IKAP proteins and methods

Details IKAP proteins and methods IKAP proteins and methods

1999-04-06

2001-01-09

Kemmerer, Elizabeth (Department: 1646)

Chemistry: natural resins or derivatives; peptides or proteins;

Proteins, i.e., more than 100 amino acid residues

C530S300000, C530S351000, C436S501000

Reexamination Certificate

active

06172195

ABSTRACT:

FIELD OF THE INVENTION
The field of this invention is proteins involved in cell signal transduction.
BACKGROUND
Cytokines trigger changes in gene expression by modifying the activity of otherwise latent transcription factors (Hill and Treisman, 1995). Nuclear factor &kgr;B (NF-&kgr;B) is a
1
prominent example of how such an external stimulus is converted into an active transcription factor (Verma et al., 1995). The NF-&kgr;B system is composed of homo- and heterodimers of members of the Re
1
family of related transcription factors that control the expression of numerous immune and inflammatory response genes as well as important viral genes (Lenardo and Baltimore, 1989; Baeuerle and Henkel, 1994). The activity of NF-&kgr;B transcription factors is regulated by their subcellular localization (Verma et al., 1995). In most cell types, NF-&kgr;B is present as a heterodimer comprising of a 50 kDa and a 65 kDa subunit. This heterodimer is sequestered in the cytoplasm in association with I&kgr;B&agr; a member of the I&kgr;B family of inhibitory proteins (Finco and Baldwin, 1995; Thanos and Maniatis, 1995; Verma et al., 1995). I&kgr;B&agr; masks the nuclear localization signal of NF-&kgr;B and thereby prevents NF-&kgr;B nuclear translocation. Conversion of NF-&kgr;B into an active transcription factor that translocates into the nucleus and binds to cognate DNA sequences requires the phosphorylation and subsequent ubiquitin-dependent degradation of I&kgr;B&agr; in the 26s proteasome. Signal-induced phosphorylation of I&kgr;B&agr; occurs at serines 32 and 36. Mutation of one or both of these serines renders I&kgr;B&agr; resistant to ubiquitination and proteolytic degradation (Chen et al., 1995); DiDonato, 1996 #370, Roff, 1996 #397.
The pleiotropic cytokines tumor necrosis factor (TNF) and interleukin-1 (IL-1) are among the physiological inducers of I&kgr;B phosphorylation and subsequent NF-&kgr;B activation (Osborn et al., 1989; Beg et al., 1993). Although TNF and IL-1 initiate signaling cascades leading to NF-&kgr;B activation via distinct families of cell-surface receptors (Smith et al., 1994; Dinarello, 1996), both pathways utilize members of the TNF receptor-associated factor (TRAF) family of adaptor proteins as signal transducers (Rothe et al., 1995; Hsu et al., 1996; Cao et al., 1996b). TRAF proteins were originally found to associate directly with the cytoplasmic domains of several members of the TNF receptor family including the 75 kDa TNF receptor (TNFR2), CD40, CD30, and the lymphotoxin-&bgr; receptor (Rothe et al., 1994; Hu et al., 1994; Cheng et al., 1995; Mosialos et al., 1995; Song and Donner, 1995; Sato et al., 1995; Lee et al., 1996; Gedrich et al., 1996; Ansieau et al., 1996). In addition, TRAF proteins are recruited indirectly to the 55 kDa TNF receptor (TNFR1) by the adaptor protein TRADD (Hsu et al., 1996). Activation of NF-&kgr;B by TNF requires TRAF2 (Rothe et al., 1995; Hsu et al., 1996). TRAF5 has also been implicated in NF-&kgr;B activation by members of the TNF receptor family (Nakano et al., 1996); Ishida, 1996 #240. In contrast, TRAF6 participates in NF-&kgr;B activation by IL-1 (Cao et al., 1996b). Upon IL-1 treatment, TRAF6 associates with IRAK, a serine-threonine kinase that binds to the IL-1 receptor complex (Cao et al., 1996a); Huang, 1997 #400.
The NF-&kgr;B-inducing kinase (NIK) is a member of the MAP kinase kinase kinase (MAP3K) family that was identified as a TRAF2-interacting protein (Malinin et al., 1997). NIK activates NF-&kgr;B when overexpressed, and kinase-inactive mutants of NIK comprising its TRAF2-interacting C-terminal domain (NIK
(624-947)
) or lacking two crucial lysine residues in its kinase domain (NIK
(KK429-430AA)
) behave as dominant-negative inhibitors that suppress TNF-, IL-1-, and TRAF2-induced NF-&kgr;B activation (Malinin et al., 1997). Recently, NIK was found to associate with additional members of the TRAF family, including TRAF5 and TRAF6. Catalytically inactive mutants of NIK also inhibited TRAF5- and TRAF6-induced NF-&kgr;B activation, thus providing a unifying concept for NIK as a common mediator in the NF-&kgr;B signaling cascades triggered by TNF and IL-1 downstream of TRAFs. Recently two NIK-interacting protein designated characterized as novel human kinase I&kgr;B Kinases, IKK-&agr; and IKK&bgr; have been reported (Woronicz et al., 1997; Mercurio et al. 1997; Maniatis, 1997). Catalytically inactive mutants of IKK suppress NF-&kgr;B activation induced by TNF and IL-1 stimulation as well as by TRAF and NIK overexpression; transiently expressed IKK associates with endogenous I&kgr;B&agr; complex; and IKK phosphorylates I&kgr;B&agr; on serines 32 and 36.
Relevant Literature
Ansieau, S., et al. (1996). Proc. Natl. Acad. Sci. USA 93, 14053-14058.
Baeuerle, P. A., and Henkel, T. (1994). Annu. Rev. Immunol. 12, 141-179.
Beg, A. A., et al. (1993). Mol. Cell. Biol. 13, 3301-3310.
Cao, Z., Henzel, W. J., and Gao, X. (1996a). Science 271, 1128-1131.
Cao, Z., et al. (1996b). Nature 383, 443-446.
Chen, Z., et al. (1995). Genes Dev. 9, 1586-1597.
Cheng, G., et al. (1995). Science 267, 1494-1498.
Connelly, M. A., and Marcu, K. B. (1995). Cell. Mol. Biol. Res. 41, 537-549.
Dinarello, C. A. (1996). Biologic basis for interleukin-1 in disease. Blood 87, 2095-2147.
Fields, S., and Song, O.-k. (1989). Nature 340, 245-246.
Finco, T. S., and Baldwin, A. S. (1995). Immunity 3, 263-272.
Gedrich, R. W., et al. (1996). J. Biol. Chem. 271, 12852-12858.
Hill, C. S., and Treisman, R. (1995). Cell 80, 199-211.
Hsu, H., Shu, H.-B., Pan, M.-P., and Goeddel, D. V. (1996). Cell 84, 299-308.
Hu, H. M., et al. (1994). J. Biol. Chem. 269, 30069-30072.
Lee, S. Y., et al. (1996). Proc. Natl. Acad. Sci. USA 93, 9699-9703.
Lenardo, M., and Baltimore, D. (1989). Cell 58, 227-229.
Malinin, N. L., et al. (1997). Nature 385, 540-544.
Maniatis (1997) Science 278, 818.
Mercurio et al. (1997) Science 278, 860.
Mock et al. (1995). Genomics 27, 348-351.
Mosialos, G., et al. (1995). Cell 80, 389-399.
Nakano, H., et al. (1996). J. Biol. Chem. 271, 14661-14664.
Osborn, L., et al. (1989). Proc. Natl. Acad. Sci. USA 86, 2336-2340.
Rothe, M., Sarma, V., Dixit, V. M., and Goeddel, D. V. (1995). Science 269, 1424-1427.
Rothe, M., Wong, S. C., Henzel, W. J., and Goeddel, D. V. (1994). Cell 78, 681-692.
Sato, T., Irie, S., and Reed, J. C. (1995). FEBS Lett. 358, 113-118.
Schindler, U., and Baichwal, V. R. (1994). Mol. Cell. Biol. 14, 5820-5831.
Smith, C. A., Farrah, T., and Goodwin, R. G. (1994). Cell 76, 959-962.
Song, H. Y., and Donner, D. B. (1995). Biochem. J. 809, 825-829.
Thanos, D., and Maniatis, T. (1995). Cell 80, 529-532.
Woronicz et al., (1997) Science 278, 866.
Verma, I. M., et al. (1995). Genes Dev. 9, 2723-2735.
SUMMARY OF THE INVENTION
The invention provides methods and compositions relating to isolated IKAP polypeptides, related nucleic acids, polypeptide domains thereof having IKAP-specific structure and activity and modulators of IKAP function, particularly NIK binding activity. IKAP polypeptides can regulate NF&kgr;B activation and hence provide important regulators of cell function. The polypeptides may be produced recombinantly from transformed host cells from the subject IKAP polypeptide encoding nucleic acids or purified from mammalian cells. The invention provides isolated IKAP hybridization probes and primers capable of specifically hybridizing with the disclosed IKAP gene, IKAP-specific binding agents such as specific antibodies, and methods of making and using the subject compositions in diagnosis (e.g. genetic hybridization screens for IKAP transcripts), therapy (e.g. IKAP inhibitors to inhibit TNF signal transduction) and in the biopharmaceutical industry (e.g. as immunogens, reagents for isolating other transcriptional regulators, reagents for screening chemical libraries for lead pharmacological agents, etc.).


REFERENCES:
patent: WO 94/01548 (1994-01-01), None
Database GenBank, National Library of Medicine, Bethesda, Maryland USA, Accession No. H19711, Hillier et al., yn60b07.rlHomo sapienscDNA clone 172789 5′. Jul. 03,

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