Fig 1: TIN2 is phosphorylated by the mitotic kinase RSK2.(A) Detection of S330 phosphorylation of TIN2 with a phosphorylation-specific antibody in cells arrested with nocodazole and treated with kinase inhibitors. HeLa cells stably expressing wild-type Flag-TIN2 were treated with DMSO, H-89, BI-D1870, BI 2536 or VX-680 in the presence of either nocodazole (Noc) or vehicle (DMSO). Derived lysates were immunoprecipitated (IP) with an anti-Flag antibody, resolved by SDS-PAGE, and immunoblotted (IB) with an anti-Phos-S330 antibody or, as a loading control, an anti-TIN2 antibody. Representative of two experiments. (B) DNA profiles of HeLa cells treated with BI-D1870. HeLa cells treated with DMSO, nocodazole (Noc), or nocodazole+ BI-D1870 were harvested, stained with propidium iodide, and subjected to fluorescence-activated cell sorting (FACS) analysis. Representative of two experiments. (C) Detection of S295 and S330 phosphorylation of TIN2 by the Phos-tag reagent in asynchronous or nocodazole arrested cells with or without the RSK2 inhibitor BI-D1870. 293T cells were either untreated or treated with nocodazole (Noc), BI-D1870, or both compounds. Derived lysates were then subjected to immunoprecipitation (IP) with an anti-Flag antibody and resolved by SDS-PAGE in the presence of the Phos-tag reagent and immunoblotted (IB) with an anti-TIN2 antibody. The supershifted bands corresponding to S295, S330, or S295 and S330 phosphorylation, as well as the unphosphorylated TIN2 (UP), are denoted on the left. Representative of two experiments. (D) Detection of S295 and S330 phosphorylation of TIN2 by the Phos-tag reagent in asynchronous cells with ectopic RSK2 and/or the RSK2 inhibitor BI-D1870. 293T cells transiently transfected with Flag-TIN2 and the Y707A constitutively active mutant form of RSK2 (Flag-RSK2Y707A) were either left untreated or treated with RSK kinase inhibitor BI-D1870. Derived lysates were split into two portions. The first portions were subjected to immunoprecipitation (IP) with an anti-Flag antibody, resolved by SDS-PAGE in the presence of the Phos-tag reagent, and immunoblotted (IB) with an anti-TIN2 antibody. The supershifted bands corresponding to S295, S330, or S295 and S330 phosphorylation, as well as the unphosphorylated TIN2 (UP), are denoted on the left (top). The second portions were resolved by normal SDS-PAGE and immunoblotted with either an anti-Phospho-S6 antibody to monitor RSK2 kinase activity, or an anti-Tubulin antibody as a loading control (bottom). Representative of two experiments. (E) Detection of TIN2 phosphorylation by RSK2 in vitro. Recombinant maltose-binding protein (MBP) or N-terminal MBP-tagged TIN2 (MBP-TIN2) in the WT, S295A, S330A, or AA mutant configuration were captured with amylose resin and eluted with maltose. No protein (-) or equal amounts of the aforementioned purified MBP-TIN2 proteins were incubated with recombinant N-terminal 6His-tagged RSK2 (6His-RSK2) in the presence of ATP32, after which the reaction products were resolved by SDS-PAGE and either (top) exposed to autographic film or (bottom) stained with Coomassie Brilliant Blue (CBB staining). Phosphorylated (P32) MBP-TIN2 and a non-specific band (*) are denoted on the left top panel. MBP-TIN2 and MBP are denoted on the left bottom panel. Representative of two experiments.