Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving transferase
Reexamination Certificate
2001-04-27
2003-06-10
Patterson, Jr., Charles L. (Department: 1652)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving transferase
C435S068100, C435S194000
Reexamination Certificate
active
06576437
ABSTRACT:
TECHNICAL FIELD
The present invention relates generally to compositions and methods useful for the study of cascades leading to the activation of nuclear factor &kgr;B (NF-&kgr;B) and for treating diseases associated with such pathways. The invention is more particularly related to a stimulus-inducible I&kgr;B kinase (IKK) signalsome, component I&kgr;B kinases and variants of such kinases. The present invention is also related to the use of a stimulus-inducible IKK signalsome or I&kgr;B kinase to identify antibodies and other agents that inhibit or activate signal transduction via the NF-&kgr;B pathway.
BACKGROUND OF THE INVENTION
Transcription factors of the NF&kgr;B/Rel family are critical regulators of genes involved in inflammation, cell proliferation and apoptosis (for reviews, see Verma et al.,
Genes Dev.
9:2723-35, 1995; Siebenlist,
Biochim. Biophys. Acta
1332:7-13, 1997; Baeuerle and Henkel,
Ann. Rev. Immunol.
12:141-79, 1994; Barnes and Karin,
New Engl. J. Med.
336, 1066-71, 1997; Baeuerle and Baltimore,
Cell
87:13-20, 1996; Grilli et al.,
NF-kB and Rel: Participants in a multiform transcriptional regulatory system
(Academic Press, Inc., 1993), vol. 143; Baichwal and Baeuerle,
Curr. Biol.
7:94-96, 1997). The prototype member of the family, NF&kgr;B, is composed of a dimer of p50 NF&kgr;B and p65 RelA (Baeuerle and Baltimore,
Cell
53:211-17, 1988; Baeuerle and Baltimore,
Genes Dev.
3:1689-98, 1989). NF-&kgr;B plays a pivotal role in the highly specific pattern of gene expression observed for immune, inflammatory and acute phase response genes, including interleukin 1, interleukin 8, tumor necrosis factor and certain cell adhesion molecules.
Like other members of the Rel family of transcriptional activators, NF-&kgr;B is sequestered in an inactive form in the cytoplasm of most cell types. A variety of extracellular stimuli including mitogens, cytokines, antigens, stress inducing agents, UV light and viral proteins initiate a signal transduction pathway that ultimately leads to NF-&kgr;B release and activation. Thus, inhibitors and activators of the signal transduction pathway may be used to alter the level of active NF-&kgr;B, and have potential utility in the treatment of diseases associated with NF-&kgr;B activation.
Activation of NF&kgr;B in response to each of these stimuli is controlled by an inhibitory subunit, I&kgr;B, which retains NF&kgr;B in the cytoplasm. I&kgr;B proteins, of which there are six known members, each contain 5-7 ankyrin-like repeats required for association with the NF&kgr;B/Rel dimer and for inhibitory activity (see Beg et al.,
Genes Dev.
7, 2064-70, 1993; Gilmore and Morin,
Trends Genet.
9, 427-33, 1993; Diaz-Meco et al.,
Mol. Cell. Biol.
13:4770-75, 1993; Haskill et al.,
Cell
65:1281-89, 1991). I&kgr;B proteins include I&kgr;B&agr; and I&kgr;B&bgr;.
NF&kgr;B activation involves the sequential phosphorylation, ubiquitination, and degradation of I&kgr;B. Phosphorylation of I&kgr;B is highly specific for target residues. For example, phosphorylation of the I&kgr;B protein I&kgr;B&agr; takes place at serine residues S32 and S36, and phosphorylation of I&kgr;B&bgr; occurs at serine residues S19 and S23. The choreographed series of modification and degradation steps results in nuclear import of transcriptionally active NF&kgr;B due to the exposure of a nuclear localization signal on NF&kgr;B that was previously masked by I&kgr;B (Beg et al.,
Genes Dev.
6:1899-1913, 1992). Thus, NF&kgr;B activation is mediated by a signal transduction cascade that includes one or more specific I&kgr;B kinases, a linked series of E1, E2 and E3 ubiquitin enzymes, the 26S proteasome, and the nuclear import machinery. The phosphorylation of I&kgr;B is a critical step in NF-&kgr;B activation, and the identification of an I&kgr;B kinase, as well as proteins that modulate its kinase activity, would further the understanding of the activation process, as well as the development of therapeutic methods.
Several protein kinases have been found to phosphorylate I&kgr;B in vitro, including protein kinase A (Ghosh and Baltimore,
Nature
344:678-82, 1990), protein kinase C (Ghosh and Baltimore,
Nature
344:678-82, 1990) and double stranded RNA-dependent protein kinase (Kumar et al.,
Proc. Natl. Acad. Sci. USA
91:6288-92, 1994). Constitutive phosphorylation of I&kgr;B&agr; by casein kinase II has also been observed (see Barroga et al.,
Proc. Natl. Acad. Sci. USA
92:7637-41, 1995). None of these kinases, however appear to be responsible for in vivo activation of NF-&kgr;B. For example, phosphorylation of I&kgr;B&agr; in vitro by protein kinase A and protein kinase C prevent its association with NF-&kgr;B, and phosphorylation by double-stranded RNA-dependent protein kinase results in dissociation of NF-&kgr;B. Neither of these conform to the effect of phosphorylation in vivo, where I&kgr;B&agr; phosphorylation at S32 and S36 does not result in dissociation from NF-&kgr;B.
Other previously unknown proteins with I&kgr;B kinase activity have been reported, but these proteins also do not appear to be significant activators in vivo. A putative I&kgr;B&agr; kinase was identified by Kuno et al.,
J. Biol. Chem.
270:27914-27919, 1995, but that kinase appears to phosphorylate residues in the C-terminal region of I&kgr;B&agr;, rather than the S32 and S36 residues known to be important for in vivo regulation. Diaz-Meco et al.,
EMBO J.
13:2842-2848, 1994 also identified a 50 kD I&kgr;B kinase, with uncharacterized phosphorylation sites. Schouten et al.,
EMBO J.
16:3133-44, 1997 identified p90
rski
as a putative I&kgr;B&agr; kinase; however, p90
rski
is only activated by TPA and phosphorylates I&kgr;B&agr; only on Ser32, which is insufficient to render I&kgr;B&agr; a target for ubiquitination. Finally, Chen et al,
Cell
84:853-862, 1996 identified a kinase that phosphorylates I&kgr;B&agr;, but that kinase was identified using a non-physiological inducer of I&kgr;B&agr; kinase activity and requires the addition of exogenous factors for in vitro phosphorylation.
Accordingly, there is a need in the art for an I&kgr;B kinase that possesses the substrate specificity and other properties of the in vivo kinase. There is also a need for improved methods for modulating the activity of proteins involved in activation of NF-&kgr;and for treating diseases associated with NF-&kgr;cB activation. The present invention fulfills these needs and further provides other related advantages.
SUMMARY OF THE INVENTION
Briefly stated, the present invention provides compositions and methods employing a large, multi-subunit IKK signalsome, or a component or variant thereof. In one aspect, the present invention provides an IKK signalsome capable of specifically phosphorylating I&kgr;B&agr; at residues S32 and S36, and I&kgr;K&bgr; at residues 19 and 23, without the addition of exogenous cofactors.
In a further related aspect, a polypeptide comprising a component of an IKK signalsome, or a variant of such a component, is provided, wherein the component has a sequence recited in SEQ ID NO:9. An isolated DNA molecule and recombinant expression vector encoding such a polypeptide, as well as a transfected host cell, are also provided.
In another aspect, methods for preparing an IKK signalsome are provided, comprising combining components of an IKK signalsome in a suitable buffer.
In yet another aspect, methods are provided for phosphorylating a substrate of an IKK signalsome, comprising contacting a substrate with an IKK signalsome or a component thereof, and thereby phosphorylating the substrate.
In a further aspect, the present invention provides a method for screening for an agent that modulates IKK signalsome activity, comprising: (a) contacting a candidate agent with an IKK signalsome, wherein the step of contacting is carried out under conditions and for a time sufficient to allow the candidate agent and the IKK signalsome to interact; and (b) subsequently measuring the ability of the candidate agent to modulate IKK signalsome activity.
Within a related aspect, the present invent
Barbosa Miguel
Li Jian Wu
Mercurio Frank
Murray Brion W.
Zhu Hengyi
Mathews, Collins, Shepher & McKay, P.A.
Patterson Jr. Charles L.
Signal Pharmaceuticals Inc.
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