title: A comparison of phosphospecific affinity reagents reveals the utility of recombinant Forkhead-associated domains in recognizing phosphothreonine-containing peptides.
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 commercially available antibodies. All FHA domains were shown to be phosphorylation-dependent and phosphothreonine-specific in their binding, unlike several commercial monoclonal and polyclonal antibodies. Both the pThr and the residue at the pT+3 position were major factors in defining the specificity of the FHA domains.
authors.affiliation: Department of Biological Sciences, University of Illinois at Chicago, Chicago, IL 60607, USA.
authors.forename: Leon A
authors.initials: LA
authors.lastname: Venegas
authors.affiliation: Department of Biological Sciences, University of Illinois at Chicago, Chicago, IL 60607, USA.
authors.forename: Kritika
authors.initials: K
authors.lastname: Pershad
authors.affiliation: Department of Biological Sciences, University of Illinois at Chicago, Chicago, IL 60607, USA.
authors.forename: Oluwadamilola
authors.initials: O
authors.lastname: Bankole
authors.affiliation: Department of Biological Sciences, University of Illinois at Chicago, Chicago, IL 60607, USA.
authors.forename: Noman
authors.initials: N
authors.lastname: Shah
authors.affiliation: Department of Biological Sciences, University of Illinois at Chicago, Chicago, IL 60607, USA. Electronic address: bkay@uic.edu.
authors.forename: Brian K
authors.initials: BK
authors.lastname: Kay
mesh_terms: D000345:Affinity Labels; D000595:Amino Acid Sequence; D000906:Antibodies; D001709:Biotechnology; D051858:Forkhead Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D006801:Humans; D008958:Models, Molecular; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D010769:Phosphothreonine / Q000737:chemistry*; D000072417:Protein Domains / Q000235:genetics / Q000276:immunology; D054730:Protein Interaction Domains and Motifs / Q000235:genetics / Q000276:immunology; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology
chemical_list: D000345:Affinity Labels; D000906:Antibodies; D051858:Forkhead Transcription Factors; D019151:Peptide Library; D010455:Peptides; D011994:Recombinant Proteins; D010769:Phosphothreonine