IKK3 kinase

Details IKK3 kinase IKK3 kinase

2001-06-21

2003-06-10

Monshipouri, M. (Department: 1652)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving transferase

C435S194000, C435S320100, C435S325000, C435S252300, C435S006120

Reexamination Certificate

active

06576439

ABSTRACT:

This application is filed pursuant to 35 U.S.C. §371 as a U.S. National Phase Application of International Application No. PCT/JP99/07286 filed Dec. 24, 1999, which claims priority from GB 9828704.8 filed Dec. 24, 1998.
TECHNICAL FIELD
This invention relates to a novel IKK kinase protein, IKK3, nucleotides coding for it, vectors and host cells containing the same and methods for screening for modulators of said IKK3 protein for treatment of conditions involving inflammation.
BACKGROUND ART
The transcription factor NF-kB controls the activation of various genes in response to pathogens and pro-inflammatory cytokines. Thus, for example, NF-kB is activated by various kinds of stimulation including tumour necrosis factor alfa (TNF alfa) and interleukin-1 (IL-1), bacterial LPS, viral infection, antigen receptor cross-linking of T and B cells, calcium ionophores, phorbol esters, UV radiation and free radicals (for reviews, see Varma et al., 1995, Genes Dev., 9, 2723-2735; Baueurerle and Baltimore, 1996, Cell, 87, 13-20), (see FIG.
2
). NF-kB in turn controls the activation of various genes in response to these stimuli. Activation of these various genes in turn may result in the production of cytokines, chemokines, leukocyte adhesion molecules, hematopoietic growth factors and may also effect development and cell death as well as cell survival (see FIG.
1
). Specifically, the transcription factor NF-kB controls the activation of various genes in response to pathogens and pro-inflammatory cytokines. The NF-kB activity is regulated through interaction with specific inhibitors, IkBs. Upon cell stimulation, the IkBs are rapidly phosphorylated and then undergo ubiquitin-mediated proteolysis, resulting in the release of active NF-kB (Baldwin, 1996, Annu. Rev. Immunol., 14, 649-681; Baueurerle and Baltimore, 1996, Cell, 87, 13-20), (see FIG.
2
). It has been reported that the 700 kDa complex specifically phosphorylated IkB&agr; at S32 and S36 (Chen et al., 1996, Cell, 84, 853-862).
Several groups found. that two kinases termed IKK1 and IKK2 (also known as IKK&agr; and IKK&bgr;), were the subunits of the kinase complex. The groups showed that the IKKs immunoprecipitates, derived from the TNF&agr; or IL-1 stimulated cells are able to phosphorylate IkB in vitro. In addition to these observations, two groups reported that IKK1 and IKK2 purified from insect cells are able to phosphorylate IkB in vitro. These results suggested that IKK directly phosphorylates IkBs. The over expression of anti-sense IKK1, kinase-inactive IKK1 or IKK2 resulted in the inhibition of NF-kB activation mediated by TNF&agr; and IL-1. These results suggest that IKKs are critical kinases in the NF-kB activation pathway (May and Ghosh, 1998, Immunol. Today 19, 80-88; Stancovski and Baltimore, 1997, Cell, 91, 299-302). It has, however, not been understood how upstream signals are transmitted to the kinase complex, or whether different kinase complexes might exist to phosphorylate distinct IkBs.
NEMO (NF-kB essential modifier) and IKK&ggr; (human homologue of the mouse NEMO) were isolated from purified IKK complex, and the inhibition of NEMO/IKK&ggr; gene expression impaired the cytokine induced NF-kB activation via IKK1 and IKK2. In NEMO deficient cells, smaller complexes of Mr 3,000-4,000 are formed, though the normal complex is Mr 7,000-9,000, suggesting that NEMO/IKK&ggr; physically link IkB kinase to upstream activators (Scheidereit, Nature, 1998, 395, 225-226).
The IKK-complex-associated protein (IKAP) was isolated from the IKK complexes. IKAP binds to IkB kinases and NIK and the complex, containing three kinases, leads to the maximum phosphorylation of IkB as compared to the complex containing one or two kinases. Accordingly, IKAP may act as scaffold proteins that link NIK or other molecules to IKK1 and IKK2 (Scheidereit, Nature, 1998, 395, 225-226). Accumulating evidence suggests that the IKK complex consists of several essential molecules, however, the molecular mechanisms that control the signalling complex were not well understood. Therefore, further association molecules were needed to complete the picture.
KIAA0151 was originally isolated from the KG-1 cDNA library (Nagase et al., 1995, DNA Res, 2, 167-174). KIAA0151 was identified as a potential Ser/Thr kinase, however, the importance of the molecule was not recognised. We have now found that KIAA0151 is similar to IKK1 and IKK2 using a computer homology analysis. KIAA0151, renamed IKK3, has a 21% homology with IKK1 and 23% with IKK2. IKK3 was able to phosphorylate IkB family proteins and directly phosphorylate IkB in vitro. The over expression of IKK3 leads to the activation of various inflammatory genes, such as IL-8, IL-6 and RANTES. These genes contain the NF-kB site in the gene regulation region. We know that IKK3 has an effect on IL-8 expression in Hela cells and also that IKK3 phosphorylates NF-kB. Moreover, it is known that the NF-kB site has an important role in IL-8 regulation. Our results suggest a correlation between IKK3 and the NF-kB site of the IL-8 promoter that has previously been identified as an endogenous NF-kB binding site, further suggesting that IKK3 plays an important role in controlling the NF-kB site of the IL-8 promoter. Specifically we have shown that IKK3 transactivates the IL-8 gene via the NF-kB binding to a site in the IL-8 promoter. These results lead to the conclusion that IKK3 is an important regulator of IL-8 gene regulation and thus activates genes that are important for the inflammatory diseases (see Table 1 below).
TABLE 1
Differences between IKK1, 2 and IKK3
IKK1, 2 (also known as
IKK&agr;, &bgr;)
IKK3
Expression (mRNA)
Constitutive
Inducible by IL-1
and TNF-alfa
Source for in vitro
Mammalian and Insect cells
Mammalian and
phosphorylation
Bacterial cells
Spectrum
Unknown
IL-8, IL-6 and
RANTES
Substrate Selectively
IkB&agr; > IkB&bgr;
IkB&egr; IkB&bgr; > IkB&agr;
Enzymatic activity
Need for IL-1 or TNF alfa
No need for
stimulation
stimulation
Using a computer homology analysis, we have now found that KIAA0151 is similar to IKK1 and IKK2. Importantly, recent experimental evidence has shown that IKK3 specifically controls various inflammatory genes, such as IL-8, IL-6 and RANTES. Moreover, IKK3 has been shown to phosphorylate various IkBs and directly phosphorylate TRIP9 (human IkB&bgr;). IKK3 has therefore been shown to have a specific role in the control of inflammation.
DISCLOSURE OF INVENTION
Accordingly this invention provides a novel kinase protein, IKK3.
Nucleotide sequence analysis of IKK3 reveals a 2148 bp open reading frame which encodes 716 amino acid protein (FIG.
3
). This deduced protein sequence shares many of the characteristics of IKK1 and IKK2. (see FIG.
5
).
One aspect of the invention therefore provides an isolated IKK3 kinase protein or a variant thereof. The amino acid sequence of this isolated IKK3 kinase protein is shown in FIG.
3
.
Included within the invention are variants of the IKK3 kinase protein. Such variants include fragments, analogues, derivatives and splice variants. The term “variant” refers to a protein or part of a protein which retains substantially the same biological function or activity as IKK3.
Fragments can include a part of IKK3 which retains sufficient identity of the original protein to be effective for example in a screen. Such fragments may be probes such as the ones described hereinafter for the identification of the full length protein. Fragments may be fused to other amino acids or proteins or may be comprised within a larger protein. Such a fragment may be comprised within a precursor protein designed for expression in a host. Therefore, in one aspect the term fragment means a portion or portions of a fusion protein or polypeptide derived from IKK3.
Fragments also include portions of IKK3 characterised by structural or functional attributes of the protein. These may have similar or improved chemical or biological activity or reduced side-effect activity. For example, fragments may comprise an alpha, alpha-helix or alpha-helix-forming region beta sheet and beta-

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