Development of Monoclonal Antibodies Specific to Ribosomal Protein S6 Kinase 2

Abstract

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 Nterminal 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.

S6K2: The Neglected S6 Kinase Family Member

“…The kinase domain is followed toward the C-terminus by a kinase extension domain and a pseudo-substrate inhibitory region. The greatest degree of divergence between S6K1 and S6K2 lies in the C-and N-terminus, a fact that has enabled the development of S6K2-specific antibodies (9). The presence in the C-terminus of S6K2 of a nuclear localization sequence (NLS) means that this isoform is predominantly localized to the nuclei of quiescent cells (10).…”

PI3K/mTOR/S6K signaling pathway – new players and new functional links

“…For analysis of S6K1 and S6K2 expression level and kinases subcellular localization we generated a set of monoclonal and polyclonal antibodies specific for Nand C-terminal parts of S6K1/2 [11][12][13]. According to our data S6K1 and S6K2 have a tissue specific expression profile with the highest level in brain and testis respectively [14].…”