Putative Substrates of AKT in Multiple Myeloma Cells.

Abstract

The serine/threonine protein kinase AKT (also known as protein kinase B) is thought to be a key mediator of signal transduction processes. The AKT kinase is activated in myeloma tumor cells. Frequent AKT activation in situ in myeloma cells of patient bone marrow has been demonstrated. IL-6 is a known growth factor of myeloma cells. Recent evidence indicates that AKT plays a critical role in IL-6-dependent expansion of multiple myeloma clones. A method to identify serine kinase substrates has been described. In that method, an antibody specific for the phosphomotif generated by the kinase is used to isolate phosphorylated substrates by immunoprecipitation, and the isolated proteins are identified by tandem mass spectrometry of peptides. This method has been adopted in identifying putative substrates of AKT in mutiple myeloma cells. AF-10 cells of myeloma cell line were treated with IL-6 to activate AKT, and the putative AKT substrates were isolated by immunoprecipitation with PAS (phosphospecific Ser/Thr AKT substrate) antibody. The isolated proteins were identified by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. In Western blot probed with PAS antibody, intensities of putative AKT substrates increased with IL-6 treatment, but decreased when the cells were preincubated with wortmannin (a specific PI 3-kinase inhibitor) then treated with IL-6. Putative AKT substrates identified include: nonmuscle myosine-9, vimentin, and tubulin beta-2. All these proteins contain appropriate motifs of AKT substrates (R/KXRXXS/T, XXRXXS/T). Conformation studies and studies of the physiological significances of these putative substrates are undergoing. Recently, it has been reported that a novel isocoumarin derivative induces mitotic phase arrest and apoptosis of human mutiple myeloma cells by affecting tubulin assembly.