The field of this invention is detecting the activity of an enzyme.
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-xcexaB. In unstimulated cells, NF-xcexaB proteins form a complex with inhibitory molecules, the IxcexaB proteins, and are rendered inactive in the cytoplasm. In response to cytokines and other stimuli, the IxcexaB proteins are phosphorylated on specific serine residues. Delineating TNF and IL-1 signaling pathways for NF-xcexaB 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-xcexaB activation. We previously disclosed a family of IxcexaB kinases including a TRAF2-associated kinase activity (designated T2K) and the translation product of the KIAA0151 gene product (also known as IKKi and IKKxcex5) that phosphorylates the IxcexaB 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 IKKxcex1 and IKKxcex2 peptide substrates. We disclose here materials and methods for assaying for these novel specificities.
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. NMxe2x80x94013254 and NPxe2x80x94037386 (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).
IKKxcex1 is described in Regnier et al. 1997, Cell 90, 373-383; Genbank Accession Nos.AF012890 and AAC51662.1 (human).
IKKxcex2 is described in U.S. Pat. No. 5,939,302; Genbank Accession Nos.AF029xcexdand AAC51860.1 (human).
Song et al., U.S. Pat. No. 5,874,230 disclose a TRAF2-associated kinase.
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 SX1X2X3SX4 (SEQ ID NO:1) wherein X1 and X4 are aliphatic residues and both of the S residues are targets of the kinase. In particular aspects, X1 and X4 are L and F, respectively; X1-X4 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 IKKxcex1 and IKKxcex2. 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 NfxcexaB 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 IKKxcex3; 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.
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 SX1X2X3SX4 (SEQ ID NO:1), wherein X1 and X4 are aliphatic residues and both of the S residues are targets of the kinase, particularly wherein X1 and X4 are L and F, more particularly wherein X1-X4 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.
In more particular embodiments the substrate comprises an IKKxcex1 fragment including serines 176 and 180 or an IKKxcex2 fragment including serines 177 and 181; hence, there are ten IKKxcex1 fragments and ten IKKxcex2 fragments 15 residues in length (IKKxcex2 resides 167-181, 168-182, etc.). Particular substrates include IKKxcex1 fragment YAKDVDQGSLCTSFVGTLQYL (SEQ ID NO:2) and IKKxcex2 fragment YAKELDQGSLCTSFVGTLQYL (SEQ ID NO:3). Alternatively, native IKKxcex1 and IKKxcex2 substrates may be used.
In another embodiment, the T2K substrate comprises an IL-1 or TNF signaling cascade component other than IxcexaB 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 NfxcexaB protein selected from the group consisting of p50, p65, p49, cRel and ReIB; a natural human protein selected from the group consisting of I-TRAF and IKKxcex3 (also known as NEMO); 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. These enumerated components are all defined in the art and readily isolated or produced recombinantly using sequence available through public repositories such as Genbank. In addition, 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.
After forming the mixture, the method involves 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. Incubation conditions and periods are for kinase activity but also minimized to facilitate rapid, high-throughput screening. For continuous assays, the rate may be expressed in terms of a dynamic activity, whereas in most applications, the assays are discontinuous (e.g. have a fixed-time endpoint) so the rate is expressed as net kinase activity over a fixed time.
In a particular embodiment, the method is used to screen for agents which modulate the activity of the kinase, wherein 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. A wide variety of homogeneous and heterogeneous (solid phase) assays may be used, including fluorescent polarization assays, homogeneous time resolved fluorescence assays (HTRF), capture assays such as described by Strulovici in U.S. Pat. No. 5,759,787, etc. In particular embodiments, 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.
The following experimental section and examples are offered by way of illustration and not by way of limitation.
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