A comparison of phosphospecific affinity reagents reveals the utility of recombinant Forkhead-associated domains in recognizing phosphothreonine-containing peptides

New Biotechnology
Leon A VenegasBrian K Kay

Abstract

Phosphorylation is an important post-translational event that has a wide array of functional consequences. With advances in the ability of various technologies in revealing and mapping new phosphosites in proteins, it is equally important to develop affinity reagents that can monitor such post-translational modifications in eukaryotic cells. While monoclonal and polyclonal antibodies have been shown to be useful in assessing the phosphoproteome, we have expanded our efforts to exploit the Forkhead-associated 1 (FHA1) domain as scaffold for generating recombinant affinity reagents that recognize phosphothreonine-containing peptides. A phage display library of FHA1 variants was screened by affinity selection with 15 phosphothreonine-containing peptides corresponding to various human transcription factors and kinases, including human Myc, calmodulin-dependent protein kinase II (CaMKII), and extracellular-signal regulated kinases 1 and 2 (ERK1/2). The library yielded binding variants against 10 targets (66% success rate); success was largely determined by what residue occurred at the +3 position (C-terminal) to the pThr moiety (i.e., pT+3). The FHA domains binding Myc, CaMKII, and ERK1/2 were characterized and compared against comm...Continue Reading

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Citations

Oct 26, 2018·International Journal of Molecular Sciences·Jennifer E McGinnisBrian K Kay
May 13, 2021·Science Signaling·Rashida Abdul-GaniyuDavid E Stone
Aug 11, 2021·Open Biology·Lucy RemnantWilliam C Earnshaw
Nov 7, 2017·New Biotechnology·Jennifer E McGinnis, Brian K Kay

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