title: Monoclonal 1- and 3-Phosphohistidine Antibodies: New Tools to Study Histidine Phosphorylation.
abstract: Histidine phosphorylation (pHis) is well studied in bacteria; however, its role in mammalian signaling remains largely unexplored due to the lack of pHis-specific antibodies and the lability of the phosphoramidate (P-N) bond. Both imidazole nitrogens can be phosphorylated, forming 1-phosphohistidine (1-pHis) or 3-phosphohistidine (3-pHis). We have developed monoclonal antibodies (mAbs) that specifically recognize 1-pHis or 3-pHis; they do not cross-react with phosphotyrosine or the other pHis isomer. Assays based on the isomer-specific autophosphorylation of NME1 and phosphoglycerate mutase were used with immunoblotting and sequencing IgG variable domains to screen, select, and characterize anti-1-pHis and anti-3-pHis mAbs. Their sequence independence was determined by blotting synthetic peptide arrays, and they have been tested for immunofluorescence staining and immunoaffinity purification, leading to putative identification of pHis-containing proteins. These reagents should be broadly useful for identification of pHis substrates and functional study of pHis using a variety of immunological, proteomic, and biological assays.
authors.affiliation: Molecular and Cell Biology Laboratory, Salk Institute for Biological Studies, La Jolla, CA 92037, USA.
authors.forename: Stephen Rush
authors.initials: SR
authors.lastname: Fuhs
authors.affiliation: Molecular and Cell Biology Laboratory, Salk Institute for Biological Studies, La Jolla, CA 92037, USA.
authors.forename: Jill
authors.initials: J
authors.lastname: Meisenhelder
authors.affiliation: Molecular and Cell Biology Laboratory, Salk Institute for Biological Studies, La Jolla, CA 92037, USA; Department of Chemical Physiology, The Scripps Research Institute, La Jolla, CA 92037, USA.
authors.forename: Aaron
authors.initials: A
authors.lastname: Aslanian
authors.affiliation: Molecular and Cell Biology Laboratory, Salk Institute for Biological Studies, La Jolla, CA 92037, USA.
authors.forename: Li
authors.initials: L
authors.lastname: Ma
authors.affiliation: Molecular Neurobiology Laboratory, Salk Institute for Biological Studies, La Jolla, CA 92037, USA.
authors.forename: Anna
authors.initials: A
authors.lastname: Zagorska
authors.affiliation: Tucson Innovation Center, Sanofi, Tucson, AZ 85755, USA.
authors.forename: Magda
authors.initials: M
authors.lastname: Stankova
authors.affiliation: Tucson Innovation Center, Sanofi, Tucson, AZ 85755, USA.
authors.forename: Alan
authors.initials: A
authors.lastname: Binnie
authors.affiliation: Tucson Innovation Center, Sanofi, Tucson, AZ 85755, USA.
authors.forename: Fahad
authors.initials: F
authors.lastname: Al-Obeidi
authors.affiliation: Tucson Innovation Center, Sanofi, Tucson, AZ 85755, USA.
authors.forename: Jacques
authors.initials: J
authors.lastname: Mauger
authors.affiliation: Molecular Neurobiology Laboratory, Salk Institute for Biological Studies, La Jolla, CA 92037, USA.
authors.forename: Greg
authors.initials: G
authors.lastname: Lemke
authors.affiliation: Department of Chemical Physiology, The Scripps Research Institute, La Jolla, CA 92037, USA.
authors.forename: John R
authors.initials: JR
authors.lastname: Yates
authors.affiliation: Molecular and Cell Biology Laboratory, Salk Institute for Biological Studies, La Jolla, CA 92037, USA. Electronic address: hunter@salk.edu.
authors.forename: Tony
authors.initials: T
authors.lastname: Hunter
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal; D018385:Centrosome; D002853:Chromatography, Liquid; D006367:HeLa Cells; D006639:Histidine / Q000378:metabolism*; D006801:Humans; D008956:Models, Chemical; D010455:Peptides / Q000032:analysis; D010766:Phosphorylation; D064047:Spindle Poles; D053719:Tandem Mass Spectrometry
chemical_list: D000911:Antibodies, Monoclonal; D010455:Peptides; D006639:Histidine