T2k kinase assays

Details T2k kinase assays T2k kinase assays

2000-03-13

2002-04-02

Leary, Louise N. (Department: 1623)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving hydrolase

C435S004000, C435S968000, C435S007100, C435S015000, C530S300000

Reexamination Certificate

active

06365366

ABSTRACT:

FIELD OF THE INVENTION
The field of this invention is detecting the activity of an enzyme.
BACKGROUND
Inflammatory cytokines IL-1 and TNF exert diverse biological activities by altering gene expression in the cells, a function mediated in part by transcription factor NF-&kgr;B. In unstimulated cells, NF-&kgr;B proteins form a complex with inhibitory molecules, the I&kgr;B proteins, and are rendered inactive in the cytoplasm. In response to cytokines and other stimuli, the I&kgr;B proteins are phosphorylated on specific serine residues. Delineating TNF and IL-1 signaling pathways for NF-&kgr;B activation has implicated the TRAF molecules as converging point for different cytokines, with TRAF2 being involved in TNF- and TRAF6 in IL-1-induced NF-&kgr;B activation. We previously disclosed a family of I&kgr;B kinases including a TRAF2-associated kinase activity (designated T2K) and the translation product of the KIAA0151 gene product (also known as IKKi and IKK&egr;) that phosphorylates the I&kgr;B molecules on the specific regulatory serine residues. We have now found that T2K and IKKi in fact have alternative substrate specificities and alternative physiologically relevant substrate targets, particularly IKK&agr; and IKK&bgr; peptide substrates. We disclose here materials and methods for assaying for these novel specificities.
RELEVANT LITERATURE
T2K (also known as TBK1) is described by Cao et al. (U.S. Pat. No. 5,776,717) and Pomerantz and Baltimore, EMBO J. 18 (23), 6694-6704, 1999; Genbank Accession No. NM
—
013254 and NP
—
037386 (human); AF191839 and AAF05990.1 (mouse).
IKKi is described by Nagase et al., DNA Res.,1995, 2, 167-174; Genbank Accession Nos. D63485 and BAA09772 (human); and by Shimada et al., Int Immunol. 1999 Aug;11(8):1357-62; Genbank Accession Nos.AB016590 and BAA85155.1 (human); AB016589 and BAA85154.1 (mouse).
IKK&agr; is described in Regnier et al. 1997, Cell 90, 373-383; Genbank Accession Nos.AF012890 and AAC51662.1 (human).
IKK&bgr; is described in U.S. Pat. No. 5,939,302; Genbank Accession Nos.AF029&ngr;and AAC51860.1 (human).
Song et al., U.S. Pat. No. 5,874,230 disclose a TRAF2-associated kinase.
SUMMARY OF THE INVENTION
The invention provides compositions and methods for detecting kinase activity. The subject compositions include in vitro mixtures comprising or consisting essentially of an isolated, active T2K kinase and an isolated, functional T2K substrate. In one embodiment, the substrate comprises or consists essentially of SX
1
X
2
X
3
SX
4
(SEQ ID NO:1) wherein X
1
and X
4
are aliphatic residues and both of the S residues are targets of the kinase. In particular aspects, X
1
and X
4
are L and F, respectively; X
1
-X
4
are L, C, T and F, respectively; the substrate comprises a sequence selected from the group consisting of YAKDVDQGSLCTSFVGTLQYL (SEQ ID NO:2) and YAKELDQGSLCTSFVGTLQYL (SEQ ID NO:3); and/or the substrate comprises a natural human kinase selected from the group consisting of IKK&agr; and IKK&bgr;. In another embodiment, the substrate comprises or consists essentially of an IL-1 or TNF signaling cascade component selected from the group consisting of: an isolated natural human IL-1 Rc superfamily receptor selected from the group consisting of IL-1RP1, IL-1RP2, IL-1RP3, IL-18Rc, and TLR2 and TLR4; natural human Nf&kgr;B protein selected from the group consisting of p50, p65, p49, cRel and RelB; a natural human protein selected from the group consisting of I-TRAF and IKK&ggr;; a natural human protein selected from the group consisting of TRAF5 and TRAF6; a natural human protein selected from the group consisting of RIP, IRAK, MYD88 and TRADD; and a natural human TNFRc1 protein selected from the group consisting of CD40 and CD30.
The subject methods for detecting kinase activity comprise the steps of forming a subject mixture, incubating the mixture under conditions whereby the kinase phosphorylates the substrate at a first rate, and detecting the first rate as an indication of the kinase activity. In particular aspects, the mixture comprises an agent and but for the presence of the agent, the kinase phosphorylates the substrate at a second rate, wherein a significant difference between the first and second rate is an indication that the agent modulates the kinase activity. In more particular aspects, the detecting step is a chemiluminescent assay comprising detecting the phosphorylated substrate with a specific, labeled probe, particularly wherein the phosphorylated substrate is immobilized and detecting the phosphorylated substrate is effected indirectly by detecting a substrate-specific primary antibody with the probe, wherein the probe is a labeled secondary antibody specific for the primary antibody.
DETAILED DESCRIPTION OF THE INVENTION
The invention provides a method for detecting kinase activity comprising the steps of (a) forming a mixture comprising an active T2K kinase and a T2K substrate; (b) incubating the mixture under conditions whereby the kinase phosphorylates the substrate at a first rate; and (c) detecting the first rate as an indication of the kinase activity. As used herein, the term T2K kinase describes the two related natural proteins T2K and natural IKKi, however insubstantial variants such as minor truncations, etc. which retain substantially the same kinase activity and specificity of the native proteins are clearly equivalents in the disclosed assays. The T2K kinases may be produced recombinantly and/or isolated from any convenient source, particularly human or murine cells, see e.g. Molecular Cloning, A Laboratory Manual (Sambrook, et al. Cold Spring Harbor Laboratory), Current Protocols in Molecular Biology (Eds. Ausubel, et al., Greene Publ. Assoc., Wiley-Interscience, NY).
The T2K substrate is of sufficient length and sequence to provide a functional substrate for the T2K kinase under assay conditions. Depending on the assay structure, the substrate is generally a peptide of at least 5, preferably at least 10, more preferably at least 15 residues in length. In many cases, cost and specificity are optimized by using peptides of fewer than 100 residues, preferably fewer than 50 residues, more preferably fewer than 25 residues, as opposed to native protein substrates.
In one embodiment, the T2K substrate comprises SX
1
X
2
X
3
SX
4
(SEQ ID NO:1), wherein X
1
and X
4
are aliphatic residues and both of the S residues are targets of the kinase, particularly wherein X
1
and X
4
are L and F, more particularly wherein X
1
-X
4
are L, C, T and F, respectively. Table 1 provides exemplary peptides providing requisite T2K substrate specificity.
Table 1. Exemplary peptides providing requisite T2K substrate specificity.
Peptide
Sequence Identifier
T2K substrate specificity
GGSLCTSF
(SEQ ID NO:4)
+++

GGSLMTSF
(SEQ ID NO:5)
+++

GGSFCTSL
(SEQ ID NO:6)
+++

DQGSFCTSL
(SEQ ID NO:7)
+++

VDQGSLAASFVGT
(SEQ ID NO:8)
+++

VDQGSMAASLVGT
(SEQ ID NO:9)
+++

VDQGSIAASFVGT
(SEQ ID NO:10)
+++

AKDVDQGSVCTSFVGTLQY
(SEQ ID NO:11)
+++

AKDVDQGSLCTSLVGTLQY
(SEQ ID NO:12)
+++

AKDVDQGSFCTSLVGTLQY
(SEQ ID NO:13)
+++
In more particular embodiments the substrate comprises an IKK&agr; fragment including serines 176 and 180 or an IKK&bgr; fragment including serines 177 and 181; hence, there are ten IKK&agr; fragments and ten IKK&bgr; fragments 15 residues in length (IKK&bgr; resides 167-181, 168-182, etc.). Particular substrates include IKK&agr; fragment YAKDVDQGSLCTSFVGTLQYL (SEQ ID NO:2) and IKK&bgr; fragment YAKELDQGSLCTSFVGTLQYL (SEQ ID NO:3). Alternatively, native IKK&agr; and IKK&bgr; substrates may be used.
In another embodiment, the T2K substrate comprises an IL-1 or TNF signaling cascade component other than I&kgr;B and TRAF2, such as a natural human IL-1Rc superfamily receptor selected from the group consisting of IL-1RP1, IL-1RP2, IL-1RP3, IL-18Rc, TLRC2 and TLR4; a natural human Nf&kgr;B protein selected from the group consisting of p50, p65, p49, cRel and ReIB

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