title: Detection and Differentiation of Threonine- and Tyrosine-Monophosphorylated Forms of ERK1/2 by Capillary Isoelectric Focusing-Immunoassay.
abstract: The extracellular signal regulated kinases ERK1/2 play important roles in the regulation of diverse cellular functions and have been implicated in several human diseases. In addition to the fully activated, diphosphorylated ERK1/2 protein, monophosphorylated forms of ERK1/2 have been observed, which may have distinct biological functions. We report here on the highly sensitive detection and differentiation of unphosphorylated, threonine-phosphorylated (pT), tyrosine-phosphorylated (pY) and diphosphorylated ERK1 and ERK2 by capillary isoelectric focusing followed by immunological detection (CIEF-immunoassay). Eight different phosphorylated and unphosphorylated forms of ERK1/2 were resolved according to charge. The unequivocal identification and differentiation of ERK1 and ERK2 forms monophosphorylated at either threonine or tyrosine was achieved by competitive blocking with specific phospho-peptides and different phosphorylation-sensitive antibodies. The suitability of the additional pT-ERK1/2 and pY-ERK1/2 differentiation for the time-resolved in-depth study of phospho-form distribution in response to specific stimuli is demonstrated in human neuroblastoma SH-SY5Y and monocytic THP-1 cell lines, and in human peripheral blood mononuclear cells.
authors.affiliation: 1] LVR-Hospital Essen, Department of Psychiatry and Psychotherapy, Faculty of Medicine, University of Duisburg-Essen, Essen, Germany [2] German Center for Neurodegenerative Diseases (DZNE), Research Site Goettingen, Germany [3] Dept. of Psychiatry and Psychotherapy, University Medical Center Goettingen (UMG), Georg-August-University Goettingen, Germany.
authors.forename: Inga
authors.initials: I
authors.lastname: Kraus
authors.affiliation: LVR-Hospital Essen, Department of Psychiatry and Psychotherapy, Faculty of Medicine, University of Duisburg-Essen, Essen, Germany.
authors.forename: Daniela
authors.initials: D
authors.lastname: Besong Agbo
authors.affiliation: Department of Neurology, University of Ulm, Germany.
authors.forename: Markus
authors.initials: M
authors.lastname: Otto
authors.affiliation: 1] Dept. of Psychiatry and Psychotherapy, University Medical Center Goettingen (UMG), Georg-August-University Goettingen, Germany [2] German Center for Neurodegenerative Diseases (DZNE), Research Site Goettingen, Germany.
authors.forename: Jens
authors.initials: J
authors.lastname: Wiltfang
authors.affiliation: 1] LVR-Hospital Essen, Department of Psychiatry and Psychotherapy, Faculty of Medicine, University of Duisburg-Essen, Essen, Germany [2] Dept. of Psychiatry and Psychotherapy, University Medical Center Goettingen (UMG), Georg-August-University Goettingen, Germany.
authors.forename: Hans
authors.initials: H
authors.lastname: Klafki
mesh_terms: D001920:Bradykinin / Q000494:pharmacology; D002460:Cell Line; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D007525:Isoelectric Focusing / Q000379:methods*; D007963:Leukocytes, Mononuclear / Q000187:drug effects / Q000378:metabolism; D019950:Mitogen-Activated Protein Kinase 1 / Q000032:analysis / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000032:analysis / Q000378:metabolism*; D016202:N-Methylaspartate / Q000494:pharmacology; D010766:Phosphorylation; D013912:Threonine / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism
chemical_list: D013912:Threonine; D014443:Tyrosine; D016202:N-Methylaspartate; C535150:MAPK1 protein, human; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D001920:Bradykinin