Development of monoclonal antibodies specific to ribosomal protein S6 kinase 2.
Ribosomal protein S6 kinase 2 (S6K2) is a serine/threonine kinase that belongs to the family of AGC kinases, which includes PKB/Akt, PKC, PDK1, and SGK1. Mammalian cells express two isoforms of S6K, termed S6K1 and S6K2. Each of these has nuclear and cytoplasmic spicing variants, which originate from different initiation start codons. Nuclear isoforms of S6K1 and S6K2 are slightly longer, as they possess additional sequences at the N-terminus with nuclear localization signals. Biochemical and genetic studies implicated S6Ks in the regulation of cell size, growth, and energy metabolism. Deregulation of S6K signaling has been linked to various human pathologies, making them excellent targets for drug discovery. The aim of this study was to produce monoclonal antibodies directed at the N-terminal regulatory region of S6K2, which shows very low homology to S6K1 or other members of the AGC family. To achieve this goal, two S6K2 fragments covering 1-64aa and 14-64aa N-terminal sequences were expressed in bacteria as GST/6His fusion proteins. Affinity purified recombinant proteins were used as antigens for immunization, hybridoma screening, and analysis of generated clones. We produced a panel of S6K2-specific antibodies, which recognized recombinant S6K2 proteins in ELISA and Western blot analysis. Further analysis of selected clones revealed that three clones, termed B1, B2, and B4, specifically recognized not only recombinant, but also endogenous S6K2 in Western blot analysis of HEK293 cell lysates. Specificity of B2 clone has been confirmed in additional commonly used immunoassays, including immunoprecipitation and immunocytochemistry. These properties make B2 MAb particularly valuable for elucidating signal transduction pathways involving S6K2 signaling under physiological conditions and in human pathologies.
Department of Cell Signalling, Institute of Molecular Biology and Genetics, NAS of Ukraine, Zabolotnogo str 150, Kyiv, Ukraine.
Lilia
L
Savinska
Oleksandr
O
Skorokhod
Olga
O
Klipa
Ivan
I
Gout
Valeriy
V
Filonenko
D000818:Animals; D058846:Antibodies, Monoclonal, Murine-Derived / Q000737:chemistry*; D015153:Blotting, Western; D005260:Female; D057809:HEK293 Cells; D006801:Humans; D006825:Hybridomas; D007150:Immunohistochemistry; D047468:Immunoprecipitation; D051379:Mice; D008807:Mice, Inbred BALB C; D010446:Peptide Fragments / Q000276:immunology*; D020033:Protein Isoforms / Q000276:immunology / Q000378:metabolism; D017434:Protein Structure, Tertiary; D038744:Ribosomal Protein S6 Kinases, 90-kDa / Q000276:immunology* / Q000378:metabolism
D058846:Antibodies, Monoclonal, Murine-Derived; D010446:Peptide Fragments; D020033:Protein Isoforms; D038744:Ribosomal Protein S6 Kinases, 90-kDa; C069330:ribosomal protein S6 kinase, 90kDa, polypeptide 3