U.S. patent application number 11/716086 was filed with the patent office on 2007-07-19 for gamma subunit of cytokine responsive lkb-alpha kinase complex.
This patent application is currently assigned to The Regents of the University of California. Invention is credited to Michael Karin, David M. Rothwarf, Ebrahim Zandi.
Application Number | 20070166812 11/716086 |
Document ID | / |
Family ID | 37833379 |
Filed Date | 2007-07-19 |
United States Patent
Application |
20070166812 |
Kind Code |
A1 |
Karin; Michael ; et
al. |
July 19, 2007 |
Gamma subunit of cytokine responsive lkB-alpha kinase complex
Abstract
The present invention provides a novel essential regulatory
subunit of the I.kappa.B kinase (IKK) complex, IKK-.gamma.. The
isolated IKK-.gamma. subunit of the invention has substantially the
same amino acid sequence as SEQ ID NO: 2 shown in FIG. 2.
Inventors: |
Karin; Michael; (La Jolla,
CA) ; Rothwarf; David M.; (La Jolla, CA) ;
Zandi; Ebrahim; (Duarte, CA) |
Correspondence
Address: |
Medlen & Carroll, LLP;Suite 350
101 Howard Street
San Francisco
CA
94105
US
|
Assignee: |
The Regents of the University of
California
Oakland
CA
94607
|
Family ID: |
37833379 |
Appl. No.: |
11/716086 |
Filed: |
March 9, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09377795 |
Aug 20, 1999 |
7189832 |
|
|
11716086 |
Mar 9, 2007 |
|
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60097418 |
Aug 20, 1998 |
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Current U.S.
Class: |
435/194 |
Current CPC
Class: |
C12N 9/1205
20130101 |
Class at
Publication: |
435/194 |
International
Class: |
C12N 9/12 20060101
C12N009/12 |
Goverment Interests
[0002] This invention was made with government support under grant
number R01AI43477 awarded by the National Institutes of Health. The
government has certain rights in the invention.
Claims
1-29. (canceled)
30. An isolated I.kappa.B kinase (IKK) subunit, IKK-.gamma.,
comprising the amino acid sequence SEQ ID NO: 2.
31. An isolated IKK-.gamma. fragment comprising amino acids 134-419
of SEQ ID NO:2.
32. An isolated IKK-.gamma. fragment comprising amino acids 1-300
of SEQ ID NO:2.
Description
[0001] This application claims the benefit of U.S. Provisional
Application Ser. No. 60/097,418, filed Aug. 20, 1998, and which is
incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] The present invention relates generally to molecular biology
and biochemistry and more specifically to a subunit of a protein
kinase, the I.kappa.B kinase, which is activated in response to
environmental stresses and proinflammatory signals to phosphorylate
inhibitors of NF-.kappa.B.
[0005] 2. Background Information
[0006] In chronic inflammatory disease including asthma, rheumatoid
arthritis, inflammatory bowel disease and psoriasis, cytokines
recruit activated immune and inflammatory cells to the site of
lesions, thereby amplifying and perpetuating the inflammatory
states. Although the causes of chronic inflammatory disease are in
large part unknown, both genetic and environmental factors
contribute to pathology. Genes such as those for HLA antigens in
rheumatoid arthritis and inflammatory bowel disease can determine a
patient's susceptibility to the disease and disease severity, and
environmental factors can determine its course. Once established, a
chronic inflammatory process is virtually impossible to disrupt;
there is no curative treatment for any chronic inflammatory
disease. Although chronic disease can be suppressed by
glucocorticoid or immunosuppressive therapy, side effects are
associated with prolonged treatment.
[0007] A ubiquitous transcription factor, nuclear factor-.kappa.B
(NF-.kappa.B), has been identified as of particular importance in
immune and inflammatory responses. NF-.kappa.B increases the
expression of the genes for many cytokines, enzymes, and adhesion
molecules in chronic inflammatory diseases. One gene regulated by
NF-.kappa.B is the gene for inducible nitric oxide synthase, the
expression of which is increased in airway epithelial cells and
macrophages in patients with asthma; in colonic epithelial cells of
patients with the inflammatory bowel disease, ulcerative colitis;
and in synovial cells in inflamed joints. The increased expression
is reflected by an increased amount of nitric oxide in the exhaled
breath of patients with asthma and in the colons of patients with
active ulcerative colitis, as well as by elevated urinary nitrite
concentrations in patients with rheumatoid arthritis.
Cyclooxygenase-2, another inducible enzyme regulated by
NF-.kappa.B, is responsible for the increased production of
prostaglandins and thromboxane in inflammatory diseases.
[0008] NF-.kappa.B also is involved in expression of adhesion
molecules, which recruit inflammatory cells such as neutrophils,
eosinophils and T lymphocytes from the circulation to the site of
inflammation in all chronic inflammatory diseases. NF-.kappa.B
regulates the expression of several genes that encode adhesion
molecules such as intercellular adhesion molecule 1, vascular cell
adhesion molecule 1, and E-selectin.
[0009] The production of pro-inflammatory cytokines also can be
regulated by NF-.kappa.B. Interleukin-1.beta., TNF-.alpha.,
interleukin-6, granulocyte-macrophage colony-stimulating factor,
and many chemotactic cytokines (chemokines) is increased in
patients with asthma, rheumatoid arthritis, psoriasis and
inflammatory bowel disease, and all have important roles in the
inflammatory process. Interleukin-1.beta. and TNF-.alpha. appear to
influence severity of disease, possibly by persistent activation of
NF-.kappa.B.
[0010] Thus, the transcription factor NF-.kappa.B plays a central
role in inflammatory disease. This role is emphasized by the
targeted disruption of an inhibitor of NF-.kappa.B in mice, which
resulted in prolonged activation of NF-.kappa.B in response to
inflammatory stimuli, and the death of these animals due to
widespread inflammation.
[0011] Unfortunately, the available therapies for chronic
inflammatory diseases such as asthma, rheumatoid arthritis and
inflammatory bowel disease are unsatisfactory. Identification of
molecules that regulate NF-.kappa.B would provide new strategies
for screening for anti-inflammatory therapeutics. The present
invention satisfies this need by providing the novel I.kappa.B
kinase subunit, IKK-.gamma., which is an essential regulatory
subunit of the I.kappa.B kinase required for NF-.kappa.B
activation. Related advantages also are provided.
SUMMARY OF THE INVENTION
[0012] The present invention provides a novel essential regulatory
subunit of the I.kappa.B kinase (IKK) complex, IKK-.gamma.. The
isolated IKK-.gamma. subunit of the invention has substantially the
same amino acid sequence as SEQ ID NO: 2, for example, an amino
acid sequence having at least 55% amino acid identity with SEQ ID
NO: 2.
[0013] Also provided by the invention is an IKK-.gamma.active
fragment that has substantially the same amino acid sequence as a
portion of the I.kappa.B kinase subunit, IKK-.gamma.. An
IKK-.gamma. active fragment of the invention can contain, for
example, ten, twenty, fifty or more contiguous amino acids of SEQ
ID NO: 2 and can have, for example, IKK-.beta. binding
activity.
[0014] The invention also provides an isolated IKK-.gamma.nucleic
acid molecule, which contains a nucleotide sequence encoding
substantially the same amino acid sequence as SEQ ID NO: 2. Such an
isolated IKK-.gamma. nucleic acid molecule of the invention can
have, for example, a nucleotide sequence encoding an amino acid
sequence having at least 55% amino acid identity with SEQ ID NO: 2.
An isolated IKK-.gamma. nucleic acid molecule of the invention can
have a nucleotide sequence encoding SEQ ID NO: 2, for example,
nucleotides 149 to 1408 of SEQ ID NO: 1 or the entire sequence of
SEQ ID NO: 1.
[0015] IKK-.gamma. polynucleotides and antisense polynucleotides
also are provided by the invention. The invention provides a
polynucleotide containing at least nine contiguous nucleotides of
SEQ ID NO: 1. The invention also provides an antisense
polynucleotide containing a nucleotide sequence complementary to at
least nine contiguous nucleotides of SEQ ID NO: 1.
[0016] The present invention also provides a method of identifying
an effective agent that modulates the specific association of an
I.kappa.B kinase .gamma. (IKK-.gamma.) subunit and a second
protein. Such agents can represent novel therapeutic agents, such
as immunosuppressant, anti-inflammatory or anti-cancer
therapeutics. The method includes the steps of contacting the
IKK-.gamma. subunit and the second protein with an agent under
conditions suitable for the specific association of the
IKK-.gamma.subunit and the second protein, and detecting an altered
association of the IKK-.gamma. subunit and the second protein in
the presence of the agent, where the altered association identifies
the agent as an effective agent that modulates the specific
association of the IKK-.gamma. subunit and the second protein. In a
method of the invention, the contacting can be, for example, in
vitro with an isolated IKK-.gamma. subunit. Alternatively, the
IKK-.gamma. subunit can be contacted, for example, in a cell such
as a mammalian cell or yeast cell in culture. In a method of the
invention, an altered association can be detected by a variety of
methods, for example, by measuring the transcriptional activity of
a reporter gene. In a preferred embodiment, the second protein is
IKK-.beta. and an effective agent is identified that modulates the
specific association of an IKK-.gamma. subunit and an IKK-.beta.
subunit.
[0017] Also provided herein is a method of modulating NF-.kappa.B
activity in a cell by contacting the cell with an effective agent
that modulates the specific association of an IKK-.gamma. subunit
and a second protein. In a method of the invention, the second
protein can be, for example, IKK-.beta.. Methods of modulating
NF-.kappa.B activity in a cell by introducing into the cell an
IKK-.gamma. antisense polynucleotide also are provided. The
IKK-.gamma. antisense polynucleotide can be expressed in the cell,
for example, in a vector.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIG. 1: Purification of the IKK complex and identification
of the IKK subunits. a. Partially purified HeLa cell extracts were
passed through an anti-IKK-.alpha. immunoaffinity column. The
input, flowthrough (FT) and bound fractions were separated by
SDS-PAGE and examined for IKK-.alpha. and IKK-.beta. content by
immunoblotting. b. The purified IKK complex was separated by
SDS-PAGE and stained with colloidal blue. The positions of the
different subunits are indicated. The inset shows a portion of a
gel run for a longer time in which better separation of
IKK-.gamma.1 and IKK-.gamma.2 is seen. c. 293 cells were labeled
with .sup.35S for 5 hrs using Pro-Mix (Amersham) followed by
incubation without or with TNF. Lysates were immunoprecipitated
(IP) with either anti-IKK-.alpha. or anti-HA antibody (used as a
control). After extensive washing, the immunecomplexes were
separated by SDS-PAGE and visualized by autoradiography.
[0019] FIG. 2: Primary and secondary structure of IKK-.gamma.. a.
The nucleotide sequence of the complete IKK-.gamma.cDNA (SEQ ID NO:
1). The initiator "ATG" is underlined. b. The amino acid sequence
of the complete IKK-.gamma. ORF (SEQ ID NO: 2). Peptide sequences
obtained by microsequencing are overlined; the leucines of the
leucine zipper are indicated by solid dots. c. Secondary structure
prediction of IKK-.gamma.. The boxes indicate .alpha.-helical
regions. "Coil" designates coiled-coil regions, and "LZ" designates
leucine zipper motif (which is a coiled-coil). The amino acid
positions that mark the approximate boundaries of these motifs are
indicated.
[0020] FIG. 3: IKK-.gamma. physically interacts with
IKK-.alpha./.beta.. a. HA-IKK-.gamma., Flag-IKK-.alpha.,
Flag-IKK-.beta. or empty expression vectors were transiently
transfected into 293 cells as indicated. After 24 hrs the cells
were lysed. Part of each lysate was precipitated with anti-HA
antibody and another part with anti-Flag antibody (M2). The levels
of Flag-IKK-.alpha., Flag-IKK-.beta. and HA-IKK-.gamma. were
determined by immunoblotting. b. HA-IKK-.gamma. or "empty" vectors
were transfected into HeLa cells. After 24 hrs the cells were left
untreated or incubated with either TNF or IL-1, lysed, and
immunoprecipitated with anti-HA antibody and immunoblotted with
anti-IKK-.alpha. and anti-HA antibody. c. HA-IKK-.gamma. or "empty"
vectors (VEC) were transfected into 293 cells that were treated and
processed as in b. d. HA-IKK-.alpha. and Flag-IKK-.beta. were
expressed in Sf9 cells using baculovirus vectors and purified as
described in Zandi et al., Science 281:1360-1363 (1998). After
incubation with or without purified recombinant Flag-IKK-.gamma.,
the proteins were immunoprecipitated with anti-IKK-.gamma. (NEMO)
antibody (Yamaoka et al., Cell 93:1231-1240 (1998)) and
immunoblotted with anti-HA and anti-Flag(M2) antibody. "IP"
designates immunoprecipitation; "IB" designates immunoblot.
[0021] FIG. 4: IKK-.gamma. is a component of the I.kappa.B kinase
complex. a. HeLa cells were transiently transfected with
HA-IKK-.gamma.. After 24 hrs, cells were treated or not with TNF or
IL-1. Part of each lysate was immunoprecipitated (IP) with anti-HA
antibody and another part with anti-IKK-.alpha.; I.kappa.B kinase
activity was determined as described in DiDonato et al., Nature
388:548-554 (1997). Levels of endogenous IKK-.alpha. were
determined by immunoblotting (IB) with anti-IKK-.alpha.. b.
Extracts of unstimulated or TNF treated 293 cells that were
transfected with an HA-IKK-.gamma. vector were fractionated on a
Superose 6 column. Fractions were immunoprecipitated with
anti-IKK-.alpha. antibody. Immunecomplex kinase assays (KA) and
immunoblotting of IKK-.alpha. and HA-IKK-.gamma. were conducted
with IKK-.alpha. and HA antibodies.
[0022] FIG. 5: IKK-.gamma. is an essential component of the
I.kappa.B kinase. a. HA-IKK-.alpha. and HA-IKK.beta. vectors were
cotransfected into HeLa cells with either "empty" or antisense
(AS)-IKK-.gamma. vectors. After 24 hrs, cells were treated or not
with TNF and lysed. Lysates were precipitated with anti-HA antibody
and IKK activity was determined by immunecomplex kinase assays
(KA). Expression of HA-IKK-.alpha. and HA-IKK-.beta. was determined
by immunoblotting (IB). Migration positions of IKK-.alpha. and
IKK-.beta. and a nonspecific (ns) band are indicated. b.
HA-IKK-.gamma., HA-IKK-.alpha. or HA-IKK-.beta. vectors were
cotransfected into 293 cells with either "empty" or AS-IKK-.gamma.
vectors as indicated. After 24 hrs the cells were lysed,
immunoprecipitated with anti-HA and immunoblotted with anti-HA
antibody. "ns" designates nonspecific bands.
[0023] c. HeLa cells were cotransfected with HA-IKK-.alpha., and
either AS-IKK-.gamma. or AS-JNKK1 vectors. After 24 hrs, cell
lysates were prepared, immunoprecipitated with anti-HA antibody and
immunoblotted with anti-HA (top panel) or anti-NEMO
(anti-IKK.gamma.) antibody (bottom panel). d. HeLa cells were
cotransfected with HA-IKK-.alpha. and either "empty" or
AS-IKK-.gamma. vectors, along with NIK or MEKK1 catalytic domain
(MEKK.DELTA.) expression vectors as indicated. After 24 hrs some
cells were treated with TNF or IL-1, lysed and immunoprecipitated
with anti-HA antibody. IKK activity was determined as above.
[0024] FIG. 6: Reduced IKK-.gamma. expression interferes with
I.kappa.B.alpha. phosphorylation, degradation and NF-.kappa.B
activation. a. Pools of 293 cells stably transfected with
AS-IKK-.gamma. vector and parental mock transfected cells were
stimulated with TNF for the indicated times, after which cells were
collected and lysed. Lysates were tested by immunoblotting for
I.kappa.B.alpha. degradation and endogenous IKK-.alpha. levels.
Basal and phosphorylated (P-I.kappa.B.alpha.) forms are indicated.
b. IKK-.alpha. immunecomplexes were isolated and immunoblotted with
anti-NEMO (anti-IKK-.gamma.) antibody. C, immunoprecipitation with
control antibody; ns, nonspecific band. c. Parental cells or pools
of 293 cells stably transfected with the AS-IKK-.gamma. vector were
incubated with TNF for the indicated times after which nuclear
extracts were prepared. The levels of NF-.kappa.B and NF-1 DNA
binding activities were determined by electrophoretic mobility
shift assay as described in DiDonato et al., Mol. Cell. Biol.
15:1302-1311 (1995) using probes corresponding to the consensus
.kappa.B (5'-AGTTGAGGGGACTTTCCCAGGC-3'; SEQ ID NO: 18) and NF-1
(5'-TTGGATTGAAGCCAATATGATA-3'; SEQ ID NO: 19) sites. d. JNK and p38
activities were determined by immunecomplex kinase assay.
[0025] FIG. 7: A C-terminal IKK-.gamma. deletion mutant is a
dominant negative inhibitor of IKK activation. a. Schematic
representation of full length (FL) IKK-.gamma. and its deletion
mutants. b. Flag-IKK-.beta. was cotransfected with wild type and
truncated IKK-.gamma. expression vectors into HeLa cells. After 24
hrs cells were incubated or not with TNF and lysed. The lysates
were immunoprecipitated with anti-Flag(M2) and IKK kinase activity
(KA) and Flag-IKK-.beta. expression were determined. c. 293 cells
were transfected with the different HA-IKK-.gamma.vectors and
treated as described above. Lysates were immunoprecipitated with
anti-IKK-.alpha. antibody and immunoblotted (IB) with
anti-IKK-.alpha. and anti-HA antibodies. d. Recombinant full length
IKK-.gamma. and its truncation mutants were expressed in E. coli,
purified and incubated with or without the crosslinking agent
ethylene glycolbis (succinimidylsuccinate) (EGS). The proteins were
separated by SDS-PAGE and visualized by immunoblotting. The
asterisk and dot mark the dimer and trimer bands, respectively. e.
Flag-p38 vector was cotransfected with either "empty" vector (VEC)
or the different IKK-.gamma. sense and antisense expression
vectors. After 24 hrs the cells were either left untreated or
incubated with TNF or IL-1. Lysates were prepared, p38 activity and
expression were determined by immunecomplex kinase assay (KA) and
immunoblotting, respectively.
DETAILED DESCRIPTION OF THE INVENTION
[0026] Proinflammatory cytokines activate the transcription factor
NF-.kappa.B by stimulating the activity of a protein kinase that
phosphorylates I.kappa.B, an inhibitor of NF-.kappa.B (DiDonato et
al., supra, 1997; Mercurio et al., Science 278:860-866 (1997);
Regnier et al., Cell 90:373-383 (1997); Woroniez et al., Science
278:866-869 (1997); Zandi et al., Cell 91:243-252 (1997)).
Phosphorylation occurs at sites that trigger ubiquitination and
degradation of I.kappa.B, resulting in nuclear translocation of
freed NF-.kappa.B dimers and activation of target gene
transcription (Verma et al., Genes Dev. 9:2723-2735 (1995) and
Baeuerle and Baltimore, Cell 87:13-20 (1996)) including
immunoregulatory target proteins (Woroniez et al., supra, 1997, and
Barnes et al., New Engl. J. Med. 336:1066-1071 (1997)). Two of the
subunits of the large cytokine-responsive I.kappa.B kinase complex
(IKK) are IKK-.alpha. and IKK-.beta., which are protein kinases
whose function is necessary for NF-.kappa.B activation by
proinflammatory stimuli.
[0027] As disclosed herein, the IKK complex is composed of similar
amounts of IKK-.alpha., IKK-.beta. and two other polypeptides,
whose partial sequences were obtained as set forth in Example I.
These polypeptides are differentially processed forms of a third
subunit, IKK-.gamma., having the amino acid sequence SEQ ID NO: 2
(see FIGS. 2a and 2b). Secondary structure prediction algorithms
indicate that IKK-.gamma. is predominantly helical with large
stretches of coiled-coil structure including a leucine zipper motif
as shown in FIG. 2c. Furthermore, the 23 carboxy-terminal residues
of IKK-.gamma. share 70% amino acid sequence identity with the
carboxy-terminus of FIP-2, including three cysteines and a
histidine, encoding a zinc finger motif (Li et al., Proc. Natl.
Acad. Sci., USA 96:1042-1047 (1999).
[0028] As further disclosed herein, IKK-.gamma. interacts directly
with IKK-.beta. in vitro (see FIG. 3d) and can associate with
IKK-.alpha. to form IKK complexes in IKK-.beta. deficient cells. In
addition, the level of TNF-induced I.kappa.B kinase activity
associated with either IKK-.alpha. or IKK-.beta. decreased upon
co-transfection with an IKK-.gamma. antisense vector as shown in
FIG. 6, indicating that IKK-.gamma. is an essential component of
the IKK complex required for its activation and that IKK-.gamma.
can function to connect the IKK complex to upstream activators.
Thus, the present invention provides a novel essential regulatory
subunit of the I.kappa.B kinase complex, IKK-.gamma., and based on
this novel regulatory subunit, provides methods of identifying new
therapeutic agents that regulate IKK activity and thereby modulate
the activity of NF-.kappa.B. Such agents can be useful as improved
immunosuppressant and anti-inflammatory drugs for treating a
variety of disorders involving NF-.kappa.B, including autoimmune
diseases, asthma, septic shock, arthritis, acquired immune
deficiency syndrome (AIDS) and cancer.
[0029] NF-.kappa.B is a member of the Rel family of transcription
factors, which are present in most if not all animal cells (Thanos
and Maniatis, Cell 80:629-532 (1995)). Rel proteins, which include,
for example, RelA (p65), c-Rel, p50, p52 and the Drosophila dorsal
and Dif gene products, are characterized by region of about 300
amino acids sharing approximately 35% to 61% homology ("Rel
homology domain"). The Rel homology domain includes DNA binding and
dimerization domains and a nuclear localization signal. Rel
proteins are grouped into one of two classes, depending on whether
the protein also contains a transcriptional activation domain
(Siebenlist et al., Ann. Rev. Cell Biol. 10:405-455 (1994)).
[0030] Rel proteins can form homodimers or heterodimers, which can
activate transcription depending on the presence of a
transactivation domain. The most common Rel/NF-.kappa.B dimer,
which is designated "NF-.kappa.B," is a p50/p65 heterodimer that
can activate transcription of genes containing the appropriate
.kappa.B binding sites. p50/p65 NF-.kappa.B is present in most cell
types and is considered the prototype of the Rel/NF-.kappa.B family
of transcription factors. Different dimers vary in their binding to
different .kappa.B elements, kinetics of nuclear translocation and
levels of expression in a tissue (Siebenlist et al., supra, 1994).
As used herein, the term "Rel/NF-.kappa.B" is used to refer
generally to the Rel family of transcription factors, and the term
"NF-.kappa.B" is used to refer specifically to the Rel/NF-.kappa.B
factor consisting of a p50/p65 heterodimer.
[0031] NF-.kappa.B originally was identified by its ability to bind
a specific DNA sequence present in the immunoglobulin .kappa. light
chain gene enhancer, the ".kappa.B element" (Sen and Baltimore,
Cell 46:705-709 (1986)). The .kappa.B element has been identified
in numerous cellular and viral promoters, including promoters
present in human immunodeficiency virus-1 (HIV-1); immunoglobulin
superfamily genes such as the MHC class 1 (H-2.kappa.) gene;
cytokine genes such as the tumor necrosis factor .alpha.
(TNF.alpha.), interleukin-1.beta. (IL-1.beta.), IL-2, IL-6 and the
granulocyte-macrophage colony stimulating factor (GM-CSF) gene;
chemokine genes such as RANTES and IL-8; and cell adhesion protein
genes such as E-selectin. The .kappa.B element exhibits dyad
symmetry; each half site of the element likely is bound by one
subunit of an NF-.kappa.B dimer.
[0032] In the absence of an appropriate signaling stimulus, a
Rel/NF-.kappa.B is maintained in the cytoplasm in an inactive form
complexed with an I.kappa.B protein. Rel/NF-.kappa.B
transcriptional activity is induced by numerous pathogenic events
or stresses, including cytokines, chemokines, viruses and viral
products, double stranded RNA, bacteria and bacterial products such
as lipopolysaccharide (LPS) and toxic shock syndrome toxin-1,
mitogens such as phorbol esters, physical and oxidative stresses,
and chemical agents such as okadaic acid and cycloheximide (Thanos
and Maniatis, supra, 1995; Siebenlist et al., supra, 1994).
Significantly, the expression of genes encoding agents such as
TNF.alpha., IL-1, IL-6, interferon-.beta. and various chemokines,
which induce NF-.kappa.B activity, are, themselves, induced by
NF-.kappa.B, resulting in amplification of their signal by a
positive, self-regulatory loop (Siebenlist et al., supra, 1994).
Phorbol esters, which activate T cells, also activate NF-.kappa.B
and immunosuppressants such as cyclosporin A inhibit activation of
T cells through T cell receptor mediated signals (Baldwin, Ann.
Rev. Immunol. 14:649-681 (1996)).
[0033] Regulation of specific genes by NF-.kappa.B can require
interaction of NF-.kappa.B with one or more other DNA binding
proteins. For example, expression of E-selectin requires an
interaction of NF-.kappa.B, the bZIP protein ATF-2 and HMG-I(Y),
and expression of the IL-2 receptor a gene requires an interaction
of NF-.kappa.B, HMG-I(Y) and the ets-like protein, ELF-1 (Baldwin,
supra, 1996).
[0034] The numerous agents that induce activation of NF-.kappa.B
likely act through various converging signal transduction pathways,
including pathways involving activation of protein kinase C, Raf
kinase and tyrosine kinases. The ability of antioxidants to inhibit
NF-.kappa.B activation by various inducing agents suggests that
reactive oxygen species are a converging point of such pathways
(Siebenlist et al., supra, 1994).
[0035] Upon activation by an appropriate inducing agent, a
Rel/NF-.kappa.B dimer is translocated into the nucleus, where it
can activate gene transcription. The subcellular localization of a
Rel/NF-.kappa.B is controlled by specific inhibitory proteins
("inhibitors of Rel/NF-.kappa.B" or "I.kappa.B's"), which
noncovalently bind the Rel/NF-.kappa.B and mask its nuclear
localization signal (NLS), thereby preventing nuclear uptake.
Various I.kappa.B's, including, for example, I.kappa.BC,
I.kappa.B.beta., Bcl-3 and the Drosophila cactus gene product, have
been identified (Baeuerle and Baltimore, supra, 1996). In addition,
Rel precursor proteins, such as p105 and p100, which are precursors
of p50 and p52, respectively, function as I.kappa.B's (Siebenlist
et al., supra, 1994). I.kappa.B.alpha. and I.kappa.B.beta. are
expressed in most cell types and generally bind p65- and
c-Rel-containing Rel/NF-.kappa.B dimers. Other I.kappa.B's appear
to be expressed in a tissue specific manner (Thompson et al., Cell
80:573-582 (1995)).
[0036] I.kappa.B proteins are characterized by the presence of 5 to
8 ankyrin repeat domains, each about 30 amino acids, and a
C-terminal PEST domain. For example, I.kappa.B.alpha. contains a 70
amino acid N-terminal domain, a 205 amino acid internal domain
containing the ankyrin repeats, and a 42 amino acid C-terminal
domain containing the PEST domain (Baldwin, supra, 1996). Although
I.kappa.B proteins interact through their ankyrin repeats with the
Rel homology domain of Rel/NF-.kappa.B dimers, binding of
particular I.kappa.B proteins with particular Rel/NF-.kappa.B
proteins appears to be relatively specific. For example,
I.kappa.B.alpha. and I.kappa.B.beta. associate primarily with RelA-
and c-Rel-containing Rel/NF-.kappa.B dimers, thereby blocking their
nuclear localization signal. The binding of an I.kappa.B to
NF-.kappa.B also interferes with the ability of NF-.kappa.B to bind
DNA. However, whereas I.kappa.B.alpha. is phosphorylated following
exposure of cells to tumor necrosis factor (TNF), IL-1, bacterial
lipopolysaccharide (LPS) or phorbol esters, I.kappa.B.beta. is
phosphorylated in certain cell types only in response to LPS or
IL-1 (Baldwin, supra, 1996). However, in other cell types,
I.kappa.B.beta. is phosphorylated in response to the same signals
that induce I.kappa.B.alpha., although with slower kinetics than
I.kappa.B.alpha. (DiDonato et al., Mol. Cell. Biol. 16:1295-1304
(1996)).
[0037] Formation of a complex between an I.kappa.B protein and a
Rel protein is due to an interaction of the ankyrin domains with a
Rel homology domain (Baeuerle and Baltimore, supra, 1996). Upon
exposure to an appropriate stimulus, the I.kappa.B portion of the
complex is rapidly degraded and the Rel/NF-.kappa.B portion becomes
free to translocate to the cell nucleus. Thus, activation of a
Rel/NF-.kappa.B does not require de novo protein synthesis and,
therefore, occurs extremely rapidly. Consequently, activation of
gene expression due to a Rel/NF-.kappa.B can be exceptionally rapid
and provides an effective means to respond to an external stimulus.
Such a rapid response of Rel/NF-.kappa.B transcription factors is
particularly important since these factors are involved in the
regulation of genes involved in the immune, inflammatory and acute
phase responses, including responses to viral and bacterial
infections and to various stresses.
[0038] Upon exposure of a cell to an appropriate inducing agent,
I.kappa.B.alpha., for example, is phosphorylated at serine residue
32 (Ser-32) and Ser-36 (Haskill et al., Cell 65:1281-1289 (1991)).
Phosphorylation of I.kappa.B.alpha. triggers its rapid
ubiquitination, which results in proteasome-mediated degradation of
the inhibitor and translocation of active NF-.kappa.B to the
nucleus (Brown et al., Science 267:1485-1488 (1995); Scherer et
al., Proc. Natl. Acad. Sci., USA. 92:11259-11263 (1995); DiDonato
et al., supra, 1996; DiDonato et al., supra, 1995; Baldi et al., J.
Biol. Chem. 271:376-379 (1996)). The same mechanism also accounts
for I.kappa.B.beta. degradation (DiDonato et al., supra, 1996).
[0039] Rel/NF-.kappa.B activation can be transient or persistent,
depending on the inducing agent and the I.kappa.B that is
phosphorylated. For example, exposure of a cell to particular
cytokines induces I.kappa.B.alpha. phosphorylation and degradation,
resulting in NF-.kappa.B activation, which induces the expression
of various genes, including the gene encoding I.kappa.B.alpha.. The
newly expressed I.kappa.B.alpha. then binds to NF-.kappa.B in the
nucleus, resulting in its export to the cytoplasm and inactivation
and, therefore, a transient NF-.kappa.B mediated response. In
comparison, bacterial LPS induces I.kappa.B.beta. phosphorylation,
resulting in NF-.kappa.B activation. However, the I.kappa.B.beta.
gene is not induced by NF-.kappa.B and, as a result, activation of
NF-.kappa.B is more persistent (Thompson et al., supra, 1995).
[0040] A large, cytokine-responsive I.kappa.B kinase complex (IKK)
has been purified, and two of its subunits molecularly cloned
(DiDonato et al., supra, 1997; Mercurio et al., supra, 1997;
Frishman and Argos, Protein Eng. 9:133-142 (1996)). The 85 and 87
kDa catalytic subunits, IKK-.alpha. and IKK-.beta., are protein
kinases whose function is necessary for NF-.kappa.B activation by
proinflammatory stimuli. The .alpha. and .beta. subunits contain an
N-terminal kinase domain and protein interaction motifs, including
a leucine zipper (LZ) and a helix-loop-helix (HLH), at their
C-terminal region (DiDonato et al., supra, 1997; Mercurio et al.,
supra, 1997; Regnier et al., supra, 1997; Woroniez et al., supra,
1997; Zandi et al., supra, 1997). The IKK-.alpha. and IKK-.beta.
subunits are rapidly activated by tumor necrosis factor (TNF) and
interleukin I (IL-1) and are necessary for NF-.kappa.B activation
(DiDonato et al., supra, 1997; Mercurio et al., supra, 1997; Zandi
et al., supra, 1997). IKK activity depends on phosphorylation, as
it is inactivated by protein phosphatase 2A (DiDonato et al.,
supra, 1997), and IKK-.alpha. and IKK-.beta. can be phosphorylated
and activated by overexpressed NF-.kappa.B activating kinase (NIK)
(Ling et al., Proc. Natl. Acad. Sci. USA 95:2791-2797 (1998)) or
MEK kinase 1 (MEKK-1) (Nakano et al., Proc. Natl. Acad. Sci. USA
95:3537-3542 (1998) and Karin et al., Proc. Natl. Acad. Sci. USA
95:9067-9069 (1998)).
[0041] As disclosed herein, the IKK complex was purified to
homogeneity from human cell lines using a monoclonal antibody to
IKK-.alpha.. IKK was composed of similar amounts of IKK-.alpha.,
IKK-.beta. and two other polypeptides, whose partial sequences were
obtained. As disclosed herein, these polypeptides are
differentially processed forms of a third subunit, IKK-.gamma.,
having the amino acid sequence SEQ ID NO: 2.
[0042] Thus, the present invention provides an isolated novel
essential regulatory subunit of the I.kappa.B kinase (IKK) complex,
IKK-.gamma.. The isolated IKK-.gamma. subunit of the invention has
substantially the same amino acid sequence as SEQ ID NO: 2, and can
have, for example, an amino acid sequence having at least 55% amino
acid identity with SEQ ID NO: 2.
[0043] As used herein, the term "isolated," when used in reference
to an IKK-.gamma. subunit of the invention, means that the subunit
is relatively free from contaminating lipids, proteins, nucleic
acids or other cellular material normally associated with an
IKK-.gamma. subunit in a cell. Thus, an isolated IKK-.gamma.
subunit is in a form that is substantially free of the IKK-.alpha.
and IKK-.beta. subunits that can be associated with IKK-.gamma. in
a cell. An isolated IKK-.gamma. subunit can be isolated, for
example, by immunoprecipitation using an antibody that binds to an
IKK catalytic subunit (IKK-.alpha. or IKK-.beta.), followed by
SDS-PAGE gel separation, for example (see Example I). In addition,
an isolated IKK-.gamma. subunit can be obtained, for example, by
expression of a recombinant nucleic acid molecule such as SEQ ID
NO: 1 (see Example II), or can be isolated from a cell by a method
comprising affinity chromatography using an anti-IKK-.gamma.
antibody.
[0044] As used herein, the term "IKK-.gamma." means a polypeptide
having substantially the same amino acid sequence as the human
IKK-.gamma. subunit (SEQ ID NO: 2) shown in FIG. 2b. The human
IKK-.gamma. subunit (SEQ ID NO: 2) is a polypeptide of 419 amino
acids containing coiled-coil and leucine zipper .alpha.-helical
regions, indicating that IKK-.gamma. can be engaged in homotypic
and heterotypic interactions. As illustrated in FIG. 1, an
IKK-.gamma. subunit can be human IKK-.gamma.1 or IKK-.gamma.2,
which are polypeptides of 52 and 50 kDa molecular weight,
respectively.
[0045] An IKK-.gamma. having substantially the same amino acid
sequence as SEQ ID NO: 2 can be a naturally occurring IKK-.gamma.
subunit such as IKK-.gamma.1 or IKK-.gamma.2 or a related
polypeptide having substantial amino acid sequence similarity to
SEQ ID NO: 2. Such related polypeptides include isotype variants,
differentially spliced or initiated forms, and species homologs of
the human IKK-.gamma. amino acid sequence shown in FIG. 2b. As used
herein, the term IKK-.gamma. subunit describes polypeptides
generally having an amino acid sequence with greater than about 35%
amino acid identity, preferably greater than about 45% amino acid
identity, more preferably greater than about 55% amino acid
identity, and also describes polypeptides having greater than about
65%, 75%, 85%, 90%, 95%, 97%, or 99% amino acid sequence identity
with SEQ ID NO: 2, said amino acid identity determined with
CLUSTALW using the BLOSUM 62 matrix with default parameters.
[0046] As used herein, the term "substantially the same amino acid
sequence," when used in reference to an IKK-.gamma. subunit, is
intended to mean a sequence as shown in FIG. 2b, or a similar,
non-identical sequence that is considered by those skilled in the
art to be a functionally equivalent amino acid sequence. For
example, an amino acid sequence that has substantially the same
amino acid sequence as human IKK-.gamma. (SEQ ID NO: 2) can have
one or more modifications such as amino acid additions, deletions
or substitutions relative to the amino acid sequence of SEQ ID NO:
2, provided that the modified polypeptide retains substantially at
least one biological activity of IKK-.gamma. such as the ability to
interact with IKK-.alpha./.beta. in cells; IKK-.beta. binding
activity; IKK-.alpha. binding activity; IKK regulatory activity,
for example, the ability to interact with a factor required for IKK
activation by TNF; or dimerization or trimerization activity,
described further below. Comparison for substantial similarity
between amino acid sequences is usually performed with sequences
between about 6 and 100 residues, preferably between about 10 and
100 residues and more preferably between about 25 and 35
residues.
[0047] Thus, it is understood that limited modifications can be
made to an IKK-.gamma. subunit, or to an active fragment thereof,
as described further below, without destroying its biological
function. A modification of an IKK-.gamma. subunit that does not
destroy biological activity, such as IKK-.beta. binding activity,
is encompassed within the meaning of the term IKK-.gamma. subunit,
as used herein. A modification can be, for example, an addition,
deletion, or conservative or non-conservative substitution of one
or more amino acid residues; substitution of a compound that mimics
amino acid structure or function; or addition of chemical moieties
such as amino or acetyl groups. The activity of a modified
IKK-.gamma. subunit can be assayed as disclosed herein (see
Examples).
[0048] In one embodiment, the invention provides an isolated
IKK-.gamma. subunit having substantially the same amino acid
sequence as SEQ ID NO: 2, provided that the subunit does not
include the amino acid sequence available as accession number
AF069542. Thus, the invention provides, for example, an isolated
IKK-.gamma. subunit having at least 35%, 45%, 55%, 65%, 75%, 85%,
90%, 95%, 97%, or 99% amino acid identity with SEQ ID NO: 2,
provided that the subunit does not include the amino acid sequence
available as accession number AF069542. The cDNA sequence of NEMO
(NF-.kappa.B Essential Modulator) is reported as accession number
AF069542 and also described in Yamaoka et al., supra, 1998.
[0049] As further disclosed herein, N- and C-terminal deletion
derivatives of IKK-.gamma. (SEQ ID NO: 2) were generated and
assayed for the ability to effect TNF-responsive and basal IKK
kinase activity. Although coexpression of
.DELTA.N-IKK-.gamma.(134-419) with FLAG-IKK-.beta. had only a
marginal effect on basal IKK activity and its response to TNF,
expression of .DELTA.C-IKK-.gamma.(1-300) inhibited activation of
IKK by TNF but not basal kinase activity (FIG. 7b). In addition,
both .DELTA.N-IKK-.gamma.(134-419) and .DELTA.C-IKK-.gamma. (1-300)
retained the ability to interact with IKK.alpha./.beta. in cells
(FIG. 7c). The ability of the C-terminally truncated IKK-.gamma.
derivative to inhibit IKK activation by upstream stimuli, while
having only a small effect on basal kinase activity, indicates that
IKK-.gamma. can function to connect the IKK complex to upstream
activators. The TNF responsive activity of the IKK can be mediated
by the C-terminal region of IKK-.gamma., while the ability to bind
IKK-.beta. can reside in the central region of the subunit.
[0050] Thus, the invention also provides IKK-.gamma. active
fragments that have substantially the same amino acid sequence as a
portion of the I.kappa.B kinase subunit, IKK-.gamma.. An
IKK-.gamma. active fragment of the invention can contain, for
example, ten, twenty, fifty or more contiguous amino acids of the
human IKK-.gamma. sequence disclosed herein as SEQ ID NO: 2.
[0051] As used herein, the term "IKK-.gamma. active fragment" means
a peptide or polypeptide which has substantially the same amino
acid sequence as a portion of an IKK-.gamma. subunit, provided that
the fragment retains at least one biological activity of the
IKK-.gamma. subunit. A portion of an IKK-.gamma. subunit generally
has an amino acid sequence of 15 to 400 contiguous residues and can
have, for example, an amino acid sequence of at least 18, 20, 25,
30, 35, 40, 50, 100, 150, 200, 250, 300, 350 or 400 contiguous
residues. An IKK-.gamma. active fragment can have a length, for
example, of 18 to 30 residues, 18 to 40 residues, 18 to 50
residues, 18 to 100 residues, 18 to 200 residues, 25 to 40
residues, 25 to 50 residues, 25 to 100 residues, 25 to 200
residues, 35 to 50 residues, 35 to 100 residues or 35 to 200
residues.
[0052] An IKK-.gamma. active fragment has one or more biological
activities of full-length IKK-.gamma. such as the ability to
interact with IKK-.alpha./.beta. in cells; IKK-.beta. binding
activity; IKK-.alpha. binding activity; IKK regulatory activity,
for example, the ability to interact with a factor required for IKK
activation by TNF; or dimerization or trimerization activity. As
disclosed herein, an IKK-.gamma. subunit can interact physically
with IKK-.alpha./.beta., as demonstrated by co-precipitation of HA
tagged IKK-.gamma. with endogenous IKK-.alpha. as disclosed in
Example II. The IKK-.gamma. deletion derivatives
.DELTA.N-IKK-.gamma.(134-419) and .DELTA.C-IKK-.gamma.(1-300),
which contain residues 134 to 419 and residues 1 to 300 of SEQ ID
NO: 2, respectively, retained the ability to interact with
IKK-.alpha./.beta. in cells as disclosed in Example IV (see FIG.
7c) and are exemplary IKK-.gamma. active fragments.
[0053] Full-length IKK-.gamma. can form dimers and trimers as
demonstrated by ethylene glycol bis (succinimidylsuccinate) (EGS)
cross-linking and shown in FIG. 7d. Dimerization activity was
retained by .DELTA.N-IKK-.gamma.(134-419) and
.DELTA.C-IKK-.gamma.(1-300), although trimerization of
.DELTA.N-IKK-.gamma.(134-419) appeared to diminish. A fragment that
has substantially the same amino acid sequence as a portion of an
IKK-.gamma. subunit and retains IKK-.gamma. dimerization or
trimerization activity also is an IKK-.gamma. active fragment of
the invention.
[0054] A biological activity of IKK-.gamma. also can be the ability
to directly bind IKK-.beta.. As disclosed in Example II,
full-length human IKK-.gamma. and the deletion derivatives
.DELTA.N-IKK-.gamma.(134-419) and .DELTA.C-IKK-.gamma.(1-300) were
shown to stably and directly bind IKK-.beta. by
co-immunoprecipitation of purified recombinant proteins. Thus, a
fragment of IKK-.gamma. that has substantially the same amino acid
sequence as a portion of an IKK-.gamma. subunit can have IKK-.beta.
binding activity and, thus, be an IKK-.gamma. active fragment as
defined herein.
[0055] A biological activity of IKK-.gamma. also can be the ability
to bind directly or indirectly to IKK-.alpha.. As disclosed herein,
an IKK-.gamma. subunit can interact physically with
IKK-.alpha./.beta.. Furthermore, IKK-.gamma. can form a complex
with IKK-.alpha. in cells even in the absence of IKK-.beta.. Thus,
an IKK-.gamma. fragment that has IKK-.alpha. binding activity also
is an active fragment of the invention.
[0056] As further disclosed herein, the level of TNF-induced
I.kappa.B kinase activity associated with either IKK subunit
decreased upon co-transfection with an IKK-.gamma. antisense vector
(FIG. 5a). Furthermore, IKK-.gamma. fragments can inhibit IKK
activation by stimuli such as TNF while negligibly effecting basal
kinase activity, indicating that one biological function of
full-length IKK-.gamma. is the ability to interact with an upstream
activator such as an activator required for TNF-inducible IKK
kinase activity. Thus, an IKK-.gamma. active fragment also can be a
fragment that has substantially the same amino acid sequence as a
portion of an IKK-.gamma. subunit and IKK kinase regulatory
activity.
[0057] In one embodiment, an "IKK-.gamma. active fragment" is a
polypeptide portion having substantially the same amino acid
sequence as a portion of an IKK-.gamma., provided that the fragment
does not consist of the identical amino acid sequence encoded by an
expressed sequence tag having accession number AA133061 and R56495,
and provided that the fragment retains at least one biological
activity of an IKK-.gamma. subunit. Such an IKK-.gamma. active
fragment can have, for example, an amino acid sequence that is
identical or substantially the same as a portion of the amino acid
sequence of human IKK-.gamma. (SEQ ID NO:2), provided that the
segment does not consist of the identical amino acid sequence
encoded by an expressed sequence tag having accession number
AA133061 and R56495, and provided that the fragment retains at
least one biological activity of an IKK-.gamma. subunit.
[0058] IKK-.gamma. active fragments can be identified by screening
a large collection, or library, of peptides of interest for
IKK-.gamma. biological activity such as one of the IKK-.gamma.
biological activities described hereinabove. For example, a panel
of peptides spanning the entire sequence of an IKK-.gamma. subunit
polypeptide such as the human IKK-.gamma.shown as SEQ ID NO: 2 can
be screened for the ability to interact with IKK-.alpha./.beta. in
cells; dimerization or trimerization activity; IKK-.beta. binding
activity; IKK-.alpha. binding activity; IKK regulatory activity; or
other IKK-.gamma.biological activity as described below. Such a
panel can be, for example, a panel of 15-mer peptides spanning the
sequence of human IKK-.gamma. (SEQ ID NO: 2), each overlapping by
three or five residue shifts using the Mimotope cleavable pin
technology (Cambridge Research Biochemicals, Wilmington, Del.), as
described by Geysen et al., Science 235:1184 (1987)). The panel is
subsequently screened for IKK-.beta. binding activity or other
IKK-.gamma.biological activity as described hereinabove. A library
of peptides to be screened also can be a population of peptides
related in amino acid sequence to SEQ ID NO: 2 but having one or
more amino acids that differ from SEQ ID NO: 2, for example, one or
more conservative or non-conservative substitutions.
[0059] Additional peptides to be screened for IKK-.gamma.
biological activity to identify active fragments include, for
example, tagged chemical libraries of peptides and peptidomimetic
molecules. Peptide libraries encompass those generated by phage
display technology, which is a technology that includes the
expression of peptide molecules on the surface of phage as well as
other methodologies by which a protein ligand is or can be
associated with the nucleic acid which encodes it. Methods for
production of phage display libraries, including vectors and
methods of diversifying the population of peptides which are
expressed, are well known in the art (see, for example, Smith and
Scott, Methods Enzymol. 217:228-257 (1993); Scott and Smith,
Science 249:386-390 (1990); and Huse, WO 91/07141 and WO 91/07149).
These or other well known methods can be used to produce a phage
display library which can be screened, for example, with one of the
disclosed assays for an IKK-.gamma. biological activity, for
example, IKK-.beta. binding activity. If desired, a population of
peptides can be assayed for activity en masse. For example, to
identify an active fragment of an IKK-.gamma. subunit with
IKK-.beta. binding activity, a population of peptides can be
assayed for the ability to specifically bind IKK-.beta.; the active
population can be subdivided and the assay repeated in order to
isolate the IKK-.gamma. active fragment from the population.
[0060] IKK-.gamma. active fragments also can be identified by
screening, for example, fragments of the polypeptide produced by
chemical or proteolytic cleavage. Methods for chemical and
proteolytic cleavage and for purification of the resultant protein
fragments are well known in the art (see, for example, Deutscher,
Methods in Enzymology, Vol. 182, "Guide to Protein Purification,"
San Diego: Academic Press, Inc. (1990)). For example, a chemical
such as cyanogen bromide or a protease such as trypsin,
chymotrypsin, V8 protease, endoproteinase Lys-C, endoproteinase
Arg-C or endoproteinase Asp-N can be used to produce convenient
fragments of an IKK-.gamma. subunit that can be screened for
biological activity using one of the assays disclosed herein.
[0061] As used herein, the term "fragment" means a peptide,
polypeptide or compound containing naturally occurring amino acids,
non-naturally occurring amino acids or chemically modified amino
acids. An IKK-.gamma.active fragment also can be a peptide mimetic,
which has a non-amino acid chemical structure that mimics the
structure of a peptide having an amino acid sequence, provided that
the mimetic retains at least one biological activity of an
IKK-.gamma. subunit. Such a mimetic generally is characterized as
exhibiting similar physical characteristics such as size, charge or
hydrophobicity in the same spatial arrangement found in its peptide
counterpart. A specific example of a peptide mimetic is a compound
in which the amide bond between one or more of the amino acids is
replaced, for example, by a carbon-carbon bond or other bond well
known in the art (see, for example, Sawyer, Peptide Based Drug
Design, ACS, Washington (1995)).
[0062] As used herein, the term "amino acid" refers to one of the
twenty naturally occurring amino acids, including, unless stated
otherwise, L-amino acids and D-amino acids. The term amino acid
also refers to compounds such as chemically modified amino acids
including amino acid analogs, naturally occurring amino acids that
are not usually incorporated into proteins such as norleucine, and
chemically synthesized compounds having properties known in the art
to be characteristic of an amino acid, provided that the compound
can be substituted within a peptide such that it retains at least
one biological activity of IKK-.gamma.. Examples of amino acids and
amino acids analogs are listed in Gross and Meienhofer, The
Peptides: Analysis, Synthesis, Biology, Academic Press, Inc., New
York (1983). An amino acid also can be an amino acid mimetic, which
is a structure that exhibits substantially the same spatial
arrangement of functional groups as an amino acid but does not
necessarily have both the .alpha.-amino and .alpha.-carboxyl groups
characteristic of an amino acid.
[0063] An IKK-.gamma. active fragment can be produced or
synthesized using methods well known in the art. Such methods
include recombinant DNA methods and chemical synthesis methods for
production of a peptide. Recombinant methods for producing a
peptide through expression of a nucleic acid sequence encoding the
peptide in a suitable host cell are well known in the art and are
described, for example, in Sambrook et al., Molecular Cloning: A
Laboratory Manual, 2nd Ed, Vols 1 to 3, Cold Spring Harbor
Laboratory Press, New York (1989). The sequence of a nucleic acid
molecule encoding a human IKK-.gamma. subunit is disclosed herein
as SEQ ID NO: 1.
[0064] An IKK-.gamma. active fragment also can be produced by
chemical synthesis, for example, by the solid phase peptide
synthesis method of Merrifield et al., J. Am. Chem. Soc. 85:2149
(1964). Standard solution methods well known in the art also can be
used to synthesize an IKK-.gamma. active fragment having IKK-.beta.
binding activity or other IKK-.gamma. biological activity as
disclosed herein (see, for example, Bodanszky, Principles of
Peptide Synthesis, Springer-Verlag, Berlin (1984) and Bodanszky,
Peptide Chemistry, Springer-Verlag, Berlin (1993)). A newly
synthesized peptide can be purified, for example, by high
performance liquid chromatography (HPLC), and can be characterized
using, for example, mass spectrometry or amino acid sequence
analysis.
[0065] Also provided herein are nucleic acid molecules encoding the
IKK-.gamma. subunit of the invention. These nucleic acid molecules
are useful, for example, in producing recombinant polypeptides and
as probes for diagnosing diseases involving aberrant expression of
an IKK-.gamma. subunit.
[0066] Thus, the invention provides isolated IKK-.gamma. nucleic
acid molecules, which contain a nucleotide sequence encoding
substantially the same amino acid sequence as SEQ ID NO: 2,
provided that the nucleic acid molecules do not consist of the
identical nucleic sequence of an expressed sequence tag having
accession number AA133061 or R56495. Such isolated IKK-.gamma.
nucleic acid molecules of the invention can have, for example, a
nucleotide sequence encoding an amino acid sequence having at least
55% amino acid identity with SEQ ID NO: 2. An isolated IKK-.gamma.
nucleic acid molecule of the invention can have a nucleotide
sequence encoding SEQ ID NO: 2, for example, nucleotides 149 to
1408 of SEQ ID NO: 1 or the entire sequence of SEQ ID NO: 1. An
exemplary IKK-.gamma. nucleic acid molecule of the invention is the
human IKK-.gamma. nucleic acid molecule provided herein as SEQ ID
NO: 1 in FIG. 2a.
[0067] In one embodiment, the invention provides an isolated
IKK-.gamma. nucleic acid molecule, which contains a nucleotide
sequence encoding substantially the same amino acid sequence as SEQ
ID NO: 2, provided that the nucleic acid molecule does not consist
of the identical nucleic sequence of a sequence having accession
number AA133061, R56495 or AF069542.
[0068] The term "isolated," as used herein in reference to a
nucleic acid molecule of the invention, means a nucleic acid
molecule that is in a form that is relatively free from
contaminating lipids, polypeptides, unrelated nucleic acids and
other cellular material normally associated with a nucleic acid
molecule in a cell.
[0069] Isolated nucleic acid molecules of the invention also
include, for example, nucleic acid molecules encoding species
homologs of human IKK-.gamma. such as bovine, monkey, rat, mouse
and other mammalian homologs, or other homologs such as chicken or
xenopus homologs. Isolated IKK-.gamma. nucleic acid molecules of
the invention also include nucleic acid molecules that encode
exactly the polypeptide of SEQ ID NO: 2 and are related but
different from SEQ ID NO:1 due to the degeneracy of the genetic
code. An IKK-.gamma. nucleic acid molecule of the invention also
can encode an IKK-.gamma. subunit having a sequence that is
different from SEQ ID NO:2, for example, containing one or more
conservative or non-conservative amino acid substitutions relative
to the amino acid sequence shown as SEQ ID NO: 2, provided that the
encoded IKK-.gamma. subunit retains at least one biological
activity of IKK-.gamma.. The invention also provides nucleic acid
molecules encoding IKK-.gamma. active fragments, described
hereinabove.
[0070] An IKK-.gamma. nucleic acid molecule of the invention can
have a nucleotide sequence of, for example, 15 to 1200 or more
nucleotides. In particular, a nucleic acid molecule of the
invention can have a sequence of at least 15, 18, 20, 25, 30, 35,
50, 100, 200, 500 or more nucleotides.
[0071] Further provided by the invention are IKK-.gamma.
polynucleotides and antisense polynucleotides, which can be useful
as probes and primers and to reduce IKK-.gamma. expression in a
cell. A polynucleotide of the invention contains at least nine
contiguous nucleotides of the human IKK-.gamma. sequence shown as
SEQ ID NO: 1. An antisense polynucleotide of the invention contains
a nucleotide sequence complementary to at least nine contiguous
nucleotides of SEQ ID NO: 1. As defined herein, a polynucleotide of
the invention is not the nucleotide sequence of accession number
AA133061 or R56495.
[0072] As used herein, the term "polynucleotide" is used in its
broadest sense to mean two or more nucleotides or nucleotide
analogs linked by a covalent bond and refers to sense and antisense
nucleotide sequences and encompasses both single-stranded and
double-stranded molecules. The term "oligonucleotide" also is used
herein to mean two or more nucleotides or nucleotide analogs linked
by a covalent bond, although those in the art will recognize that
oligonucleotides generally are less than about fifty nucleotides in
length and, therefore, are a subset within the broader meaning of
the term "polynucleotide."
[0073] In general, the nucleotides comprising a polynucleotide are
naturally occurring deoxyribonucleotides, such as adenine,
cytosine, guanine or thymine linked to 2'-deoxyribose, or
ribonucleotides such as adenine, cytosine, guanine or uracil linked
to ribose. However, a polynucleotide also can comprise nucleotide
analogs, including non-naturally occurring synthetic nucleotides or
modified naturally occurring nucleotides. Such nucleotide analogs
are well known in the art and commercially available, as are
polynucleotides containing such nucleotide analogs (Lin et al.,
Nucl. Acids Res. 22:5220-5234 (1994); Jellinek et al., Biochemistry
34:11363-11372 (1995); Pagratis et al., Nature Biotechnol. 15:68-73
(1997)). The covalent bond linking the nucleotides of a
polynucleotide generally is a phosphodiester bond. However, the
covalent bond also can be any of numerous other bonds, including a
thiodiester bond, a phosphorothioate bond, a peptide-like bond or
any other bond known to those in the art as useful for linking
nucleotides to produce synthetic polynucleotides (see, for example,
Tam et al., Nucl. Acids Res. 22:977-986 (1994); Ecker and Crooke,
BioTechnology 13:351360 (1995)).
[0074] Where it is desired to synthesize a polynucleotide of the
invention, the artisan will know that the selection of particular
nucleotides or nucleotide analogs and the covalent bond used to
link the nucleotides will depend, in part, on the purpose for which
the polynucleotide is prepared. For example, where a polynucleotide
will be exposed to an environment containing substantial nuclease
activity, the artisan will select nucleotide analogs or covalent
bonds that are relatively resistant to the nucleases. A
polynucleotide containing naturally occurring nucleotides and
phosphodiester bonds can be chemically synthesized or can be
produced using recombinant DNA methods using an appropriate
polynucleotide as a template. In comparison, a polynucleotide
containing nucleotide analogs or covalent bonds other than
phosphodiester bonds generally will be chemically synthesized,
although an enzyme such as T7 polymerase can incorporate certain
types of nucleotide analogs and, therefore, can be used to produce
such a polynucleotide recombinantly from an appropriate template
(Jellinek et al., supra, 1995).
[0075] The polynucleotides of the invention can specifically
hybridize to a nucleic acid molecule encoding IKK-.gamma. or can
hybridize to a related molecule. Such hybridizing polynucleotides
are useful, for example, as probes, which can hybridize to a
nucleic acid molecule encoding an IKK-.gamma. subunit and allow the
identification of the nucleic acid molecule in a sample. A
polynucleotide of the invention is characterized, in part, in that
it contains at least nine contiguous nucleotides of the sequence
shown as SEQ ID NO: 1, such sequences being particularly useful as
primers for the polymerase chain reaction (PCR). A polynucleotide
of the invention also can have, for example, at least 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or 40 contiguous nucleotides
of SEQ ID NO: 1, such polynucleotides being useful as hybridization
probes and primers for PCR. The polynucleotides of the invention
are particularly useful in methods of diagnosing a pathology, for
example, a human disease, characterized by aberrant IKK-.gamma.
expression. For convenience, such polynucleotides can be packaged
in a kit, which can be made commercially available and can provide
a standardized diagnostic assay.
[0076] The invention also provides antisense polynucleotides, which
are complementary to a portion of a nucleic acid molecule encoding
an IKK-.gamma. subunit and can bind to and inhibit expression of
the IKK-.gamma. in a cell. As disclosed herein, expression of an
antisense molecule complementary to the nucleotide sequence shown
in SEQ ID NO: 1 inhibited expression of HA-IKK-.gamma. but had no
effect on expression of either HA-IKK-.alpha. or HA-IKK-.beta.
(Example III; FIG. 5B). Furthermore, the level of TNF-induced
I.kappa.B kinase activity associated with either kinase subunit
decreased upon co-transfection with an antisense molecule
complementary to SEQ ID NO: 1. Thus, an antisense molecule of the
invention can be useful for decreasing inducible IKK activity in a
cell, thereby reducing or inhibiting the level of NF-.kappa.B
mediated gene expression and ameliorating the severity of a disease
involving NF-.kappa.B as described further below.
[0077] An antisense nucleic acid molecule of the invention can
contain a sequence complementary to the entire coding sequence of
an IKK-.gamma. subunit such as a sequence complementary to SEQ ID
NO: 1, provided the antisense sequence is not complementary in its
entirety to the sequence of GenBank Accession number AA133061 or
R56495.
[0078] Antisense methods involve introducing the nucleic acid
molecule, which is complementary to and can hybridize to the target
nucleic acid molecule, into a cell. An antisense nucleic acid
molecule can be a chemically synthesized polynucleotide, which can
be introduced into the target cells by methods of transfection, or
can be expressed from a plasmid or viral vector, which can be
introduced into the cell and stably or transiently expressed using
well known methods (see, for example, Sambrook et al., Molecular
Cloning: A laboratory manual (Cold Spring Harbor Laboratory Press
1989); Ausubel et al., Current Protocols in Molecular Biology
(Green Publ., NY 1989)). One in the art understands that the
ability of an antisense polynucleotide sequence to specifically
hybridize to a target nucleic acid sequence depends, for example,
on the degree of complementarity shared between the sequences, the
GC content of the hybridizing molecules, and the length of the
antisense polynucleotide, which can be at least ten nucleotides in
length, generally at least thirty nucleotides in length or at least
fifty nucleotides in length, and can be up to the full length of a
nucleotide sequence of SEQ ID NO: 1 (see Sambrook et al., supra,
1989).
[0079] The present invention also provides a method of identifying
an effective agent that modulates the specific association of an
IKK-.gamma. subunit and a second protein. Such agents can represent
novel therapeutic agents with immunosuppressant, anti-inflammatory
or anti-cancer activity. The method includes the steps of
contacting the IKK-.gamma. subunit and the second protein with an
agent under conditions suitable for the specific association of the
IKK-.gamma. subunit and the second protein, and detecting an
altered association of the IKK-.gamma. subunit and the second
protein in the presence of the agent, where the altered association
identifies the agent as an effective agent that modulates the
specific association of the IKK-.gamma. subunit and the second
protein. In a method of the invention, the contacting can be, for
example, in vitro with an isolated IKK-.gamma. subunit.
Alternatively, the IKK-.gamma. subunit can be contacted, for
example, in a cell such as a mammalian cell or yeast cell in
culture. In a method of the invention, an altered association can
be detected by a variety of methods, for example, by measuring
transcriptional activity of a reporter gene. In one embodiment, the
second protein is IKK-.beta. and an effective agent is identified
that modulates the specific association of an IKK-.gamma. subunit
and an IKK-.beta. subunit. In another embodiment, the second
protein is IKK-.alpha. and an effective agent is identified that
modulates the specific association of an IKK-.gamma. subunit and an
IKK-.alpha. subunit.
[0080] The ability of the human IKK-.gamma. subunit to associate
with IKK-.alpha./.beta. was demonstrated by co-precipitation of
IKK-.beta. with transiently expressed HA-IKK-.gamma.. As further
disclosed herein, purified recombinant IKK-.gamma. binds to
purified IKK-.beta., indicating that IKK-.gamma. directly binds the
.beta. subunit of IKK. In addition, IKK-.gamma. associates with
IKK-.alpha. in a cell in the absence of IKK-.beta.. The ability of
an IKK-.gamma. subunit to associate with other proteins also is
indicated, for example, by the presence in the IKK-.gamma. subunit
of two different protein binding domains, a helix-loop-helix domain
and a leucine zipper domain (see FIG. 2c).
[0081] A screening assay of the invention provides a means to
identify an effective agent that modulates the specific association
of an IKK-.gamma. subunit and a second protein. As used herein, the
term "modulate" or "alter" when used in reference to the specific
association of an IKK-.gamma. subunit and a second protein, means
that the affinity of the association is increased or decreased with
respect to a control level of association, which is the level of
association in the absence of an agent. Agents that can alter the
specific association of an IKK-.gamma. subunit with a second
protein can be useful for modulating the level of phosphorylation
of I.kappa.B in a cell, which, in turn, modulates the activity of
NF-.kappa.B in the cell and the expression of one or more genes
regulated by NF-.kappa.B.
[0082] A screening assay of the invention is particularly useful to
identify, from among a diverse population of agents, those
effective agents that modulate the specific association of an
IKK-.gamma. subunit and a second protein. Methods for producing
libraries containing diverse populations of molecules, including
chemical or biological molecules such as simple or complex organic
molecules, peptides, proteins, peptidomimetics, glycoproteins,
lipoproteins, polynucleotides, antibodies and the like, are well
known in the art (Huse, U.S. Pat. No. 5,264,563, issued Nov. 23,
1993; Blondelle et al., Trends Anal. Chem. 14:83-92 (1995); York et
al., Science 274:1520-1522 (1996); Gold et al., Proc. Natl. Acad.
Sci., USA 94:59-64 (1997); Gold, U.S. Pat. No. 5,270,163, issued
Dec. 14, 1993). Such libraries also can be obtained from commercial
sources.
[0083] A particularly useful effective agent can be an agent that
mimics the IKK-.gamma. recognition site present in the final 70
amino acids of IKK-.gamma.. Such a structural analog of this
IKK-.gamma. region, or an active subpart thereof, can prevent an
IKK complex from being activated by an upstream component that
results in inducible kinase activity such as TNF-inducible
activity. A particularly useful effective agent also can be agent
such as an antibody or other binding agent that binds to the
carboxy-terminal 70 amino acids of IKK-.gamma. or otherwise
prevents this region from being activated by an upstream component
that results in inducible IKK kinase activity.
[0084] Since libraries of diverse molecules can contain as many as
10.sup.14 to 10.sup.15 different molecules, a screening assay of
the invention provides a simple means for identifying those
effective agents in the library that can modulate the specific
association of an IKK-.gamma. subunit and a second protein. In
particular, a screening assay of the invention can be automated,
which allows for high through-put screening of randomly designed
libraries of agents to identify those particular agents that can
modulate the ability of an IKK-.gamma. subunit and a second protein
to associate specifically.
[0085] A drug screening assay of the invention utilizes an
IKK-.gamma. subunit, which, when used in isolated form, can be
prepared recombinantly, for example, by expressing a nucleic acid
molecule encoding the amino acid sequence shown in SEQ ID NO: 2; or
can be purified as disclosed herein using immunoaffinity
chromatography on immobilized anti-IKK-.alpha.; or can utilize an
IKK-.gamma. subunit fusion protein such as an hemagglutinin (HA),
glutathione-5-transferase (GST) or histidine.sub.6 (HIS6) fusion
protein, wherein the HA, GST or HIS6 is linked to the IKK-.gamma.
subunit and comprises a tag (see Example II). Such a IKK-.gamma.
subunit fusion protein is characterized, in part, by having an
affinity for a solid substrate as well as having the ability to
specifically associate with a second protein such as IKK-.beta..
For example, where an HA-IKK-.gamma. fusion protein is used in such
a screening assay, the solid substrate can contain covalently
attached anti-HA antibody, which is bound by the HA tag component
of the fusion protein.
[0086] As used herein, the term "IKK-.beta. " means a polypeptide
that phosphorylates I.kappa.B.alpha. on serine-32 and serine-36 and
has an apparent molecular mass of about 87 kDa. An IKK-.beta. can
have, for example, substantially the IKK-.beta. amino acid sequence
shown in Zandi et al., supra, 1997.
[0087] The term "IKK-.alpha.," as used herein, means a polypeptide
that phosphorylates I.kappa.B.alpha. on serine-32 and serine-36 and
has an apparent molecular mass of about 85 kDa. An IKK-.alpha. can
have, for example, substantially the IKK-.alpha. amino acid
sequence shown in Zandi et al., supra, 1997.
[0088] A variety of in vivo and in vitro screening assays for
detecting an altered association are well known in the art. A drug
screening assay to identify an effective agent that modulates the
specific association of an IKK-.gamma. subunit and a second protein
can be performed by allowing, for example, the IKK-.gamma. subunit,
which can be a fusion protein, to bind to a solid support, then
adding the second protein, for example IKK-.beta., and an agent to
be tested, under conditions suitable for the association of the
IKK-.beta. with IKK-.gamma. in the absence of a drug (see Example
II). As appropriate, the IKK-.gamma. subunit can be activated, for
example by TNF, or inactivated as disclosed herein and, typically,
the component not bound to a solid support is detectably labeled so
as to facilitate identification of the association. Control
reactions, which contain or lack either the IKK-.gamma. subunit,
second protein, or the agent also are performed. Following
incubation of the reaction mixture, the amount of second protein
specifically bound to the IKK-.gamma. subunit in the presence of an
agent can be determined and compared to the amount of binding in
the absence of the agent so that effective agents that modulate the
specific association can be identified.
[0089] In an in vitro screening assay of the invention, a component
conveniently can be detectably labeled with a radionuclide,
fluorescent label, enzyme, peptide epitope or other such moiety
that facilitates a determination of the amount of association in a
reaction. By comparing the amount of specific binding of an
IKK-.gamma. subunit to a second protein in the presence of an agent
as compared to a control level of binding, an effective agent that
increases or decreases specific binding of the IKK-.gamma. subunit
to a second protein can be identified. Thus, the disclosed drug
screening assays provide a rapid and simple method for selecting
effective agents that desirably modulate the specific association
of an IKK-.gamma. subunit and a second protein such as an
IKK-.beta. subunit. Such agents can be useful, for example, for
decreasing the activity of NF-.kappa.B in a cell and, therefore,
can be useful as medicaments for the treatment of a pathology due,
at least in part, to aberrant NF-.kappa.B activity.
[0090] A two-hybrid system such as the yeast two hybrid system also
can be used to screen a panel of agents to detect an altered
association of an IKK-.gamma. subunit with a second protein. Using
a two hybrid system, an effective agent is identified by an altered
level of transcription of a reporter gene (see Fields and Song,
Nature 340:245-246 (1989)). For example, the level of transcription
of a reporter gene due to the bridging of a DNA-binding
domain/IKK-.gamma. subunit hybrid and a transactivation
domain/second protein hybrid can be determined in the absence and
presence of an agent. An effective agent, which alters the specific
association of an IKK-.gamma. subunit and a second protein, can be
identified by an altered level of reporter gene transcription as
compared to a control level of transcription in the absence of the
agent.
[0091] In some cases, an agent may not be able to cross the yeast
cell wall and, therefore, cannot enter a yeast cell to alter the
association of an IKK-.gamma. subunit and a second protein. The use
of yeast spheroplasts, which are yeast cells that lack a cell wall,
can circumvent this problem (Smith and Corcoran, In Current
Protocols in Molecular Biology (ed. Ausubel et al.; Greene Publ.,
NY 1989)). In addition, an agent, upon entering a cell, may require
"activation" by a cellular mechanism that may not be present in
yeast. Activation of an agent can include, for example, metabolic
processing of the agent or a modification such as phosphorylation
of the agent, which can be necessary to convert the agent into an
effective agent. A yeast two hybrid system can be adapted for use
in mammalian cells using well known methods as described, for
example, in Fearon et al., Proc. Natl. Acad. Sci., USA 89:7958-7962
(1992); Sambrook et al., supra, 1989; and Ausubel et al., supra,
1989.
[0092] One skilled in the art understands that an effective agent
can function directly or indirectly and by a variety of mechanisms
to alter the specific association of a a IKK-.gamma. subunit and a
second protein. An effective agent can function, for example, as a
competitor of the binding interaction between an IKK-.gamma.
subunit and a second protein such as IKK-.beta. or a protein
required for TNF inducible IKK kinase activity. For example, a
peptide or peptidomimetic that mimics the structure of the
IKK-.beta. binding region of an IKK-.gamma. subunit can be an
effective agent that decreases the association of an IKK-.gamma.
subunit and a second protein. A truncated IKK-.gamma. subunit that
retains the ability to specifically associate with IKK-.beta. or a
protein required for TNF inducible IKK kinase activity is an
example of an effective agent that can decrease the specific
association of an IKK-.gamma. subunit with a second protein by
acting as a competitor of the binding interaction. A fragment of
IKK-.beta. or a protein required for TNF inducible IKK kinase
activity also can be useful as an effective agent, providing that
the fragment can alter the association of an IKK-.gamma. subunit
with a second protein. Such fragments, which can be peptides as
small as about five amino acids, can be identified, for example, by
screening a peptide library (see, for example, Ladner et al., U.S.
Pat. No. 5,223,409) using one of the assays described herein.
[0093] An effective agent also can bind to an IKK-.gamma. at a site
distant from the site of interaction, thereby altering the
three-dimensional conformation of the polypeptide such that the
association of IKK-.gamma. and a second protein is increased or
decreased. An effective agent also can produce an altered
association by promoting a modification such as phosphorylation of
an IKK-.gamma. subunit. In addition, an effective agent can
sequester or alter the subcellular localization of an IKK-.gamma.
subunit, thereby modulating the effective concentration of the
subunit and the extent to which it can associate with a second
protein.
[0094] The present invention also provides a method of modulating
NF-.kappa.B activity in a cell by contacting the cell with an
effective agent that modulates the specific association of an
IKK-.gamma. subunit and a second protein. In a method of the
invention, the second protein can be, for example, IKK-.beta..
Further provided are methods of modulating NF-.kappa.B activity in
a cell by introducing into the cell an IKK-.gamma. antisense
polynucleotide. The IKK-.gamma. antisense polynucleotide can be
expressed in the cell, for example, in a vector.
[0095] Inappropriate regulation of Rel/NF-.kappa.B transcription
factors is associated with various human diseases. For example,
many viruses, including human immunodeficiency virus-1 (HIV-1),
herpes simplex virus-1 (HSV-1) and cytomegalovirus (CMV) contain
genes regulated by a .kappa.B regulatory element and these viruses,
upon infecting a cell, utilize cellular Rel/NF-.kappa.B
transcription factors to mediate viral gene expression (Siebenlist
et al., supra, 1994). Tat-mediated transcription from the HIV-1
enhancer, for example, is decreased if the NF-.kappa.B and SP1
binding sites are deleted from the enhancer/promoter region,
indicating that Tat interacts with NF-.kappa.B, SP1 or other
transcription factors bound at this site to stimulate transcription
(Roulston et al., Microbiol. Rev. 59:481-505 (1995)). In addition,
chronic HIV-1 infection, and progression to AIDS, is associated
with the development of constitutive NF-.kappa.B DNA binding
activity in myeloid cells (Roulston et al., supra, 1995). Thus, a
positive autoregulatory loop is formed, whereby HIV-1 infection
results in constitutively active NF-.kappa.B, which induces
expression of HIV-1 genes (Baeuerle and Baltimore, supra, 1996).
Constitutive NF-.kappa.B activation also may protect cells against
apoptosis, preventing clearance of virus-infected cells by the
immune system (Liu et al., Cell 87:565-576 (1996)).
[0096] An effective agent that modulates the specific association
of an IKK-.gamma. subunit and a second protein such that I.kappa.B
phosphorylation is decreased can be useful for reducing the
severity of a viral infection such as HIV-1 infection in an
individual by providing increased levels of unphosphorylated
I.kappa.B in virus-infected cells. The unphosphorylated I.kappa.B
then can bind to NF-.kappa.B in the cell, thereby preventing
nuclear translocation of the NF-.kappa.B and viral gene expression.
In this way, the rate of expansion of the virus population can be
limited, thereby providing a therapeutic advantage to an individual
infected with HIV.
[0097] In addition, decreased levels of NF-.kappa.B activity can
allow virus-infected cell to undergo apoptosis, resulting in a
decrease in the viral load in the individual. As such, it can be
particularly useful to treat virus-infected cells ex vivo with an
agent identified using a method of the invention. For example,
peripheral blood mononuclear cells (PBMCs) can be collected from an
HIV-1 infected individual and treated in culture with an effective
agent that decreases the specific association of an IKK-.gamma.
subunit and a second protein. Such a treatment can be useful to
purge the PBMCs of the virus-infected cells by allowing apoptosis
to proceed. The purged population of PBMCs then can be expanded, if
desired, and readministered to the individual.
[0098] Rel/NF-.kappa.B proteins also are involved in a number of
different types of cancer. For example, the adhesion of cancer
cells to endothelial cells is increased due to treatment of the
cancer cells with IL-1, indicating that NF-.kappa.B induces
expression of cell adhesion molecules, which mediate adherence of
tumor cells to endothelial cells; agents such as aspirin, which
decrease NF-.kappa.B activity, block adhesion by inhibiting
expression of the cell adhesion molecules (Tozawa et al., Cancer
Res. 55:4162-4167 (1995)). These results indicate that an agent
that decreases the specific association of an IKK-.gamma. subunit
and a second protein can be useful for reducing the likelihood of
metastasis of a tumor in an individual.
[0099] As discussed above for virus-infected cells, constitutive
NF-.kappa.B activation also can protect tumor cells against
programmed cell death as well as apoptosis induced by
chemotherapeutic agents (Liu et al., supra, 1996; Baeuerle and
Baltimore, supra, 1996). Thus, an agent that decreases the specific
association of an IKK-.gamma. subunit and a second protein can be
useful in combination with an apoptosis inducing anti-cancer
therapeutic, such as a chemotherapeutic agent, for allowing
programmed cell death to occur in a tumor cell by increasing the
level of unphosphorylated I.kappa.B, which can bind NF-.kappa.B and
decrease the level of active NF-.kappa.B in the tumor cell. One
skilled in the art understands that such effective agents can be
particularly useful in treating tumors having elevated NF-.kappa.B
activity.
[0100] NF-.kappa.B also plays a role in a variety of inflammatory
diseases, for example, asthma, rheumatoid arthritis, inflammatory
bowel diseases and psoriasis. In regard to asthma, for example,
induction of the NF-.kappa.B target gene iNOS has been demonstrated
in airway epithelium (Hamid et al., Lancet 342:1510-1513 (1993));
the exhaled air of asthmatic patients has a considerably higher NO
content than that of normal individuals (Kharitonov et al., Lancet
343:133-135 (1994)). Furthermore, COX-2, also regulated by
NF-.kappa.B, is expressed in higher levels at sites of
inflammation, accounting for increased production of prostaglandins
and thromboxanes (Barnes et al., supra, 1997). Asthma is associated
with increased production of cytokines and chemokines that are
activated by NF-.kappa.B, including IL-1.beta., TNF, IL-6, GM-CSF,
and IL-8. Asthma attacks are triggered by viral infections and
exposure to inhaled oxidants that activate NF-.kappa.B in the nasal
epithelium and lungs of experimental animals (Haddad et al., FEBS
Lett. 379:265-268 (1996)). Nuclear NF-.kappa.B was detected in
inflammatory cells isolated from sputum and bronchial biopsies of
asthmatic patients (Barnes et al., supra, 1997), and exposure of
human lung tissue to proinflammatory cytokines resulted in
prolonged NF-.kappa.B activation (Adcock et al., Eur. Res. J.
7:2117-2123 (1994)). In addition, glucocorticoids, which inhibit
NF-.kappa.B activation, are an effective class of anti-inflammatory
drugs for the treatment of asthma. Thus, NF-.kappa.B plays a role
in inflammatory diseases such as asthma, and effective agents of
the invention, which decrease IKK kinase activity and thereby
decreased NF-.kappa.B activity, can be valuable therapeutics for
the treatment of asthma.
[0101] As in asthma, NF-.kappa.B plays a role in the etiology of
rheumatoid arthritis (RA). NF-.kappa.B is activated in synoviocytes
of RA patients (Handel et al., Arthritis Rheum. 38:1762-1770
(1995); Marok et al., Arthritis Rheum. 39:583-591 (1996)).
Transgenic mice that expression the potent NF-.kappa.B activator
human T-cell leukemia virus (HTLV)-1 Tax protein develop RA-like
symptoms (Nishioka et al., Arthritis Rheum. 39:1410-1418 (1996)).
RA also involves increased synthesis of inflammatory mediators
regulated by NF-.kappa.B and of the cell adhesion molecules ICAM-1,
VCAM-1 and E-selectin, which are synthesized when NF-.kappa.B is
activated (Arent et al., Arthritis Rheum. 38:151-160 (1995);
Firestein et al., J. Clin. Invest. 96:1631-1638 (1995); McMurray,
Semin. Arthritis Rheum. 25:215-233 (1996)). NF-.kappa.B inhibitors
including glucocorticoids and salicylates are effective
anti-arthritic drugs. Thus, NF-.kappa.B activation promotes RA, and
effective agents of the invention identified as disclosed herein,
can be useful as therapeutics for reducing the severity of
inflammatory diseases such as rheumatoid arthritis.
[0102] Glucocorticoids are potent anti-inflammatory and
immunosuppressive agents that are used clinically to treat various
pathologic conditions, including autoimmune diseases such as
rheumatoid arthritis, systemic lupus erythematosis and asthma.
Glucocorticoids suppress the immune and inflammatory responses, at
least in part, by increasing the rate of I.kappa.B.alpha.
synthesis, resulting in increased cellular levels of
I.kappa.B.alpha., which bind to and inactivate NF-.kappa.B
(Scheinman et al., Science 270:283-286 (1995); Auphan et al.,
Science 270:286-290 (1995)). Thus, glucocorticoids suppress
NF-.kappa.B mediated expression of genes encoding, for example,
cytokines, thereby suppressing the immune, inflammatory and acute
phase responses. However, glucocorticoids and glucocorticoid-like
steroids also are produced physiologically and are required for
normal growth and development. Unfortunately, prolonged treatment
of an individual with higher than physiological amounts of
glucocorticoids produces clinically undesirable side effects. Thus,
the invention provides alternative agents that can decrease the
specific association of an IKK-.gamma. subunit and a second protein
and can provide a means for selectively decreasing NF-.kappa.B
activity without producing some of the undesirable side effects
associated with glucocorticoid treatment.
[0103] The methods of the invention involve contacting a cell with
an effective agent that alters the specific association of an
IKK-.gamma. subunit with a second protein and can be performed by
contacting cells in vitro or in vivo. Where the methods is
performed with cells in vivo, the effective agent is administered
to a subject. Based on the above, the skilled artisan will
recognize that the methods of the invention are useful to treat,
for example, patients with HIV-infection, cancer, asthma,
rheumatoid arthritis or inflammatory bowel disease.
[0104] In the claimed methods of modulating NF-.kappa.B activity in
a cell, an effective agent can be formulated, if desired, with a
pharmaceutically acceptable carrier to produce a pharmaceutical
composition, which can be administered to an individual such as a
human or other mammal. A pharmaceutically acceptable carrier can
be, for example, water, sodium phosphate buffer, phosphate buffered
saline, normal saline or Ringer's solution or other physiologically
buffered saline, or other solvent or vehicle such as a glycol,
glycerol, an oil such as olive oil or an injectable organic
ester.
[0105] A pharmaceutically acceptable carrier can contain, if
desired, one or more physiologically acceptable compounds that act,
for example, to stabilize or increase absorption of the effective
agent. Such physiologically acceptable compounds include, for
example, carbohydrates such as glucose, sucrose or dextrans;
antioxidants such as ascorbic acid or glutathione; divalent metal
ions such as calcium or magnesium; low molecular weight proteins;
and other stabilizers or excipients. One skilled in the art would
know that the choice of a pharmaceutically acceptable carrier,
including a physiologically acceptable compound, depends, for
example, on the route of administration of the composition.
[0106] A pharmaceutical composition containing an effective agent
can be administered to an individual by various routes, including
by intravenous, subcutaneous, intramuscular, intrathecal or
intraperitoneal injection; orally, as an aerosol spray; or by
intubation. If desired, the effective agent can be incorporated
into a liposome, a non-liposome lipid complex, or other polymer
matrix, which further can have incorporated therein, for example, a
second drug useful for treating the individual. Liposomes, which
consist of phospholipids or other lipids, are nontoxic,
physiologically acceptable and metabolizable carriers that are
relatively simple to make and administer (Gregoriadis, Liposome
Technology, Vol. 1 (CRC Press, Boca Raton Fla., 1984)). The skilled
artisan will select a particular route and method of administration
based, for example, on the disease to be treated in a subject, and
the specific effective agent that is administered.
[0107] In the methods of the invention for modulating NF-.kappa.B
activity, an effective amount of the effective agent is
administered. As used herein, the term "effective amount" refers to
the amount of an effective agent that alters the specific
association of an IKK-.gamma. subunit with a second protein. In
general, an effective amount of an effective agent produces minimal
side effects, although the level of an acceptable deleterious
effect is weighed against the benefit caused by the effective agent
in treating, for example, a subject with HIV, cancer, asthma,
rheumatoid arthritis or inflammatory bowel disease.
[0108] An effective agent can be administered to a subject such as
a human systemically at a dose ranging from 1 to 100 mg/kg body
weight, for example, at a dose of about 10 to 80 mg/kg,
particularly about 10 to 50 mg/kg. An effective agent also can be
incorporated into liposomes, if desired, in which case the total
amount administered to a subject generally can be reduced.
Furthermore, an effective agent can be administered orally to a
subject at a dose ranging from about 1 to 100 mg/kg body weight,
for example at a dose of about 10 to 200 mg/kg, in particular about
20 to 100 mg/kg. In addition, an effective agent can be
administered topically to an environment, which can be a human
subject, or can be placed in a solution, at a concentration of
about 0.1 to 10 mg/ml, for example, at a concentration of about 0.5
to 5 mg/ml. The skilled artisan will recognize that the level of
any side effects must be considered in prescribing a treatment
regimen and must be monitored during the treatment period, and will
adjust the amount of the effective agent that is administered
accordingly.
[0109] The following examples are intended to illustrate but not
limit the present invention.
EXAMPLE I
Isolation and Characterization of IKK.gamma.
[0110] This example describes the isolation and characterization of
a novel subunit of the IKK complex.
A. Purification of IKK-.gamma.
[0111] To purify IKK to homogeneity, monoclonal antibodies specific
for IKK-.alpha. were prepared. Using anti-IKK-o as an
immunoaffinity reagent, the IKK complex was shown to contain nearly
equal amounts of IKK-.alpha. and IKK-.beta. in both HeLa and Jurkat
cells (FIG. 1). Furthermore, passing partially purified IKK
fractions through the anti-IKK-.alpha. column resulted in
quantitative recovery of IKK-.beta. (FIG. 1a), with which the
antibody does not crossreact.
[0112] Large scale purification by combining the protocol described
in DiDonato et al., supra, 1997, with immunoaffinity chromatography
on immobilized anti-IKK-.alpha. revealed that, in addition to
IKK-.alpha. and IKK-.beta., purified IKK contained two polypeptides
of 50 and 52 kDa in size (FIG. 1b). Similar results were obtained
by immunoprecipitation of .sup.35S-labeled cell lysates (FIG. 1c).
TNF treatment did not change the relative amounts of these
polypeptides. Microsequencing of these polypeptides yielded the
peptide sequence data shown in Table 1 and indicated that they are
either differentially processed or initiated forms of the same
protein, designated IKK-.gamma.. TABLE-US-00001 TABLE 1 Peptide
sequence data for IKK.sub..gamma. Sequence ID No. Peptide Sequence
Source 3 IVMETVPVLK HeLa GF 4 KELLQEQLEQLQREYSK HeLa GF 5
ELLQEQLEQLQREYSK HeLa GF 6 RHVEVSQAPLPPAPAYLSSP HeLa GF 7
LAQLQVAYHQLEQEYDNHIK HeLa GF 8 XQYQAPDMDTL HeLa GF 9
XQPSGGPAADQDVLGEE HeLa GF 10 QQLQQAEEALVAK HeLa GF 11 EQALREVEHLK
HeLa GF 12 LVERLGLEK HeLa GF 13 KELLQEQLEQLQREY JURKAT IA LOWER 14
XXVTSLLGFLQESQ JURKAT IA LOWER 15 XXLQQAEEALVAK HeLa IA LOWER 16
XQVTXLLXELQEXQQ HeLa IA LOWER 17 XAQLQVAYHQLFQEYDNHIK HeLa IA LOWER
.sup.a Peptides generated by endoproteinase Lys-C digest .sup.b
Source of digested protein listing cell type, method of
purification, and band location: GF-gel filtration; IA -
immunoaffinity. LOWER and UPPER refer to the two bands labeled
IKK-.gamma. in FIG. 1.
[0113] Purification of IKK-.gamma. was performed as follows. The
IKK complex was purified from HeLa and Jurkat cells as described in
DiDonato et al., supra, 1997, except for substituting affinity
chromatography on an I.kappa.B.alpha.(1-54) column with affinity
chromatography on immobilized monoclonal anti-IKK-.alpha. antibody
(B78-743), generated against full length recombinant IKK-.alpha.
and available from PharMingen (San Diego, Calif.). Purified
antibody (0.5 mg) was coupled to 0.3 ml of CNBr-activated Sepharose
4B (Pharmacia). Active IKK fractions, after gel filtration on
Superose 6, were pooled (1.6 ml) and applied batchwise to the
immunoaffinity resin (0.1 ml). The mixture was rotated at 4.degree.
C. for 4 hrs and then centrifuged. The beads were washed with 20 ml
of Buffer A prepared as in DiDonato et al, supra, 1997, containing
500 mM NaCl, 1% Triton X-100 followed by 5 ml of Buffer A
containing 2 M urea. Bound protein was eluted with 0.5% SDS and
separated by SDS-PAGE. Bands identified as IKK-.gamma. were
transferred to a PVDF membrane, stained with colloidal blue and
digested with Lys-C. The IKK-.gamma.1 band also was obtained in
pure form after gel filtration and SDS-PAGE without requiring the
immunoaffinity step. This material was transferred to a PVDF
membrane and digested with Lys-C. A total of 15 peptide sequences
derived from the 52 kDa (IKK-.gamma.1) and 50 kDa (IKK-.gamma.2)
forms were obtained. All of these sequences were contained within
the IKK-.gamma.open reading frame (FIG. 2b). The peptide maps
generated by Lys-C digestion of IKK-.gamma.1 and IKK-.gamma.2 were
very similar.
[0114] These results demonstrate that, in addition to the
IKK-.alpha. and IKK-.beta. subunits, IKK contains two other major
polypeptides, IKK-.gamma.1 and IKK-.gamma.2, that are derived from
the same transcript.
B. Isolation of the IKK-.gamma. cDNA
[0115] Screening of Genebank for identical or similar sequences
revealed two overlapping expressed sequence tags (ESTs) with
accession numbers AA133061 and R56495. A cDNA probe corresponding
to these ESTs was used to isolate a cDNA clone containing the
entire IKK-.gamma. open reading frame from a HeLa library (FIGS. 2a
and b). The complete IKK-.gamma. nucleotide sequence is available
under accession number AF074382. As shown in FIG. 2, the predicted
IKK-.gamma. polypeptide is a glutamine rich protein 419 amino acids
in length with a predicted secondary structure containing two
extended coil-coil motifs and a leucine zipper, each of which can
function in protein-protein interactions (Berger et al., Proc.
Natl. Acad. Sci. USA 92:8259-8263 (1995); Rost B., Meth. Enzymol.
266:525-539 (1996); Frishman and Argos, supra, 1996).
EXAMPLE II
IKK-.gamma. Binds the IKK-.beta. Component of IKK and Associates
with IKK-.alpha. in the Absence of IKK-.beta.
[0116] This example demonstrates that IKK-.gamma. directly binds
IKK-.beta. and that IKK-.gamma. can form a complex with IKK-.alpha.
in IKK-.beta. deficient cells.
A. IKK-.gamma. Interacts Physically with IKK-.alpha./.beta.
[0117] To confirm that the cloned IKK-.gamma. protein interacts
with IKK-.alpha./.beta. subunits in cells, an expression vector for
N-terminally hemagglutinin (HA)-tagged IKK-.gamma. was
cotransfected into HeLa cells with expression vectors for either
Flag-IKK-.alpha. or Flag-IKK-.beta.. Transfected cell lysates were
immunoprecipitated with anti-HA followed by immunoblotting with
anti-Flag. As shown in FIG. 3a, IKK-.gamma. interacted efficiently
with IKK-.alpha. and IKK-.beta.. Similar results were obtained when
the immunoprecipitating antibody was directed to the Flag epitope
and the immunecomplexes were immunoblotted with anti-HA antibody
recognizing the epitope on IKK-.gamma.. Immunoprecipitation of
transiently expressed HA-IKK-.gamma. resulted in isolation of
endogenous IKK-.alpha.X (FIG. 3b). In addition, immunoprecipitation
of endogenous IKK-.alpha. coprecipitated HA-IKK-.gamma. (FIG. 3c).
The interaction between IKK-.gamma. and IKK-.alpha. was not
modulated by cytokines (FIGS. 1c, 3b).
[0118] Cell culture, transfections, immunoprecipitation and
immunoblotting were performed essentially as follows. The various
expression vectors were constructed using standard recombinant DNA
procedures. The .beta.-actin promoter was used to drive expression
of all sense IKK-.gamma. constructs. Cell culture and transfections
were as described in DiDonato et al., supra, 1997, except that
Lipofectamine Plus (GIBCO) was used.
[0119] Immunoprecipitation and immunoblotting were performed as
described in DiDonato et al., supra, 1997, and Zandi et al., supra,
1997. The monoclonal anti-IKK-.alpha. antibody, which does not
cross-react with IKK-.beta., was used for immunoblotting and
immunoprecipitation. TNF-.alpha. and IL-1 were used at 20 ng/ml and
10 ng/ml, respectively. Induction times were 10 min except where
otherwise indicated in the Brief Description of Drawings.
B. Purified IKK-.gamma. Directly Binds IKK-.beta.
[0120] IKK-.alpha. and IKK-.beta. are known to form very stable
heterodimers (Zandi et al, supra, 1998). Accordingly, the
co-immunoprecipitation results indicate that IKK-.gamma. binds
directly to IKK-.alpha., or directly to IKK-.beta., or directly to
both proteins. The interaction of IKK-.gamma. with IKK-.alpha. and
IKK-.beta. therefore was analyzed using purified recombinant
proteins. As shown in FIG. 3d, direct and stable binding of
IKK-.gamma. to IKK-.beta. was detected, although binding to
IKK-.alpha. was not evident, potentially as a result of
interference of epitope tags. These results indicate that
IKK-.gamma. directly binds IKK-.beta..
[0121] Recombinant proteins were expressed and purified as follows.
Recombinant HA-IKK-.alpha. and Flag-IKK-.beta. were expressed in
Sf9 cells using recombinant baculovirus vectors and purified to
homogeneity as described in Zandi et al., supra, 1998. Recombinant
hexahistidine tagged IKK-.gamma. proteins were expressed in E. coli
and purified by nickel affinity chromatography.
C. IKK-.gamma. Associates with IKK-.alpha. in the Absence of
IKK-.beta.
[0122] Lysates of IKK-.beta..sup.+/+, IKK-.beta..sup.+/- and
IKK-.beta..sup.-/- cells were prepared as described in Li et al.,
J. Exp. Med. 189:1839-1845 (1999). The lysates were
immunoprecipitated with either anti-IKK-.alpha. or anti-IKK-.gamma.
antibodies; dissolved in SDS loading buffer and separated by
SDS-PAGE. After transfer to an Immobilon membrane, the proteins
were analyzed by immunoblotting with anti-IKK-.alpha. antibody. A
3T3 cell lysate was used as a control.
[0123] The co-immunoprecipitation results showed that, in contrast
to the results obtained with recombinant proteins, very efficient
co-precipitation of IKK-.alpha. was achieved with anti-IKK-.gamma.
antibodies using lysates of IKK-.beta..sup.-/- cells as starting
material, similar to the co-precipitation observed with
IKK-.beta..sup.+/+ cell lysates. These results indicate that
IKK-.gamma. can associate directly or indirectly with IKK-.alpha.
and that IKK-.beta. is not required for this association.
EXAMPLE III
IKK-.gamma. is an Essential Component of the I.kappa.B Kinase
[0124] This example uses antisense methodology to demonstrate that
IKK-.gamma. is an essential component of the I.kappa.B kinase
(IKK).
A. IKK-.gamma. is an Essential Component of the I.kappa.B
Kinase
[0125] Immunoprecipitation of HA-IKK-.gamma. from transiently
transfected cells resulted in isolation of an I.kappa.B kinase
activity that was stimulated by either TNF or IL-1 (FIG. 4a) as was
seen when anti-IKK-.alpha. was used to isolate the IKK complex.
These results were identical to those obtained when
anti-IKK-.alpha. was used to isolated the IKK complex. This
similarity was due to efficient interaction between transiently
expressed HA-IKK-.gamma. and other IKK components. Gel filtration
analysis indicated that HA-IKK-.gamma. was incorporated into the
large 900 kDa IKK complex, precisely coeluting with IKK-.alpha.
(FIG. 4b).
[0126] The role of IKK-.gamma. in IKK activation was analyzed using
expression of IKK-.gamma. antisense constructs made in
pcDNA3.1/Myc-His (Invitrogen). Whereas cotransfection of an
IKK-.gamma. sense vector had no effect on I.kappa.B kinase activity
associated with either IKK-.alpha. or IKK-.beta. (see below), the
level of TNF-induced I.kappa.B kinase activity associated with
either IKK subunit decreased upon cotransfection with the
IKK-.gamma. antisense vector (FIG. 5a). Antisense IKK-.gamma.
reduced expression of HA-IKK-.gamma., but had no effect on
expression of either HA-IKK-.alpha. or HA-IKK-.beta. (FIG. 5b).
Using anti-NEMO antibodies (Yamaoka et al., supra, 1998), transient
expression of antisense IKK-.gamma. RNA was shown to reduce
expression of endogenous IKK-.gamma. but not IKK-.alpha. (FIG. 5c)
or IKK-.beta..
[0127] Antisense IKK-.gamma. also reduced the extent of IKK
activation by IL-1 or transiently transfected MEKKI or NIK vectors,
although the inhibition of the response to overexpressed NIK was
considerably weaker than that of the other responses (FIG. 5d).
Cotransfection with antisense vectors for the kinases JNKK-1 or
MKK-3 had no effect on IKK activity (DiDonato et al., supra, 1997),
and antisense IKK-.gamma. did not inhibit activation of
p38.sup.MAPK by either TNF or IL-1 (FIG. 7e). Transient expression
of antisense IKK-.gamma. prevented TNF-induced nuclear entry of the
RelA (p65) subunit of NF-.kappa.B.
B. Reduced IKK-.gamma. Expression Interferes with IkB.alpha.
Phosphorylation and Degradation and NF-.kappa.B Activation
[0128] Stably transfected pools of 293 cells harboring the
antisense IKK-.gamma. expression vector were also established.
Cells in these pools expressed lower levels of IKK-.gamma. (FIG.
6b) and, in comparison to the parental cells, exhibited lower
levels of TNF-induced I.kappa.B.alpha. phosphorylation and
degradation (FIG. 6a) and NF-.kappa.B activation (FIG. 6c). This
inhibitory effect was specific as IKK-.alpha. expression was not
decreased (FIG. 6a) and the DNA binding activity of the
constitutive transcription factor NF-1 was actually elevated in
cells transfected with antisense IKK-.gamma. (FIG. 6c). In
addition, the antisense IKK-.gamma. transfected cells exhibited
normal activation of the kinases JNK and p38.sup.MAPK (FIG.
6d).
[0129] These results indicate that reduced IKK-.gamma. expression
interferes with I.kappa.B.alpha. phosphorylation and degradation
and NF-.kappa.B activation.
EXAMPLE IV
Dominant Negative Inhibitors of IKK Activation
[0130] This example demonstrates that a C-terminal IKK-.gamma.
deletion mutant is a dominant negative inhibitor of IKK
activation.
[0131] To study the function of IKK-.gamma., N- and C-terminal
deletion mutants were constructed as described in Zandi et al.,
supra, 1997) and analyzed for possible dominant inhibitory activity
(FIG. 7a). Coexpression of .DELTA.N-IKK-.gamma.(134-419) with
FLAG-IKK-.beta. had only a marginal effect on basal IKK activity
and its response to TNF. However, expression of
.DELTA.C-IKK-.gamma.(1-300) or .DELTA.C-IKK-.gamma.(1-349)
inhibited activation of IKK by TNF but not basal kinase activity
(FIG. 7b). .DELTA.N-IKK-.gamma.(134-419), .DELTA.C-IKK-.gamma.
(1-300) and .DELTA.C-IKK-.gamma.(1-349) interacted with
IKK.alpha./.beta. in cells (FIG. 7c), but only full length
IKK-.gamma. and .DELTA.N-IKK-.gamma.(134-419) were able to
coprecipitate IKK activity stimulated by TNF, IL-1, MEKK-1 or NIK.
Crosslinking experiments using recombinant proteins indicated that
IKK-.gamma. can form dimers and trimers and that the C-terminal
truncation had no effect on this activity, although the N-terminal
truncation may have reduced the efficiency of trimerization.
Neither full-length IKK-.gamma. nor its truncation mutants
inhibited activation of p38.sup.MAPK by TNF or IL-1 (FIG. 7e).
[0132] These results indicate that the IKK-.gamma. region
responsible for interaction with an upstream factor required for
TNF-inducible kinase activity resides in the 70 carboxy-terminal
amino acids of IKK-.gamma..
[0133] All journal article, reference and patent citations provided
above, in parentheses or otherwise, whether previously stated or
not, are incorporated herein by reference in their entirety.
[0134] Although the invention has been described with reference to
the examples provided above, it should be understood that various
modifications can be made without departing from the spirit of the
invention. Accordingly, the invention is limited only by the
claims.
Sequence CWU 1
1
19 1 1994 DNA Homo sapiens CDS (149)..(1408) 1 ggcacgagca
tggcccttgt gatccaggtg gggaaactaa ggcccagaga agtgaggacc 60
ccgcagacta tcaatcccag tctcttcccc tcactccctg tgaagctctc cagcatcatc
120 gaggtcccat cagcccttgc cctgttgg atg aat agg cac ctc tgg aag agc
172 Met Asn Arg His Leu Trp Lys Ser 1 5 caa ctg tgt gag atg gtg cag
ccc agt ggt ggc ccg gca gca gat cag 220 Gln Leu Cys Glu Met Val Gln
Pro Ser Gly Gly Pro Ala Ala Asp Gln 10 15 20 gac gta ctg ggc gaa
gag tct cct ctg ggg aag cca gcc atg ctg cac 268 Asp Val Leu Gly Glu
Glu Ser Pro Leu Gly Lys Pro Ala Met Leu His 25 30 35 40 ctg cct tca
gaa cag ggc gct cct gag acc ctc cag cgc tgc ctg gag 316 Leu Pro Ser
Glu Gln Gly Ala Pro Glu Thr Leu Gln Arg Cys Leu Glu 45 50 55 gag
aat caa gag ctc cga gat gcc atc cgg cag agc aac cag att ctg 364 Glu
Asn Gln Glu Leu Arg Asp Ala Ile Arg Gln Ser Asn Gln Ile Leu 60 65
70 cgg gag cgc tgc gag gag ctt ctg cat ttc caa gcc agc cag agg gag
412 Arg Glu Arg Cys Glu Glu Leu Leu His Phe Gln Ala Ser Gln Arg Glu
75 80 85 gag aag gag ttc ctc atg tgc aag ttc cag gag gcc agg aaa
ctg gtg 460 Glu Lys Glu Phe Leu Met Cys Lys Phe Gln Glu Ala Arg Lys
Leu Val 90 95 100 gag aga ctc ggc ctg gag aag ctc gat ctg aag agg
cag aag gag cag 508 Glu Arg Leu Gly Leu Glu Lys Leu Asp Leu Lys Arg
Gln Lys Glu Gln 105 110 115 120 gct ctg cgg gag gtg gag cac ctg aag
aga tgc cag cag cag atg gct 556 Ala Leu Arg Glu Val Glu His Leu Lys
Arg Cys Gln Gln Gln Met Ala 125 130 135 gag gac aag gcc tct gtg aaa
gcc cag gtg acg tcc ttg ctc ggg gag 604 Glu Asp Lys Ala Ser Val Lys
Ala Gln Val Thr Ser Leu Leu Gly Glu 140 145 150 ctg cag gag agc cag
agt cgc ttg gag gct gcc act aag gaa tgc cag 652 Leu Gln Glu Ser Gln
Ser Arg Leu Glu Ala Ala Thr Lys Glu Cys Gln 155 160 165 gct ctg gag
ggt cgg gcc cgg gcg gcc agc gag cag gcg cgg cag ctg 700 Ala Leu Glu
Gly Arg Ala Arg Ala Ala Ser Glu Gln Ala Arg Gln Leu 170 175 180 gag
agt gag cgc gag gcg ctg cag cag cag cac agc gtg cag gtg gac 748 Glu
Ser Glu Arg Glu Ala Leu Gln Gln Gln His Ser Val Gln Val Asp 185 190
195 200 cag ctg cgc atg cag ggc cag agc gtg gag gcc gcg ctc cgc atg
gag 796 Gln Leu Arg Met Gln Gly Gln Ser Val Glu Ala Ala Leu Arg Met
Glu 205 210 215 cgc cag gcc gcc tcg gag gag aag agg aag ctg gcc cag
ttg cag gtg 844 Arg Gln Ala Ala Ser Glu Glu Lys Arg Lys Leu Ala Gln
Leu Gln Val 220 225 230 gcc tat cac cag ctc ttc caa gaa tac gac aac
cac atc aag agc agc 892 Ala Tyr His Gln Leu Phe Gln Glu Tyr Asp Asn
His Ile Lys Ser Ser 235 240 245 gtg gtg ggc agt gag cgg aag cga gga
atg cag ctg gaa gat ctc aaa 940 Val Val Gly Ser Glu Arg Lys Arg Gly
Met Gln Leu Glu Asp Leu Lys 250 255 260 cag cag ctc cag cag gcc gag
gag gcc ctg gtg gcc aaa cag gag gtg 988 Gln Gln Leu Gln Gln Ala Glu
Glu Ala Leu Val Ala Lys Gln Glu Val 265 270 275 280 atc gat aag ctg
aag gag gag gcc gag cag cac aag att gtg atg gag 1036 Ile Asp Lys
Leu Lys Glu Glu Ala Glu Gln His Lys Ile Val Met Glu 285 290 295 acc
gtt ccg gtg ctg aag gcc cag gcg gat atc tac aag gcg gac ttc 1084
Thr Val Pro Val Leu Lys Ala Gln Ala Asp Ile Tyr Lys Ala Asp Phe 300
305 310 cag gct gag agg cag gcc cgg gag aag ctg gcc gag aag aag gag
ctc 1132 Gln Ala Glu Arg Gln Ala Arg Glu Lys Leu Ala Glu Lys Lys
Glu Leu 315 320 325 ctg cag gag cag ctg gag cag ctg cag agg gag tac
agc aaa ctg aag 1180 Leu Gln Glu Gln Leu Glu Gln Leu Gln Arg Glu
Tyr Ser Lys Leu Lys 330 335 340 gcc agc tgt cag gag tcg gcc agg atc
gag gac atg agg aag cgg cat 1228 Ala Ser Cys Gln Glu Ser Ala Arg
Ile Glu Asp Met Arg Lys Arg His 345 350 355 360 gtc gag gtc tcc cag
gcc ccc ttg ccc ccc gcc cct gcc tac ctc tcc 1276 Val Glu Val Ser
Gln Ala Pro Leu Pro Pro Ala Pro Ala Tyr Leu Ser 365 370 375 tct ccc
ctg gcc ctg ccc agc cag agg agg agc ccc ccc gag gag cca 1324 Ser
Pro Leu Ala Leu Pro Ser Gln Arg Arg Ser Pro Pro Glu Glu Pro 380 385
390 cct gac ttc tgc tgt ccc aag tgc cag tat cag gcc cct gat atg gac
1372 Pro Asp Phe Cys Cys Pro Lys Cys Gln Tyr Gln Ala Pro Asp Met
Asp 395 400 405 acc ctg cag ata cat gtc atg gag tgc att gag tag
ggccggccag 1418 Thr Leu Gln Ile His Val Met Glu Cys Ile Glu 410 415
tgcaaggcca ctgcctgccc gaggacgtgc ccgggaccgt gcagtctgcg ctttcctctc
1478 ccgcctgcct agcccaggat gaagggctgg gtggccacaa ctgggatgcc
acctggagcc 1538 ccacccagga gctggccgcg gcaccttacg cttcagctgt
tgatccgctg gtcccctctt 1598 ttggggtaga tgcggccccg atcaggcctg
actcgctgct ctttttgttc ccttctgtct 1658 gctcgaacca cttgcctcgg
gctaatccct ccctcttcct ccacccggca ctggggaagt 1718 caagaatggg
gcctggggct ctcagggaga actgcttccc ctggcagagc tgggtggcag 1778
ctcttcctcc caccggacac cgacccgccc gccgctgtgc cctgggagtg ctgccctctt
1838 accatgcaca cgggtgctct ccttttgggc tgcatgctat tccattttgc
agccagaccg 1898 atgtgtattt aaccagtcac tattgatgga catttgggtt
gtttcccatc tttttgttac 1958 cataaataat ggcatagtaa aaaaaaaaaa aaaaaa
1994 2 419 PRT Homo sapiens 2 Met Asn Arg His Leu Trp Lys Ser Gln
Leu Cys Glu Met Val Gln Pro 1 5 10 15 Ser Gly Gly Pro Ala Ala Asp
Gln Asp Val Leu Gly Glu Glu Ser Pro 20 25 30 Leu Gly Lys Pro Ala
Met Leu His Leu Pro Ser Glu Gln Gly Ala Pro 35 40 45 Glu Thr Leu
Gln Arg Cys Leu Glu Glu Asn Gln Glu Leu Arg Asp Ala 50 55 60 Ile
Arg Gln Ser Asn Gln Ile Leu Arg Glu Arg Cys Glu Glu Leu Leu 65 70
75 80 His Phe Gln Ala Ser Gln Arg Glu Glu Lys Glu Phe Leu Met Cys
Lys 85 90 95 Phe Gln Glu Ala Arg Lys Leu Val Glu Arg Leu Gly Leu
Glu Lys Leu 100 105 110 Asp Leu Lys Arg Gln Lys Glu Gln Ala Leu Arg
Glu Val Glu His Leu 115 120 125 Lys Arg Cys Gln Gln Gln Met Ala Glu
Asp Lys Ala Ser Val Lys Ala 130 135 140 Gln Val Thr Ser Leu Leu Gly
Glu Leu Gln Glu Ser Gln Ser Arg Leu 145 150 155 160 Glu Ala Ala Thr
Lys Glu Cys Gln Ala Leu Glu Gly Arg Ala Arg Ala 165 170 175 Ala Ser
Glu Gln Ala Arg Gln Leu Glu Ser Glu Arg Glu Ala Leu Gln 180 185 190
Gln Gln His Ser Val Gln Val Asp Gln Leu Arg Met Gln Gly Gln Ser 195
200 205 Val Glu Ala Ala Leu Arg Met Glu Arg Gln Ala Ala Ser Glu Glu
Lys 210 215 220 Arg Lys Leu Ala Gln Leu Gln Val Ala Tyr His Gln Leu
Phe Gln Glu 225 230 235 240 Tyr Asp Asn His Ile Lys Ser Ser Val Val
Gly Ser Glu Arg Lys Arg 245 250 255 Gly Met Gln Leu Glu Asp Leu Lys
Gln Gln Leu Gln Gln Ala Glu Glu 260 265 270 Ala Leu Val Ala Lys Gln
Glu Val Ile Asp Lys Leu Lys Glu Glu Ala 275 280 285 Glu Gln His Lys
Ile Val Met Glu Thr Val Pro Val Leu Lys Ala Gln 290 295 300 Ala Asp
Ile Tyr Lys Ala Asp Phe Gln Ala Glu Arg Gln Ala Arg Glu 305 310 315
320 Lys Leu Ala Glu Lys Lys Glu Leu Leu Gln Glu Gln Leu Glu Gln Leu
325 330 335 Gln Arg Glu Tyr Ser Lys Leu Lys Ala Ser Cys Gln Glu Ser
Ala Arg 340 345 350 Ile Glu Asp Met Arg Lys Arg His Val Glu Val Ser
Gln Ala Pro Leu 355 360 365 Pro Pro Ala Pro Ala Tyr Leu Ser Ser Pro
Leu Ala Leu Pro Ser Gln 370 375 380 Arg Arg Ser Pro Pro Glu Glu Pro
Pro Asp Phe Cys Cys Pro Lys Cys 385 390 395 400 Gln Tyr Gln Ala Pro
Asp Met Asp Thr Leu Gln Ile His Val Met Glu 405 410 415 Cys Ile Glu
3 10 PRT Homo sapiens 3 Ile Val Met Glu Thr Val Pro Val Leu Lys 1 5
10 4 17 PRT Homo sapiens 4 Lys Glu Leu Leu Gln Glu Gln Leu Glu Gln
Leu Gln Arg Glu Tyr Ser 1 5 10 15 Lys 5 16 PRT Homo sapiens 5 Glu
Leu Leu Gln Glu Gln Leu Glu Gln Leu Gln Arg Glu Tyr Ser Lys 1 5 10
15 6 20 PRT Homo sapiens 6 Arg His Val Glu Val Ser Gln Ala Pro Leu
Pro Pro Ala Pro Ala Tyr 1 5 10 15 Leu Ser Ser Pro 20 7 20 PRT Homo
sapiens 7 Leu Ala Gln Leu Gln Val Ala Tyr His Gln Leu Phe Gln Glu
Tyr Asp 1 5 10 15 Asn His Ile Lys 20 8 11 PRT Homo sapiens
misc_feature (1)..(1) Xaa can be any naturally occurring amino acid
8 Xaa Gln Tyr Gln Ala Pro Asp Met Asp Thr Leu 1 5 10 9 17 PRT Homo
sapiens misc_feature (1)..(1) Xaa can be any naturally occurring
amino acid 9 Xaa Gln Pro Ser Gly Gly Pro Ala Ala Asp Gln Asp Val
Leu Gly Glu 1 5 10 15 Glu 10 13 PRT Homo sapiens 10 Gln Gln Leu Gln
Gln Ala Glu Glu Ala Leu Val Ala Lys 1 5 10 11 11 PRT Homo sapiens
11 Glu Gln Ala Leu Arg Glu Val Glu His Leu Lys 1 5 10 12 9 PRT Homo
sapiens 12 Leu Val Glu Arg Leu Gly Leu Glu Lys 1 5 13 15 PRT Homo
sapiens 13 Lys Glu Leu Leu Gln Glu Gln Leu Glu Gln Leu Gln Arg Glu
Tyr 1 5 10 15 14 14 PRT Homo sapiens misc_feature (1)..(2) Xaa can
be any naturally occurring amino acid 14 Xaa Xaa Val Thr Ser Leu
Leu Gly Glu Leu Gln Glu Ser Gln 1 5 10 15 13 PRT Homo sapiens
misc_feature (1)..(2) Xaa can be any naturally occurring amino acid
15 Xaa Xaa Leu Gln Gln Ala Glu Glu Ala Leu Val Ala Lys 1 5 10 16 15
PRT Homo sapiens misc_feature (1)..(1) Xaa can be any naturally
occurring amino acid misc_feature (5)..(5) Xaa can be any naturally
occurring amino acid misc_feature (8)..(8) Xaa can be any naturally
occurring amino acid misc_feature (13)..(13) Xaa can be any
naturally occurring amino acid 16 Xaa Gln Val Thr Xaa Leu Leu Xaa
Glu Leu Gln Glu Xaa Gln Gln 1 5 10 15 17 20 PRT Homo sapiens
misc_feature (1)..(1) Xaa can be any naturally occurring amino acid
17 Xaa Ala Gln Leu Gln Val Ala Tyr His Gln Leu Phe Gln Glu Tyr Asp
1 5 10 15 Asn His Ile Lys 20 18 22 DNA Artificial Sequence
Synthetic 18 agttgagggg actttcccag gc 22 19 22 DNA Artificial
Sequence Synthetic 19 ttggattgaa gccaatatga ta 22
* * * * *