Abstract] [Full Text] [PDF]
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R. Mahajan, L. Gerace, and F. Melchior Molecular Characterization of the SUMO-1 Modification of RanGAP1 and Its Role in Nuclear Envelope Association J. Cell Biol., January 26, 1998; 140(2): 259 - 270. [Abstract] [Full Text] [PDF]
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C. Sears, J. Olesen, D. Rubin, D. Finley, and T. Maniatis NF-kappa B p105 Processing via the Ubiquitin-Proteasome Pathway J. Biol. Chem., January 16, 1998; 273(3): 1409 - 1419. [Abstract] [Full Text] [PDF]
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R. M. Attar, H. Macdonald-Bravo, C. Raventos-Suarez, S. K. Durham, and R. Bravo Expression of Constitutively Active Ikappa Bbeta in T Cells of Transgenic Mice: Persistent NF-kappa B Activity Is Required for T-Cell Immune Responses Mol. Cell. Biol., January 1, 1998; 18(1): 477 - 487. [Abstract] [Full Text]
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M. Barkett, D. Xue, H. R. Horvitz, and T. D. Gilmore Phosphorylation of Ikappa B-alpha Inhibits Its Cleavage by Caspase CPP32 in Vitro J. Biol. Chem., November 21, 1997; 272(47): 29419 - 29422. [Abstract] [Full Text] [PDF]
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L. Ghoda, X. Lin, and W. C. Greene The 90-kDa Ribosomal S6 Kinase (pp90rsk) Phosphorylates the N-terminal Regulatory Domain of Ikappa Balpha and Stimulates Its Degradation in Vitro J. Biol. Chem., August 22, 1997; 272(34): 21281 - 21288. [Abstract] [Full Text] [PDF]
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K. Tashiro, M. P. Pando, Y. Kanegae, P. M. Wamsley, S. Inoue, and I. M. Verma Direct involvement of the ubiquitin-conjugating enzyme Ubc9/Hus5 in the degradation of Ikappa Balpha PNAS, July 22, 1997; 94(15): 7862 - 7867. [Abstract] [Full Text] [PDF]
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R. Weil, C. Laurent-Winter, and A. Israel Regulation of Ikappa Bbeta Degradation. SIMILARITIES TO AND DIFFERENCES FROM Ikappa Balpha J. Biol. Chem., April 11, 1997; 272(15): 9942 - 9949. [Abstract] [Full Text] [PDF]
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M. Gueguen and E. O. Long Presentation of a cytosolic antigen by major histocompatibility complex class II molecules requires a long-lived form of the antigen PNAS, December 10, 1996; 93(25): 14692 - 14697. [Abstract] [Full Text] [PDF]
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M. Trofimova, A. B. Sprenkle, M. Green, T. W. Sturgill, M. G. Goebl, and M. A. Harrington Developmental and Tissue-specific Expression of Mouse Pelle-like Protein Kinase J. Biol. Chem., July 26, 1996; 271(30): 17609 - 17612. [Abstract] [Full Text] [PDF]
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M. P. Pando and I. M. Verma Signal-dependent and -independent Degradation of Free and NF-kappa B-bound Ikappa Balpha J. Biol. Chem., July 7, 2000; 275(28): 21278 - 21286. [Abstract] [Full Text] [PDF]
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M. Prigent, I. Barlat, H. Langen, and C. Dargemont Ikappa Balpha and Ikappa Balpha /NF-kappa B Complexes Are Retained in the Cytoplasm through Interaction with a Novel Partner, RasGAP SH3-binding Protein 2 J. Biol. Chem., November 10, 2000; 275(46): 36441 - 36449. [Abstract] [Full Text] [PDF]
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A. Leonardi, A. Chariot, E. Claudio, K. Cunningham, and U. Siebenlist CIKS, a connection to Ikappa B kinase and stress-activated protein kinase PNAS, September 12, 2000; 97(19): 10494 - 10499. [Abstract] [Full Text] [PDF]
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