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Biochem J. 2018 Jan 11;475(1):329-340. doi: 10.1042/BCJ20170579.

Assaying kinase activity of the TPL-2/NF-κB1 p105/ABIN-2 complex using an optimal peptide substrate.

Author information

Crick-GSK Biomedical LinkLabs, GlaxoSmithKline, Stevenage SG1 2NY, U.K.
Immune Cell Signalling Laboratory, The Francis Crick Institute, London NW1 1AT, U.K.
Bioinformatics and Biostatistics, The Francis Crick Institute, London NW1 1AT, U.K.
Structural Biology, Science Technology Platforms, The Francis Crick Institute, London NW1 1AT, U.K.
RD Platform Technology & Science, GlaxoSmithKline, Stevenage SG1 2NY, U.K.
Immune Cell Signalling Laboratory, The Francis Crick Institute, London NW1 1AT, U.K.


The MKK1/2 kinase tumour progression locus 2 (TPL-2) is critical for the production of tumour necrosis factor alpha (TNFα) in innate immune responses and a potential anti-inflammatory drug target. Several earlier pharmaceutical company screens with the isolated TPL-2 kinase domain have identified small-molecule inhibitors that specifically block TPL-2 signalling in cells, but none of these have progressed to clinical development. We have previously shown that TPL-2 catalytic activity regulates TNF production by macrophages while associated with NF-κB1 p105 and ABIN-2, independently of MKK1/2 phosphorylation via an unknown downstream substrate. In the present study, we used a positional scanning peptide library to determine the optimal substrate specificity of a complex of TPL-2, NF-κB1 p105 and ABIN-2. Using an optimal peptide substrate based on this screen and a high-throughput mass spectrometry assay to monitor kinase activity, we found that the TPL-2 complex has significantly altered sensitivities versus existing ATP-competitive TPL-2 inhibitors than the isolated TPL-2 kinase domain. These results imply that screens with the more physiologically relevant TPL-2/NF-κB1 p105/ABIN-2 complex have the potential to deliver novel TPL-2 chemical series; both ATP-competitive and allosteric inhibitors could emerge with significantly improved prospects for development as anti-inflammatory drugs.


TPL-2; high-throughput assay; inflammation; kinase

[Indexed for MEDLINE]
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