The erythroid differentiation of K562 cells depends on the nuclear translocation of Akt

Previous findings have demonstrated that Erythropoietin (EPO) treatment during erythroid differentiation of human erythroleukaemia cells activates PI3K (1,2) and that an active PI3K translocates into the nucleus (3). When PI3K activity is inhibited, cellular differentiation is blocked, showing the requirement of this kinase during cell differentiation (3). It is known that in the PI3K signalling pathway, the Ser/Thr kinase Akt is a downstream enzyme involved in many cellular processes including differentiation (4). Therefore we wanted to analyze the role of nuclear Akt during the erythroid differentiation process in K562 erythroleukaemia cells, following EPO treatment. Materials and methods: K562 cells coltures, EPO treatment, preparation of whole cell homogenates, isolation of nuclei, immunoblotting, Akt kinase assay and immunocytochemistry were accomplished as previously reported (3). Results: In whole cells homogenates after 10 min of EPO exposure we observed the highest level of Ser473 phosphorylated Akt. This kinase increases rapidly and transiently in response to EPO treatment its intranuclear amount, with a delay when compared with the cell homogenates. Infact we observed that into the nucleus Ser473 phosphorylated Akt was observable at highest levels after 15 min. In situ analysis by means of immunocytochemistry showed the nuclear translocation of Akt and of its phosphorylated form on Ser473 that peaked at 15 min and returned to control conditions after 30 min. Enzyme translocation and erythroid differentiation were blocked by the specific Akt pharmacological inhibitor acting on its PH activation domain, 1L-6-Hydroxymethyl-chiro-inositol 2-[(R)-2-O-methyl-3-O-octadecylcarbonate] (from Alexis). Almost all Akt kinase observed into the nucleus was phosphorylated on Serine 473 of the hydrophobic regulative activation domain, as demonstrated by using double round of immunoprecipitation (IP) experiments in which the first round of IP was performed with anti phospho- Akt (Ser473) antibody and the second round with total anti Akt antibody. Using kinase assay we observed a peak of Akt activity after 10 min of EPO treatment in whole cell homogenates and an intranuclear peak of activity after 15 min of EPO exposure, that were both blocked by pre-treatment of cells by Akt inhibitor. Conclusions: EPO induces the nuclear translocation of Akt in a time dependent and ordered manner. Furthermore, when cells were treated with an Akt inhibitor, the nuclear translocation of the enzyme was blocked as well as the differentiation process. These findings strongly suggest the requirement of active Akt translocation into the nucleus as an important step in the PI3K/Akt signaling pathway to enter into EPO-mediated erythroid differentiation.