Production and characterisation of the TPL-2 / ABIN-2 / NF-κB1 p105 complex

TPL-2 functions as a MEK1/2 kinase that is an essential component of an ERK1/2 MAP kinase pathway activated by Toll-like receptors and members of the IL-1 and TNF receptor families. TPL-2 is required for production of the pro-inflammatory cytokine TNF by macrophages after lipopolysaccharide stimulation. Recent work from the Ley laboratory has demonstrated that TPL-2 induces TNF secretion by macrophages independently of MEK1/2 and ERK1/2 phosphorylation. However, the target protein that TPL-2 phosphorylates to control TNF production is not known. The aim of this project was to determine the primary amino acid sequence specificity of TPL-2 by peptide library screening to allow bioinformatic identification of novel TPL-2 substrates. To do this, a strategy was developed to purify TPL-2, complexed with its cellular binding partners ABIN-2 and NF-κB1 p105, from lysates of transiently transfected HEK293 cells. This method generated large amounts of highly purified TPL-2 complex, which was soluble, stable, and catalytically active. Interestingly, mass spectrometric analysis of highly purified TPL-2 complex identified several novel binding partners, including mTORC2, IRAK1, and TRIM25, which may be functionally linked to the TPL-2/ABIN-2/NF-κB1 p105 complex. Screening of a positional scanning peptide library demonstrated that TPL-2 complex had an unusual amino acid specificity, in which negatively charged amino acids were preferred at all five positions N-terminal to the phosphorylation site. A synthetic peptide based on this optimal motif was specifically phosphorylated by the TPL-2 complex much more efficiently than a positive control MEK peptide, strongly suggesting that the screen had worked. A Scansite search of protein sequence databases with the positional-specific scoring matrix for TPL-2 identified a number of potential novel substrates, including several proteins involved in intracellular membrane trafficking. A strategy for future validation of potential TPL-2 substrates and the possibility of using an optimal TPL-2 substrate peptide for high throughput inhibitor screening are discussed