Sam68 interacts with IRS1

Sam68 (Src associated in mitosis) is a RNA binding protein that links cellular signaling to RNA processing. In previous studies we found that insulin promotes Sam68 relocalization in the cytoplasm allowing Sam68 to associate with p85PI3K, Grb2, GAP and probably the insulin receptor (IR), modulating insulin action positively. In the present work, we wanted to define the role of Sam68 in the first stages of IR signaling. Both BRET and co-immunoprecipitation assays have been used for the study of Sam68 binding to IR, IRS1 and p85-PI3K. BRET saturation experiments indicated, for the first time, that Sam68 associates with IRS1 in basal condition. To map the region of Sam68 implicated in the interaction with IRS1, different Sam68 mutants deleted in the proline-rich domains were used. The deletion of P0, P1 and P2 proline rich domains in N-terminus as well as P4 and P5 in C-terminus of Sam68 increased BRET 50, thus indicating that the affinity of Sam68 for IRS1 is lower when these domains are missing. Moreover, in IR-transfected HEK-293 cells, BRET saturation experiment indicated that insulin increases the affinity between Sam68-Rluc and IRS1-YFP. In conclusion, our data indicate that Sam68 interacts with IRS-1 in basal conditions, and insulin increases the affinity between these two partners.