SGK and 14-3-3 protein are involved in the serine/threonine phosphorylation mechanism for TPO/MPL signal transduction

Thrombopioetin (TPO), the critical regulator of platelet production, acts b y binding to its cell surface receptor, c-Mpl. Yeast two-hybrid screening w as performed to isolate the proteins interacting with the cytoplasmic domai n of c-Mpl. 48 positive clones were isolated from 5 X 10(6) independent tra nsformants. The results of sequence analysis demonstrate that they represen t 13 different protein encoding sequences. Among them there are a partial c oding sequence of serine/threonine protein kinase SGK (serum and glucocorti coid-inducible kinase) and 14-3-3 theta protein partial coding sequence. GS T-pull-down assay and co-immunoprecipitation in mammal cells have confirmed the Interaction between these two proteins and c-Mpl. By constructing a se ries of deleted c-Mpl cytoplasmic domain, the interaction region in c-Mpl c ytoplasmic tail was localized In amino acids 523-554. At the same time, the directed interaction between SGK and 14-3-3 proteins also has been verifie d by yeast two-hybrid assay. The present note is the first time to report t hat two proteins act with c-Mpl at the same time and put forward that SGK a nd 14-3-3 protein may be Involved in the serine/threonine phosphorylation m echanism for signal transduction.