IKK-&agr; proteins, nucleic acids and methods

Details IKK-&agr; proteins, nucleic acids and methods IKK-&agr; proteins, nucleic acids and methods

1998-02-26

2001-05-22

Patterson, Jr., Charles L. (Department: 1652)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving transferase

Reexamination Certificate

active

06235492

ABSTRACT:

INTRODUCTION
1. Field of the Invention
The field of this invention is proteins involved in transcription factor activation.
2. 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 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 Rel 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);
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). 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).
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.
Here, we disclose a novel human kinase I&kgr;B Kinase, IKK-&agr;, as a NIK-interacting protein. IKK-&agr; has sequence similarity to the conceptual translate of a previously identified open reading frame (SEQ ID NO:5) postulated to encode a serine-threonine kinase of unknown function (‘Conserved Helix-loop-helix Ubiquitous Kinase’ or CHUK, Connelly and Marcu, 1995; Mock et al., 1995). Catalytically inactive mutants of IKK-&agr; are shown to suppress NF-&kgr;B activation induced by TNF and IL-1 stimulation as well as by TRAF and NIK overexpression; transiently expressed IKK-&agr; is shown to associate with the endogenous I&kgr;B&agr; complex; and IKK-&agr; is shown to phosphorylate I&kgr;B&agr; on serines 32 and 36.
SUMMARY OF THE INVENTION
The invention provides methods and compositions relating to isolated IKK-&agr; polypeptides, related nucleic acids, polypeptide domains thereof having IKK-&agr;-specific structure and activity and modulators of IKK-&agr; function, particularly I&kgr;B kinase activity. IKK-&agr; 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 IKK-&agr; polypeptide encoding nucleic acids or purified from mammalian cells. The invention provides isolated IKK-&agr; hybridization probes and primers capable of specifically hybridizing with the disclosed IKK-&agr; gene, IKK-&agr;-specific binding agents such as specific antibodies, and methods of making and using the subject compositions in diagnosis (e.g. genetic hybridization screens for IKK-&agr; transcripts), therapy (e.g. IKK-&agr; kinase 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.).
DETAILED DESCRIPTION OF THE INVENTION
The nucleotide sequence of a natural cDNA encoding a human IKK-&agr; polypeptide is shown as SEQ ID NO:3, and the full conceptual translate is shown as SEQ ID NO:4. The IKK-&agr; polypeptides of the invention include incomplete translates of SEQ ID NO:3 which translates and deletion mutants of SEQ ID NO:4 have human IKK-&agr;-specific amino acid sequence, binding specificity or function and comprise at least one of Cys30, Leu604, Thr679, Ser680, Pro684, Thr686 and Ser678. Preferred translates/deletion mutants comprise at least a 6 residue Cys30, Leu604, Thr679, Ser680, Pro684, Thr686 or Ser687-containing domain of SEQ ID NO:4, preferably including at least 8, more preferably at least 12, most preferably at least 20 contiguous residues which immediately flank said residue, with said residue preferably residing within said contigous residues, see, e.g. Table I; which mutants provide hIKK-&agr; specific epitopes and immunogens.
TABLE I
Exemplary Deletion Mutants
&Dgr;1
SEQ ID NO: 4, residues 22-31
&Dgr;2
SEQ ID NO: 4, residues 1-30
&Dgr;3
SEQ ID NO: 4, residues 599-608
&Dgr;4
SEQ ID NO: 4, residues 601-681
&Dgr;5
SEQ ID NO: 4, residues 604-679
&Dgr;6
SEQ ID NO: 4, residues 670-687
&Dgr;7
SEQ ID NO: 4, residues 679-687
&Dgr;8
SEQ ID NO: 4, residues 680-690
&Dgr;9
SEQ ID NO: 4, residues 684-695
&Dgr;10
SEQ ID NO: 4, residues 686-699
The subject domains provide IKK-&agr; domain specific activity or function, such as IKK-&agr;-specific kinase or kinase inhibitory activity, NIK-binding or binding inhibitory activity, I&kgr;B-binding or binding inhibitory activity, NF&kgr;B activating or inhibitory activity or antibody binding. Preferred domains phosphorylate at least one and preferably both the serine 32 and 36 of I&kgr;B (Verma, I. M., et al. (1995)). As used herein, Ser32 and Ser36 of I&kgr;B refers collectively to the two serine residues which are part of the consensus sequence DSGL/IXSM/L (e.g. ser 32 and 36 in I&kgr;B&agr;, ser 19 and 23 in I&kgr;B&egr;, and ser 157 and 161, or 1

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